Repeated short read sequencing within cell samples

Abstract

The present disclosure provides methods for in situ multiplex and multi-omics detection and identification using coded padlock probes. The method includes detecting both RNA and polypeptide in a cell sample using both an RNA-specific padlock probe and a polypeptide-specific padlock probe. The two types of probes include barcodes that uniquely identify the RNA or polypeptide detected by the padlock probe. The two types of probes also include batch-specific sequencing primer binding sites to enable sequencing of a subset of desired concatemer template molecules. The use of batch-specific sequencing primers reduces over-crowded signals and images to produce strong and distinguishable optical images. Multiple rounds of sequencing are performed on the same cell sample by using different batches of specific sequencing primers, so that multiple and multi-omics sequencing can reveal numerous target RNAs and polypeptides encoded by the target RNAs.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 332,690, filed April 20, 2022, and U.S. Provisional Application No. 63 / 334,023, filed April 22, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named 52933-752_601_SL.xml, which was created on April 17, 2023 and is 12,924 bytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. Technical Field

[0005] The present disclosure provides compositions, devices, and methods for performing iterative short-read sequencing within a cell sample. In some embodiments, iterative short-read sequencing can be used to discover the RNA content of a cell sample. Summary of the Invention

[0006] In one aspect, provided herein is a method for in situ detection of at least two target nucleic acid sequences in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, the method comprising: (a) providing a biological sample comprising: (i) a first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or a reverse complement thereof or a portion thereof; and (ii) a second nucleic acid molecule comprising the second target nucleic acid sequence or a portion thereof, or a reverse complement thereof or a portion thereof; (b) determining in situ the sequence of: (i) the first nucleic acid molecule or a portion thereof in the biological sample to generate a nucleic acid sequence that is complementary to and In some embodiments, the biological sample is fixed and permeabilized. In some embodiments, the method further comprises generating in situ: (a) a first complementary DNA (cDNA) molecule by reverse transcription of a first messenger RNA (mRNA) molecule within a biological sample, wherein the first cDNA molecule or the first mRNA molecule comprises a first target nucleic acid sequence or a reverse complement of the first target nucleic acid sequence; and / or (b) a second cDNA molecule by reverse transcription of a second mRNA molecule within the biological sample, wherein the second cDNA molecule or the second mRNA molecule comprises a second target nucleic acid sequence or a reverse complement of the second target nucleic acid sequence. In some embodiments, the method further comprises: (a) contacting the first target nucleic acid sequence or its reverse complement in situ with a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target nucleic acid sequence or its reverse complement, so that the first oligonucleotide forms a circular structure having a gap between the first end and the second end; and / or (b) contacting the second target nucleic acid sequence or its reverse complement in situ with a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target nucleic acid sequence or its reverse complement, so that the second oligonucleotide forms a circular structure having a gap between the first end and the second end. In some embodiments, wherein: (a) the first target nucleic acid sequence comprises a first cDNA molecule or a first mRNA molecule; and / or (b) the second target nucleic acid sequence comprises a second cDNA molecule or a second mRNA molecule.In some embodiments, the gap of the first oligonucleotide or the second oligonucleotide has the size of one nucleotide. In some embodiments, the gap of the first oligonucleotide or the second oligonucleotide has the size of at least two nucleotides. In some embodiments, the first oligonucleotide further includes a first identification sequence for identifying the first target nucleic acid sequence, and the second oligonucleotide further includes a second identification sequence for identifying the second target nucleic acid sequence. In some embodiments, the first oligonucleotide and the second oligonucleotide further include a nucleic acid sequence that is a complementary sequence to the nucleic acid sequence of a sequencing primer. In some embodiments, the first oligonucleotide and the second oligonucleotide further include a nucleic acid sequence that is a complementary sequence to the nucleic acid sequence of a primer for nucleic acid amplification. In some embodiments, the primer for nucleic acid amplification is a primer for rolling circle amplification (RCA) for producing concatemers, wherein the concatemer comprises at least two repetitions of the target nucleic acid sequence in at least two target nucleic acid sequences or a portion thereof or its reverse complementary sequence. In some embodiments, wherein the first oligonucleotide and the second oligonucleotide further comprise a reverse complement of a compacted oligonucleotide, wherein: (a) the first segment of the compacted oligonucleotide is complementary to and binds to the first portion of the concatemer; and (b) the second segment of the compacted oligonucleotide is complementary to and binds to the second portion of the concatemer to cause a reduction in size or a change in shape of the concatemer.

[0007] In some embodiments, the method further comprises: (a) joining the first end and the second end of the first oligonucleotide in situ to produce a first circular oligonucleotide in the biological sample; and (b) joining the first end and the second end of the second oligonucleotide in situ to produce a second circular oligonucleotide in the biological sample. In some embodiments, wherein: (a) joining the first end and the second end of the first oligonucleotide comprises joining the first end and the second end of the first oligonucleotide by a first nucleic acid enzyme; and (b) joining the first end and the second end of the second oligonucleotide comprises joining the first end and the second end of the second oligonucleotide by a second nucleic acid enzyme, wherein the first nucleic acid enzyme and the second nucleic acid enzyme are the same type of enzyme. In some embodiments, wherein the first nucleic acid enzyme and the second nucleic acid enzyme comprise a nucleic acid ligase, a nucleic acid ligation enzyme, a nucleic acid polymerase, a nucleic acid polymerase, or a combination thereof. In some embodiments, the method further comprises amplifying in situ: (a) the first circular oligonucleotide to produce a first nucleic acid molecule; and / or (b) the second circular oligonucleotide to produce a second nucleic acid molecule. In some embodiments, wherein the amplification comprises rolling circle amplification (RCA), wherein the first nucleic acid molecule comprises a first concatemer and the second nucleic acid molecule comprises a second concatemer. In some embodiments, wherein: (a) the first concatemer comprises at least two repeats of a first unit nucleic acid sequence, the first unit nucleic acid sequence comprising a first target nucleic acid sequence or a portion thereof, or a reverse complement sequence of the first target nucleic acid sequence or a portion thereof; and / or (b) the second concatemer comprises at least two repeats of a second unit nucleic acid sequence, the second unit nucleic acid sequence comprising a second target nucleic acid sequence or a portion thereof, or a reverse complement sequence of the second target nucleic acid sequence or a portion thereof. In some embodiments, wherein the first concatemer further comprises a first identification sequence for identifying the first target nucleic acid sequence, or a sequencing primer or a reverse complement sequence thereof, wherein the second concatemer further comprises a second identification sequence for identifying the second target nucleic acid sequence, or a sequencing primer or a reverse complement sequence thereof. In some embodiments, wherein the first concatemer further comprises a first compacted oligonucleotide, wherein: (a) the first segment of the first compacted oligonucleotide is complementary to and binds to the first portion of the first concatemer; and (b) the second segment of the first compacted oligonucleotide is complementary to and binds to the second portion of the first concatemer, thereby causing the first concatemer to decrease in size or change in shape. In some embodiments, wherein the second concatemer further comprises a second compacted oligonucleotide, wherein: (a) the first segment of the second compacted oligonucleotide is complementary to and binds to the first portion of the second concatemer; and (b) the second segment of the second compacted oligonucleotide is complementary to and binds to the second portion of the second concatemer, resulting in a reduction in size or a change in shape of the second concatemer.In some embodiments, wherein determining comprises: (a) determining the sequence of a first nucleic acid molecule or a portion thereof, wherein the first nucleic acid molecule or a portion thereof consists of 2-30 nucleotides; and / or (b) determining the sequence of a second nucleic acid molecule or a portion thereof, wherein the second nucleic acid molecule or a portion thereof consists of 2-30 nucleotides. In some embodiments, wherein the 2-30 nucleotides in the first nucleic acid molecule or a portion thereof comprise a first identification sequence, and the 2-30 nucleotides in the second nucleic acid molecule or a portion thereof comprise a second identification sequence. In some embodiments, wherein the 2-30 nucleotides in the first nucleic acid molecule or a portion thereof further comprise at least a portion of the sequence of a first cDNA molecule or a first mRNA molecule, and the 2-30 nucleotides in the second nucleic acid molecule or a portion thereof further comprise at least a portion of the sequence of a second cDNA molecule or a second mRNA molecule. In some embodiments, the determining comprises: (a) contacting a first concatemer with a polymerase, a plurality of nucleotides, and a primer sequence that is complementary to a portion of the first concatemer under conditions sufficient to form a binding complex, the binding complex comprising a polymerase, a nucleotide from the plurality of nucleotides that is complementary to and bound to a nucleotide unit of the first concatemer, and the first concatemer hybridized to the primer sequence, wherein the nucleotide comprises a fluorescent label and a removable blocking group at the 3' carbon position of the sugar moiety; (b) incorporating the nucleotide into the 3' end of the primer sequence; and (c) identifying the nucleobase of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide. In some embodiments, the determining comprises: (a) contacting the second concatemer with a polymerase, a plurality of nucleotides, and a primer sequence that is complementary to a portion of the second concatemer under conditions sufficient to form a binding complex, the binding complex comprising the polymerase, a nucleotide from the plurality of nucleotides that is complementary to and bound to a nucleotide unit of the second concatemer, and the second concatemer hybridized to the primer sequence, wherein the nucleotide comprises a fluorescent label and a removable blocking group at the 3' carbon position of the sugar moiety; (b) incorporating the nucleotide into the 3' end of the primer sequence; and (c) identifying the nucleobase of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide.

[0008] In some embodiments, wherein determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar portion. In some embodiments, wherein the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, wherein a plurality of nucleotides are composed of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of one type of nucleotide in the group is different from the wavelength of the light emitted by the fluorescent label of another type of nucleotide in the group. In some embodiments, wherein the plurality of nucleotides comprises at least two types of nucleotides, wherein the at least two types of nucleotides are selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, wherein the fluorescent label of one type of nucleotide in the group emits light at a wavelength that is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group. In some embodiments, wherein determining comprises: (a) contacting the two first concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to portions of the first concatemers under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two first concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein each of the at least two nucleotide portions is complementary to and binds to a nucleotide in each of the two first concatemers; (b) detecting the multivalent binding complex via the label of the nucleotide conjugates; and (c) identifying the nucleobases of the nucleotides in the two first concatemers, each of which is complementary to and binds to each of two of the at least two nucleotide portions of the nucleotide conjugates. In some embodiments, the determining comprises: (a) contacting the two second concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences that are complementary to portions of the second concatemers under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two second concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein each of the at least two nucleotide portions is complementary to and binds to a nucleotide in each of the two second concatemers; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; and (c) identifying the nucleobases of the nucleotides in the two second concatemers, each of which is complementary to and bound to each of two of the at least two nucleotide portions of the nucleotide conjugate.In some embodiments, the conditions inhibit at least two nucleotide moieties of the nucleotide conjugate from being incorporated into two first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide moiety is attached to one nucleotide arm in the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, the detection comprises imaging the fluorescent label. In some embodiments, the determination further comprises: (a) removing two polymerases and the nucleotide conjugate from the two first concatemers; (b) contacting each of the two first concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, the binding complex comprising each of the two second polymerases, each of the two first concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides; and (c) incorporating each of two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and bound to the nucleotide of each of the two first concatemers, wherein the unlabeled nucleotides in the plurality of unlabeled nucleotides are included in a removable blocking group at the 3' carbon of the sugar moiety. In some embodiments, the determining further comprises: (a) removing the two polymerases and the nucleotide conjugate from the two second concatemers; (b) contacting each of the two second concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions sufficient to form a binding complex, the binding complex comprising each of the two second polymerases, each of the two second concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides; and incorporating each of the two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two second concatemers, and wherein an unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

[0009] In some embodiments, the determining comprises: (a) contacting two first concatemers with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to a first portion of the first concatemer, and a second primer sequence complementary to a second portion of the first concatemer under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, the first portion of the first concatemer hybridized to the first primer, and the second portion of the first concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein each of the at least two nucleotide portions is complementary to and binds to a nucleotide in the first portion and the second portion of the first concatemer; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; and (c) identifying the nucleobases of the nucleotides in the first and second portions of the first concatemer, each of which is complementary to and binds to each of two of the at least two nucleotide portions of the nucleotide conjugate. In some embodiments, determining comprises: (a) contacting the two second concatemers with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to a first portion of the second concatemer, and a second primer sequence complementary to a second portion of the second concatemer under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, a first portion of the second concatemer hybridized to the first primer sequence, and a second portion of the second concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein each of the at least two nucleotide portions is complementary to and binds to a nucleotide in the first portion and the second portion of the second concatemer; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; and (c) identifying the nucleobases of the nucleotides in the first and second portions of the second concatemer, each of which is complementary to and binds to each of two of the at least two nucleotide portions of the nucleotide conjugate.

[0010] In some embodiments, the condition inhibits the incorporation of at least two nucleotide moieties of the nucleotide conjugate into two first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide moiety is attached to one nucleotide arm in the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, wherein the detection comprises imaging the fluorescent label. In some embodiments, a plurality of nucleotides are composed of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of one type of nucleotide in the group is different from the wavelength of the fluorescent label emitted light of another type of nucleotide in the group. In some embodiments, a plurality of nucleotides include at least two types of nucleotides, wherein at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of one type of nucleotide in the group is different from the wavelength of the fluorescent label emitted light of another type of nucleotide in the group. In some embodiments, wherein determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar portion. In some embodiments, the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the first target nucleic acid sequence and the second target nucleic acid sequence correspond to two separate parts of the same mRNA or cDNA molecule. In some embodiments, the first target nucleic acid sequence and the second target nucleic acid sequence correspond to two different mRNA or cDNA molecules. In some embodiments, the repeating comprises repeating at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or at least 50 times. In some embodiments, determining comprises detecting the first or second sequencing product nucleic acid molecule in a biological sample in situ by imaging. In some embodiments, determining comprises detecting the first and second sequencing product nucleic acid molecules in a biological sample simultaneously in situ by imaging. In some embodiments, imaging comprises fluorescence imaging. In some embodiments, the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, an intact tissue, a tumor, an intact tumor, an organ, an organism, a protozoa, an algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal. In some embodiments, the biological sample comprises a fresh sample, a processed sample, a fresh frozen sample, a sectioned sample, or a formalin-fixed paraffin-embedded (FFPE) sample.In some embodiments, the biological sample comprises a fresh cell sample, a fresh frozen cell sample, a slice cell sample or a FFPE cell sample. In some embodiments, at least two target nucleic acid sequences include a target DNA sequence. In some embodiments, at least two target nucleic acid sequences include a target RNA sequence. In some embodiments, at least two target nucleic acid sequences include a first target RNA sequence and a second target RNA sequence. In some embodiments, the first target RNA sequence includes coding RNA, non-coding RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small interfering RNA (siRNA), antisense RNA, mature microRNA or immature microRNA. In some embodiments, the second target RNA sequence includes coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, at least two target nucleic acid sequences include a first target DNA sequence and a second target DNA sequence. In some embodiments, the first target DNA sequence comprises complementary DNA (cDNA), genomic DNA (gDNA), non-coding DNA or coding DNA. In some embodiments, the second target DNA sequence comprises cDNA, gDNA, non-coding DNA or coding DNA. In some embodiments, the at least two target nucleic acid sequences comprise a target RNA sequence and a target DNA sequence. In some embodiments, the target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, the target DNA sequence comprises cDNA, gDNA, non-coding DNA or coding DNA. In some embodiments, the biological sample comprises a human sample, a monkey sample, a monkey sample, a dog sample, a cat sample, a cow sample, a horse sample, a mouse sample, a pig sample, a goat sample, a lupine sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a viral sample, a protozoan sample or a fungal sample. In some embodiments, the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, an intact tissue, a tumor, an intact tumor, an organ, or an organism. In some embodiments, the biological sample is immobilized on a surface. In some embodiments, the surface comprises the inner surface of a flow cell.

[0011] In another aspect, provided herein is a method for in situ detection of at least two target nucleic acid molecules and at least two target polypeptides in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, wherein the at least two target polypeptides include a first target polypeptide encoded by the first target nucleic acid sequence or its reverse complement and a second target polypeptide encoded by the second target nucleic acid sequence or its reverse complement, the method comprising: (a) providing a biological sample comprising: (i) a first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or the reverse complement of the first target nucleic acid sequence or a portion thereof; (ii) a second nucleic acid molecule comprising the second target nucleic acid sequence or a portion thereof, or the reverse complement of the second target nucleic acid sequence or a portion thereof; (iii) a third nucleic acid molecule comprising a third target nucleic acid sequence or a portion thereof, or the reverse complement of the third target nucleic acid sequence or a portion thereof, wherein the presence of the third target nucleic acid sequence or its reverse complement identifies the presence of the first target polypeptide in the biological sample; and (iv) a fourth nucleic acid molecule comprising a fourth target nucleic acid sequence or a portion thereof, or the reverse complement of the fourth target nucleic acid sequence or a portion thereof (b) determining in situ the sequences of: (i) a first nucleic acid molecule or a portion thereof in the biological sample to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first nucleic acid molecule or a portion thereof; and (ii) a third nucleic acid molecule or a portion thereof in the biological sample to generate a third sequencing product nucleic acid molecule that is complementary to and binds to the third nucleic acid molecule or portion thereof; and (c) identifying in situ the sequences of: (i) a second nucleic acid molecule or a portion thereof in the biological sample to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second nucleic acid molecule or a portion thereof; and (ii) a fourth nucleic acid molecule or a portion thereof in the biological sample to generate a fourth sequencing product nucleic acid molecule that is complementary to and binds to the fourth nucleic acid molecule or portion thereof, wherein the full sequence of the first target nucleic acid sequence and the full sequence of the second target nucleic acid sequence differ by at least one nucleotide, wherein the full sequence of the first target polypeptide differs by at least one amino acid from the full sequence of the second target polypeptide, and wherein step (b) is performed under conditions that prevent step (c) from being performed.

[0012] In another aspect, provided herein is a method for in situ detection of at least two target nucleic acid sequences and at least two target polypeptides in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, wherein the at least two target polypeptides include a first target polypeptide encoded by the first target nucleic acid sequence or its reverse complement sequence and a second target polypeptide encoded by the second target nucleic acid sequence or its reverse complement sequence, the method comprising: (a) providing a biological sample comprising: (i) a first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or the reverse complement sequence of the first target nucleic acid sequence or a portion thereof; (ii) a second nucleic acid molecule comprising the second target nucleic acid sequence or a portion thereof, (b) determining in situ the sequence of: (i) the first nucleic acid molecule or portion thereof in the biological sample to generate a sequence that is identical to the first target polypeptide; (ii) a third nucleic acid molecule comprising the third target nucleic acid sequence or portion thereof, or the reverse complement of the third target nucleic acid sequence or portion thereof, wherein the presence of the third target nucleic acid sequence or its reverse complement identifies the presence of the first target polypeptide in the biological sample; and (iv) a fourth nucleic acid molecule comprising the fourth target nucleic acid sequence or portion thereof, or the reverse complement of the fourth target nucleic acid sequence or portion thereof, wherein the presence of the fourth target nucleic acid sequence or its reverse complement identifies the presence of the second target polypeptide in the biological sample. (c) removing the first sequencing product nucleic acid molecule from the first nucleic acid molecule and removing the third sequencing product nucleic acid molecule from the third nucleic acid molecule, wherein after the removal, the first nucleic acid molecule and the third nucleic acid molecule are located in the biological sample; (d) repeating (b) and (c); (e) identifying in situ the sequence of The method comprises the steps of: (i) removing a second nucleic acid molecule or a portion thereof from a biological sample to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second nucleic acid molecule or the portion thereof, wherein the second nucleic acid molecule or the portion thereof consists of 2-30 nucleotides; and (ii) removing a fourth nucleic acid molecule or a portion thereof from a biological sample to generate a fourth sequencing product nucleic acid molecule that is complementary to and binds to the fourth nucleic acid molecule or the portion thereof, wherein the fourth nucleic acid molecule or the portion thereof consists of 2-30 nucleotides; and (f) removing the second sequencing product nucleic acid molecule from the second nucleic acid molecule and removing the fourth sequencing product nucleic acid molecule from the fourth nucleic acid molecule, wherein after the removal, the second nucleic acid molecule and the fourth nucleic acid molecule are located in the biological sample.and (g) repeating (e) and (f), wherein the entire sequence of the first target nucleic acid sequence differs from the entire sequence of the second target nucleic acid sequence by at least one nucleotide, wherein the entire sequence of the first target polypeptide differs from the entire sequence of the second target polypeptide by at least one amino acid, and wherein step (b) is performed under conditions that prevent step (e) from being performed.

[0013] In another aspect, provided herein is a method for in situ detection of at least two target RNA sequences and at least two target polypeptides in a biological sample, the method comprising: (a) providing a biological sample immobilized on a surface, permeabilized, and fixed, wherein the biological sample comprises: (i) a first target RNA sequence of the at least two target RNA sequences and a first target polypeptide of the at least two polypeptides, wherein the first target polypeptide is encoded by the first target RNA sequence or its reverse complement; and (ii) a second target RNA sequence of the at least two target RNA sequences and a second target polypeptide of the at least two polypeptides, wherein the second target polypeptide is encoded by the second target RNA sequence or its reverse complement; (b) generating a first target cDNA sequence by reverse transcription of the first target RNA sequence, and The second target cDNA sequence is produced by reverse transcription of the second target RNA sequence; (c) contacting the following: (i) the first target cDNA sequence with a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target cDNA sequence, so that the first oligonucleotide forms a circular structure with a gap between the first end and the second end, wherein the first oligonucleotide comprises a first identification sequence for identifying the first target RNA sequence, a first sequencing primer, and a nucleic acid amplification primer; (ii) the second target cDNA sequence with a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target cDNA sequence. (iii) a first target polypeptide and a first oligonucleotide conjugate comprising a first short nucleic acid and a first binding portion that specifically binds to the first target polypeptide, wherein the first short nucleic acid comprises a first tag sequence and a second tag sequence, wherein the first oligonucleotide conjugate specifically binds to the first target polypeptide through the first binding portion to form a first binding complex, wherein the first and second tag sequences identify the first binding portion; and (iv) ) a second target polypeptide and a second oligonucleotide conjugate comprising a second short nucleic acid and a second binding portion that specifically binds to the second target polypeptide, wherein the second short nucleic acid comprises a third tag sequence and a fourth tag sequence, wherein the second oligonucleotide conjugate specifically binds to the second target polypeptide through the second binding portion to form a second binding complex, wherein the third and fourth tag sequences identify the second binding portion; (d) contacting the following: (i) the first binding complex and a third oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the third oligonucleotide are complementary to and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, such that the third oligonucleotide forms a ring structure with a gap between the first end and the second end;and (ii) a second binding complex and a fourth oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the fourth oligonucleotide are complementary to and bind to the third tag sequence and the fourth tag sequence, such that the fourth oligonucleotide forms a circular structure having a gap between the first end and the second end; and (e) joining the first end and the second end of the first oligonucleotide to produce a first circular oligonucleotide, joining the first end and the second end of the second oligonucleotide to produce a second circular oligonucleotide, joining the first end and the second end of the third oligonucleotide to produce a third circular oligonucleotide, and joining the first end and the second end of the fourth oligonucleotide to produce a third circular oligonucleotide. end-joining to produce a fourth circular oligonucleotide, wherein the first circular oligonucleotide and the third circular oligonucleotide comprise a first sequencing primer or its reverse complement, wherein the second circular oligonucleotide and the fourth circular oligonucleotide comprise a second sequencing primer or its reverse complement, and wherein the sequences of the first and second sequencing primers differ by at least one nucleotide; (f) amplifying: (i) the first circular oligonucleotide to produce a first concatemer comprising a plurality of first circular oligonucleotides, said amplification being by rolling circle amplification; (ii) the second circular oligonucleotide to produce a second concatemer comprising a plurality of second circular oligonucleotides, said amplification being by rolling circle amplification; (iii) a third circular oligonucleotide to generate a third concatemer comprising a plurality of third circular oligonucleotides, wherein the amplification is by rolling circle amplification; and (iv) a fourth circular oligonucleotide to generate a fourth concatemer comprising a plurality of fourth circular oligonucleotides, wherein the amplification is by rolling circle amplification; (g) determining in situ the sequence of: (i) a first concatemer or a portion thereof to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first concatemer, wherein the sequence of the first concatemer or the portion thereof consists of 2-30 nucleotides; and (ii) a third concatemer or a portion thereof to generate a third sequencing product nucleic acid molecule that is complementary to and binds to the third concatemer, wherein the first concatemer or the portion thereof consists of 2-30 nucleotides; The sequence of the triple concatemer or portion thereof consists of 2-30 nucleotides, wherein step (g) is performed under conditions that prevent step (j) from being performed; (h) removing the first sequencing product nucleic acid molecule from the first concatemer and removing the third sequencing product nucleic acid molecule from the third concatemer, wherein after the removal, the first concatemer and the third concatemer are located in the biological sample; (i) repeating (g) and (h) at least once; (j) determining in situ the sequence of: (i) a second concatemer or portion thereof to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second concatemer, wherein the sequence of the second concatemer or portion thereof consists of 2-30 nucleotides;and (ii) a fourth concatemer or a portion thereof to generate a fourth sequencing product nucleic acid molecule that is complementary to and binds to the fourth concatemer, wherein the sequence of the fourth concatemer or the portion thereof consists of 2-30 nucleotides, wherein step (j) is performed under conditions that prevent step (g) from being performed, wherein the entire sequence of the first target RNA sequence and the entire sequence of the second target RNA sequence differ by at least one nucleotide, and wherein the entire sequence of the first target polypeptide and the entire sequence of the second target polypeptide differ by at least one amino acid; (k) removing the second sequencing product nucleic acid molecule from the second concatemer and removing the fourth sequencing product nucleic acid molecule from the fourth concatemer, wherein after removal, the second concatemer and the fourth concatemer are located in the biological sample; (l) repeating (j) and (k) at least once.;

[0014] In some embodiments, determining comprises imaging the first or third sequencing product nucleic acid molecule, wherein identifying comprises imaging the second or fourth sequencing product nucleic acid molecule and the fourth sequencing product nucleic acid molecule. In some embodiments, the method further comprises simultaneously imaging the first sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule to analyze the spatial distribution of the first and second sequencing product nucleic acid molecules within the biological sample. In some embodiments, the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, a complete tissue, a tumor, a complete tumor, an organ, an organism, a protozoa, an algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal. In some embodiments, the biological sample comprises a fresh sample, a processed sample, a fresh frozen sample, a sectioned sample, or a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the first target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA, and / or wherein the first target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, the second target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA, and / or wherein the second target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, the biological sample comprises a human sample, a simian sample, an ape sample, a canine sample, a feline sample, a bovine sample, a horse sample, a mouse sample, a porcine sample, a goat sample, a lupin sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a viral sample, a protozoan sample, or a fungal sample. In some embodiments, the first, second, third, or fourth concatemer further comprises a compaction oligonucleotide, wherein: (a) the first segment of the compaction oligonucleotide is complementary to and binds to the first portion of the first, second, third, or fourth concatemer; and (b) the second segment of the compaction oligonucleotide is complementary to and binds to the second portion of the first, second, third, or fourth concatemer to cause a size reduction or shape change in the second concatemer. In some embodiments, the first sequencing product nucleic acid molecule comprises a first identification sequence that identifies a first target RNA sequence, and the second sequencing product nucleic acid molecule comprises a second identification sequence that identifies a second target RNA sequence.In some embodiments, the first sequencing product nucleic acid molecule comprises a first identification sequence that identifies a first target RNA sequence and a portion of the first target RNA sequence, wherein the second sequencing product nucleic acid molecule comprises a second identification sequence that identifies a second target RNA sequence and a portion of the second target RNA sequence. In some embodiments, determining comprises: (a) contacting the first, second, third, or fourth concatemer with a polymerase, a plurality of nucleotides, and a primer sequence that is complementary to a portion of the first, second, third, or fourth concatemer under conditions sufficient to form a binding complex, the binding complex comprising a polymerase, a nucleotide from a plurality of nucleotides that are complementary to and bind to a nucleotide unit in the first, second, third, or fourth concatemer, and the first, second, third, or fourth concatemer hybridized to the primer sequence, wherein the nucleotide comprises a fluorescent label and a removable blocking group at the 3' carbon position of the sugar portion; (b) incorporating the nucleotide into the 3' end of the primer sequence; and (c) identifying the nucleobase of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide. In some embodiments, determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar portion. In some embodiments, the blocking group includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, a plurality of nucleotides include a type of nucleotide selected from dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, a plurality of nucleotides include a mixture of any combination of two or more types of nucleotides selected from dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, determining comprises: (a) contacting two first, second, third, or fourth concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences that are complementary to portions of the first, second, third, or fourth concatemers under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two first, second, third, or fourth concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein each of the at least two nucleotide portions is complementary to and binds to a nucleotide in each of the two first concatemers; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; and (c) identifying the nucleobases of the nucleotides in the two first concatemers, each of which is complementary to and bound to each of two of the at least two nucleotide portions of the nucleotide conjugates.

[0015] In some embodiments, determining further comprises: (a) removing two polymerases and a nucleotide conjugate from the two first, second, third, or fourth concatemers; and (b) contacting each of the two first, second, third, or fourth concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, the binding complex comprising each of the two second polymerases, each of the two first, second, third, or fourth concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides, and incorporating each of the two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two first, second, third, or fourth concatemers, and wherein an unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

[0016] In some embodiments, determining comprises: (a) contacting two first, second, third, or fourth concatemers with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to a first portion of the first, second, third, or fourth concatemer, and a second primer sequence complementary to a second portion of the first, second, third, or fourth concatemer under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, a first portion of the first, second, third, or fourth concatemer hybridized to the first primer sequence, and a second portion of the first, second, third, or fourth concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein each of the at least two nucleotide portions is complementary to and binds to a nucleotide in the first portion and the second portion of the first, second, third, or fourth concatemer; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; and (c) identifying the nucleobases of the nucleotides in the first and second portions of the first, second, third, or fourth concatemer that are each complementary to and bound to each of two of the at least two nucleotide portions in the nucleotide conjugate. In some embodiments, the condition inhibits at least two nucleotide moieties of the nucleotide conjugate from being incorporated into two first, second, third or fourth concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide moiety is attached to a nucleotide arm in the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, detection comprises imaging the fluorescent label. In some embodiments, a plurality of nucleotides are composed of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the light emitted by the fluorescent label of the nucleotides of another type in the group. In some embodiments, a plurality of nucleotides include at least two types of nucleotides, wherein at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the light emitted by the fluorescent label of the nucleotides of another type in the group. In some embodiments, determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar moiety. In some embodiments, the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.In some embodiments, the plurality of nucleotides consists of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, wherein at least two types of nucleotides are selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group. In some embodiments, the first target RNA sequence comprises coding RNA, non-coding RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small interfering RNA (siRNA), antisense RNA, mature microRNA, or immature microRNA. In some embodiments, the second target RNA sequence comprises a coding RNA, a non-coding RNA, an mRNA, a tRNA, an rRNA, a miRNA, a gRNA, a snRNA, an siRNA, an antisense RNA, a mature microRNA, or an immature microRNA. In some embodiments, the biological sample comprises a human sample, a monkey sample, a simian sample, a dog sample, a cat sample, a cow sample, a horse sample, a mouse sample, a pig sample, a goat sample, a lupin sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a virus sample, a protozoan sample, or a fungal sample. In some embodiments, the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, an intact tissue, a tumor, an intact tumor, an organ, or an organism. In some embodiments, the biological sample is immobilized on a surface. In some embodiments, the surface comprises the inner surface of a flow cell.

[0017] On the other hand, the present invention provides a system for in situ detection of at least two target nucleic acid sequences in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, the system comprising: (a) a biological sample comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a first target nucleic acid sequence or its reverse complement sequence or a portion thereof, and wherein the second nucleic acid molecule comprises a second target nucleic acid sequence or its reverse complement sequence or a portion thereof; (b) a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target nucleic acid sequence or its reverse complement sequence or a portion thereof, such that the first oligonucleotide forms a first circular oligonucleotide within the biological sample, wherein the first circular oligonucleotide comprises a gap between the first end and the second end; and (c) a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target nucleic acid sequence or its reverse complement sequence or a portion thereof, such that the second oligonucleotide forms a second circular oligonucleotide within the biological sample, wherein the second circular oligonucleotide comprises a gap between the first end and the second end. In some embodiments, the first circular oligonucleotide further comprises a first nucleic acid enzyme configured to join the first end and the second end of the first oligonucleotide, and the second circular oligonucleotide further comprises a second nucleic acid enzyme configured to join the first end and the second end of the second oligonucleotide. In some embodiments, the first nucleic acid enzyme and the second nucleic acid enzyme comprise a nucleic acid ligase, a nucleic acid ligase, a nucleic acid polymerase, a nucleic acid polymerase, or a combination thereof. In some embodiments, the system further comprises a first amplicon of the first circular oligonucleotide and a second amplicon of the second circular oligonucleotide. In some embodiments, the first amplicon comprises a first concatemer and / or the second amplicon comprises a second concatemer. In some embodiments, (a) the first concatemer comprises at least two repeats of a first unit nucleic acid sequence, the first unit nucleic acid sequence comprising a first target nucleic acid sequence or a portion thereof, or a reverse complement sequence or a portion thereof; and / or (b) the second concatemer comprises at least two repeats of a second unit nucleic acid sequence, the second unit nucleic acid sequence comprising a second target nucleic acid sequence or a portion thereof, or a reverse complement sequence or a portion thereof.

[0018] In some embodiments, the first concatemer further comprises a first identification sequence that identifies the first target nucleic acid sequence, or a sequencing primer or reverse complement thereof, wherein the second concatemer further comprises a second identification sequence that identifies the second target nucleic acid sequence, or a sequencing primer or reverse complement thereof. In some embodiments, the first concatemer further comprises a first compacted oligonucleotide, wherein: (a) the first segment of the first compacted oligonucleotide is complementary to and binds to the first portion of the first concatemer; and (b) the second segment of the first compacted oligonucleotide is complementary to and binds to the second portion of the first concatemer, resulting in a reduction in size or a change in shape of the first concatemer. In some embodiments, the second concatemer further comprises a second compacted oligonucleotide, wherein: (a) the first segment of the second compacted oligonucleotide is complementary to and binds to the first portion of the second concatemer; and (b) the second segment of the second compacted oligonucleotide is complementary to and binds to the second portion of the second concatemer, resulting in a reduction in size or a change in shape of the second concatemer. In some embodiments, the system further comprises a polymerase, a plurality of nucleotides, and a primer sequence that is complementary to at least a portion of the first concatemer or the second concatemer under conditions sufficient to form a binding complex. In some embodiments, the system further includes a reagent configured to remove blocking groups from the nucleotides in a plurality of nucleotides and generate 3'OH groups on the sugar portion of the nucleotides. In some embodiments, the blocking group includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, a plurality of nucleotides include fluorescent markers. In some embodiments, a plurality of nucleotides are made up of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent marker emitted light of the nucleotides of a type in the group is different from the wavelength of the light emitted by the fluorescent markers of the nucleotides of another type in the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, wherein the at least two types of nucleotides are selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, wherein the fluorescent label of one type of nucleotide in the group emits light at a wavelength that is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group. In some embodiments, the system further comprises a plurality of nucleotide conjugates, wherein the nucleotide conjugates in the plurality of nucleotide conjugates are configured to form a multivalent binding complex comprising two or more polymerases, a nucleotide conjugate, and at least two target nucleic acid sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide moieties that are each complementary to and bind to a nucleotide in each of the at least two target nucleic acid sequences.In some embodiments, the system further includes a first oligonucleotide conjugate comprising a first short nucleic acid and a first binding portion that specifically binds to a first target polypeptide, wherein the first short nucleic acid comprises a first tag sequence and a second tag sequence, wherein the first oligonucleotide conjugate specifically binds to the first target polypeptide in the biological sample through the first binding portion to form a first binding complex, wherein the first and second tag sequences identify the first binding portion in a nucleic acid sequence reaction. In some embodiments, the system further includes a second oligonucleotide conjugate comprising a second short nucleic acid and a second binding portion that specifically binds to a second target polypeptide, wherein the second short nucleic acid comprises a third tag sequence and a fourth tag sequence, wherein the second oligonucleotide conjugate specifically binds to the second target polypeptide in the biological sample through the second binding portion to form a second binding complex, wherein the third and fourth tag sequences identify the second binding portion in a nucleic acid sequence reaction. In some embodiments, the first or second binding portion is an antibody or an antigen-binding fragment thereof. In some embodiments, the system further includes a third oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the third oligonucleotide are complementary to and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, such that the third oligonucleotide forms a ring structure having a gap between the first end and the second end. In some embodiments, the system further includes a fourth oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the fourth oligonucleotide are complementary to and bind to the third tag sequence and the fourth tag sequence so that the fourth oligonucleotide forms a circular structure with a gap between the first end and the second end. In some embodiments, the system further includes a third circular oligonucleotide produced by joining the first end and the second end of the third oligonucleotide and a fourth circular oligonucleotide produced by joining the first end and the second end of the fourth oligonucleotide, wherein the first circular oligonucleotide and the third circular oligonucleotide include a first sequencing primer or its reverse complementary sequence, wherein the second circular oligonucleotide and the fourth circular oligonucleotide include a second sequencing primer or its reverse complementary sequence, wherein the sequences of the first and second sequencing primers have a difference of at least one nucleotide, wherein the joining is performed by a first or second nuclease. In some embodiments, the system further includes a third amplicon of the third circular oligonucleotide and a fourth amplicon of the fourth circular oligonucleotide. In some embodiments, the third amplicon includes a third concatemer and / or the fourth amplicon includes a fourth concatemer. In some embodiments, (a) the third concatemer comprises at least two repeats of a third unit nucleic acid sequence comprising a third circular oligonucleotide or a portion thereof, or a reverse complement thereof or a portion thereof; and / or (b) the fourth concatemer comprises at least two repeats of a second unit nucleic acid sequence comprising a fourth circular oligonucleotide or a portion thereof, or a reverse complement thereof or a portion thereof.In some embodiments, the third concatemer further comprises a first or second tag sequence or its reverse complement that identifies the first binding moiety, wherein the fourth concatemer further comprises a third or fourth tag sequence or its reverse complement that identifies the second binding moiety. In some embodiments, the third concatemer further comprises a third compacted oligonucleotide, wherein: (a) the first segment of the third compacted oligonucleotide is complementary to and binds to the first portion of the third concatemer; and (b) the second segment of the third compacted oligonucleotide is complementary to and binds to the second portion of the third concatemer, resulting in a reduction in size or a change in shape of the third concatemer.

[0019] In some embodiments, the fourth concatemer further comprises a fourth compacted oligonucleotide, wherein: (a) the first segment of the fourth compacted oligonucleotide is complementary to and binds to the first portion of the fourth concatemer; and (b) the second segment of the fourth compacted oligonucleotide is complementary to and binds to the second portion of the fourth concatemer, thereby causing the fourth concatemer to decrease in size or change in shape. In some embodiments, the system further comprises a second polymerase, a second plurality of nucleotides, and a second primer sequence that is complementary to at least a portion of the third concatemer or the fourth concatemer under conditions sufficient to form a binding complex, the binding complex comprising the third or fourth concatemer hybridized to the second primer sequence, a second polymerase, and a second nucleotide of the second plurality of nucleotides that is complementary to and binds to a nucleotide of the third or fourth concatemer. In some embodiments, the system further comprises a second reagent configured to remove a second blocking group from a second nucleotide of the second plurality of nucleotides and generate a 3'OH group on the sugar portion of the second nucleotide. In some embodiments, the second blocking group includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, the second plurality of nucleotides comprises a second fluorescent marker. In some embodiments, the second plurality of nucleotides is composed of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the second fluorescent marker emission of one type of nucleotide in the group is different from the wavelength of the light emitted by the second fluorescent marker of another type of nucleotide in the group. In some embodiments, the second plurality of nucleotides includes at least two types of nucleotides, wherein at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the second fluorescent marker emission of one type of nucleotide in the group is different from the wavelength of the light emitted by the second fluorescent marker of another type of nucleotide in the group. In some embodiments, the system further includes a second plurality of nucleotide conjugates, wherein the second nucleotide conjugate in the second plurality of nucleotide conjugates is configured to form a multivalent binding complex, the multivalent binding complex comprising two or more second polymerases, the second nucleotide conjugate in the second plurality of nucleotide conjugates, and at least two first short nucleic acids or short nucleic acids or portions thereof, wherein the second nucleotide conjugate comprises a tag and at least two nucleotide portions, each of the at least two nucleotide portions being complementary to and binding to the nucleotides of each of the at least two first short nucleic acids or short nucleic acids or portions thereof. In some embodiments, the system further includes a solid surface comprising a biological sample fixed to the solid surface. In some embodiments, the biological sample is permeabilized.In some embodiments, the solid surface further comprises a hydrophilic polymer coating coupled thereto. In some embodiments, the hydrophilic polymer coating has a water contact angle of less than 50 degrees. In some embodiments, the system further comprises an optical imaging module configured to image the biological sample coupled to the solid surface to detect at least two target nucleic acid sequences and / or at least two target polypeptides in the biological sample in situ. In some embodiments, the first target polypeptide is encoded by a first target nucleic acid molecule or its reverse complement sequence, and the second target polypeptide is encoded by a second target nucleic acid molecule or its reverse complement sequence.

[0020] In another aspect, provided herein is a computer-implemented system comprising a computing device including at least one processor, an operating system configured to execute executable instructions, a memory, and a computer program comprising instructions executable by the computing device, wherein the instructions comprise the method disclosed herein.

[0021] In another aspect, provided herein is a non-transitory computer-readable storage medium encoded with a computer program comprising instructions executable by one or more processors, wherein the instructions comprise the methods disclosed herein.

[0022] In another aspect, provided herein is a kit for in situ detection of at least two target nucleic acid sequences in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, the kit comprising: (a) a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target nucleic acid sequence or its reverse complement or a portion thereof, such that the first oligonucleotide forms a first circular oligonucleotide within the biological sample, wherein the first circular oligonucleotide comprises a gap between the first end and the second end, wherein the first oligonucleotide comprises a first identification sequence for identifying the first target nucleic acid sequence, and a first sequencing primer or its reverse complement. , a compacted oligonucleotide or its reverse complement, or a primer for nucleic acid amplification or its reverse complement, (b) a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of a second target nucleic acid sequence or its reverse complement, or a portion thereof, such that the second oligonucleotide forms a second circular oligonucleotide within the biological sample, wherein the second circular oligonucleotide comprises a gap between the first end and the second end, wherein the second oligonucleotide comprises a second identification sequence for identifying the second target nucleic acid sequence, a second sequencing primer or its reverse complement, a compacted oligonucleotide or its reverse complement, or a primer for nucleic acid amplification or its reverse complement; (c) (i) a first nucleic acid enzyme configured to join the first end and the second end of the first oligonucleotide and generate a first circular oligonucleotide, or to join the first end and the second end of the second oligonucleotide and generate a second circular oligonucleotide; (ii) a second nucleic acid enzyme configured to amplify the first circular oligonucleotide to generate a first concatemer or amplify the second circular oligonucleotide to generate a second concatemer; (iii) a third nucleic acid enzyme, a sequencing primer complementary to a portion of the first concatemer or a portion of the second concatemer, a plurality of nucleotides, or a plurality of nucleotide conjugates, wherein the nucleotide conjugates in the plurality of nucleotide conjugates comprise a core and two nucleotide portions attached to the core, or (ii) wherein at least two third nucleases, nucleotide conjugates, sequencing primers, and first or second concatemers are configured to form a multivalent binding complex comprising a third nuclease, a nucleotide conjugate, and a first or second concatemer hybridized to a sequencing primer, wherein two nucleotide portions are complementary to and bind to two nucleotide units of the first or second concatemer, and each of the two first concatemers or second concatemers hybridizes to each of the two sequencing primers; and (f) a fourth nuclease,It is configured to: (i) add nucleotides from a plurality of nucleotides to the end of a sequencing primer hybridized to a first concatemer and generate a first sequencing product nucleic acid molecule, wherein the nucleotides are unlabeled and comprise a blocking group; or (ii) add nucleotides from a plurality of nucleotides to the end of a sequencing primer hybridized to a second concatemer and generate a second sequencing product nucleic acid molecule, wherein the nucleotides are unlabeled and comprise a blocking group; and (g) a dissociation reagent configured to remove the first sequencing product nucleic acid molecule from the first concatemer or remove the second sequencing product nucleic acid molecule from the second concatemer. In some embodiments, the kit is further configured for in situ detection of at least two target RNA sequences and at least two target polypeptides including a first target polypeptide and a second target polypeptide in a biological sample, the kit comprising: a first target polypeptide encoded by a first target nucleic acid molecule or its reverse complement sequence, wherein the second target polypeptide is encoded by a second target nucleic acid molecule or its reverse complement sequence, wherein the full sequence of the first target RNA sequence differs from the full sequence of the second target RNA sequence by at least one nucleotide, and wherein the full sequence of the first target polypeptide differs from the full sequence of the second target polypeptide by at least one amino acid. In some embodiments, the first, second, third or fourth nucleic acid enzyme comprises a nucleic acid ligase, a nucleic acid ligase, a nucleic acid polymerase, a nucleic acid polymerase or a combination thereof. In some embodiments, the nucleotides in a plurality of nucleotides comprise a fluorescent marker. In some embodiments, the nucleotides in a plurality of nucleotides comprise a removable blocking group at the 3' carbon position of the sugar moiety. In some embodiments, a plurality of nucleotides are composed of at least two nucleotides of the same type comprising a fluorescent marker, wherein the nucleotides of the same type are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent marker emission of one type of nucleotide in the group is different from the wavelength of the fluorescent marker emission of another type of nucleotide in the group. In some embodiments, a plurality of nucleotides comprise at least two types of nucleotides, wherein the types in at least two types of nucleotides comprise a fluorescent marker, wherein at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent marker emission of one type of nucleotide in the group is different from the wavelength of the fluorescent marker emission of another type of nucleotide in the group. In some embodiments, the nucleotide conjugate comprises a detectable label. In some embodiments, the nucleotide conjugate comprises a fluorescent marker. In some embodiments, the biological sample comprises a human sample, a simian sample, an ape sample, a canine sample, a feline sample, a bovine sample, a horse sample, a mouse sample, a porcine sample, a goat sample, a lupine sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a viral sample, a protozoan sample, or a fungal sample. In some embodiments, the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, an intact tissue, a tumor, an intact tumor, an organ, or an organism. In some embodiments,The biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, a complete tissue, a tumor, a complete tumor, an organ, an organism, a protozoa, an algae, a bacterium, a virus, a plant, a fungus, an insect or an animal. In some embodiments, the biological sample comprises a fresh sample, a processed sample, a fresh frozen sample, a slice sample or a formalin fixed paraffin embedded (FFPE) sample. In some embodiments, the first target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA, and / or wherein the first target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, the second target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA, and / or wherein the second target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, the kit further comprises: (a) a first oligonucleotide conjugate comprising a first short nucleic acid and a first binding portion that specifically binds to a first target polypeptide, wherein the first short nucleic acid comprises a first tag sequence, a second tag sequence, wherein the first oligonucleotide conjugate specifically binds to the first target polypeptide through the first binding portion to form a first binding complex, wherein the first and second tag sequences identify the first binding portion; or (b) a second oligonucleotide conjugate comprising a second short nucleic acid and a second binding portion that specifically binds to a second target polypeptide, wherein the second short nucleic acid comprises a third tag sequence, a fourth tag sequence, wherein the second oligonucleotide conjugate specifically binds to the second target polypeptide through the second binding portion to form a second binding complex, wherein the third and fourth tag sequences identify the second binding portion. In some embodiments, the kit further comprises: (a) a third oligonucleotide comprising a first end and a second end,wherein the first end and the second end of the third oligonucleotide are complementary to and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, so that the third oligonucleotide forms a ring structure with a gap between the first end and the second end; or (b) a fourth oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the fourth oligonucleotide are complementary to and bind to the third tag sequence and the fourth tag sequence, so that the fourth oligonucleotide forms a ring structure with a gap between the first end and the second end. In some embodiments, the third oligonucleotide comprises a third identification sequence for identifying the first binding portion, and wherein the fourth oligonucleotide comprises a fourth identification sequence for identifying the second binding portion. In some embodiments, the first oligonucleotide and the third oligonucleotide comprise the same first sequencing primer or its reverse complement, wherein the second oligonucleotide and the fourth oligonucleotide comprise the same second sequencing primer or its reverse complement. In some embodiments, the kit further comprises a reagent that reacts with the reactive group of the 3' carbon of the sugar portion in the nucleotide portion of the nucleotide conjugate. In some embodiments, the kit further comprises a reagent for nucleotide binding reaction. In some embodiments, the reagent comprises a cation. In some embodiments, the kit further comprises a reverse transcriptase, a primer for reverse transcription, a sequencing primer, a reagent configured to permeabilize a biological sample, a reagent configured to fix a biological sample, unblocked nucleotides, blocked nucleotides, a reagent for a nucleotide incorporation reaction, a solution comprising a cation, one or more unlabeled nucleotides, one or more buffers for reverse transcription, one or more buffers for nucleic acid binding, one or more buffers for nucleic acid amplification, or one or more buffers for nucleic acid dissociation. In some embodiments, the kit further comprises instructions for using the kit to detect at least two target nucleic acid sequences in a biological sample in situ. In some embodiments, the kit further comprises instructions for using the kit to detect at least two target nucleic acid sequences and at least two target polypeptides in a biological sample in situ. In some embodiments, the instructions comprise sequencing by synthesis reaction. In some embodiments, the instructions comprise sequencing by binding reaction, wherein the in situ detection is performed at different times from the nucleotide incorporation step. In some embodiments, the kit further comprises instructions for using the kit using the methods disclosed herein. ,

[0023] Incorporation by reference

[0024] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the inventive concept are set forth with particularity in the appended claims. A better understanding of the features and advantages of the inventive concept will be obtained by reference to the following detailed description of illustrative embodiments in which the principles of the inventive concept are utilized, and the accompanying drawings, in which:

[0026] Figure 1 is a schematic diagram showing a non-limiting example workflow for generating concatemers within a cell sample. Target RNA carried by the cell sample is hybridized with a reverse transcription primer (RT primer) and reverse transcribed to synthesize a first-strand cDNA. The first-strand cDNA is hybridized with a target-specific padlock probe to generate a circularized padlock probe having a gap (solid downward triangle) between the first and second ends of the hybridized padlock probe. The padlock probe carries an adapter for a universal compacting oligonucleotide binding site, an adapter for a universal sequencing primer binding site, and a target barcode sequence corresponding to a given target cDNA. The gap in the circularized padlock probe is connected to form a covalently closed circular padlock probe that carries a cDNA sequence corresponding to the target RNA. The sequence of a non-limiting example padlock probe that selectively binds to GAPDH cDNA is Figure 1 Bottom panel. Covalently closed circular padlock probes can be subjected to rolling circle amplification within a cell sample to generate concatemer molecules. Concatemer molecules can be sequenced repeatedly within a cell sample.

[0027] Figure 2 Is displayed for Figure 1 Schematic diagram of a non-limiting example workflow for sequencing concatemers generated within cells as shown in . Figure 2The concatemer depicted in includes tandem repeat units, wherein each unit includes: (i) a universal sequencing primer binding site (Seq), (ii) a universal compacted oligonucleotide binding site (CO), (iii) an insert sequence corresponding to a given target cDNA and (iv) a target barcode sequence (BC) corresponding to a given target cDNA. In some embodiments, a universal sequencing primer (solid arrow) hybridizes with the universal sequencing primer binding site, and performs no more than 30 sequencing cycles to generate a plurality of first sequencing read products (dashed arrows), wherein the first sequencing read product only includes the target barcode sequence. Multiple first sequencing read products are removed from the concatemer, and sequencing is repeated, wherein no more than 30 sequencing cycles are performed to generate another plurality of first sequencing read products (dashed arrows), wherein the first sequencing read product only includes the target barcode sequence. Multiple first sequencing read products are removed from the concatemer, and sequencing is repeated again, wherein no more than 30 sequencing cycles are performed to generate another plurality of first sequencing read products (dashed arrows), wherein the first sequencing read product only includes the target barcode sequence. In some embodiments, repeated sequencing can be performed up to 50 times. The sequences of all first sequencing reads can be determined and aligned to a first reference sequence (eg, a reference barcode sequence) to confirm the presence of the first target RNA molecule in the cell sample.

[0028] Figure 3 Is displayed for Figure 1 Schematic diagram of a non-limiting example workflow for sequencing concatemers generated within cells as shown in . Figure 3The concatemer depicted in includes tandem repeat units, wherein each unit includes: (i) a universal sequencing primer binding site (Seq), (ii) a universal compacted oligonucleotide binding site (CO), (iii) an insert sequence corresponding to a given target cDNA, and (iv) a target barcode sequence (BC) corresponding to a given target cDNA. In some embodiments, a universal sequencing primer (solid arrow) hybridizes with the universal sequencing primer binding site, and no more than 30 sequencing cycles are performed to generate a plurality of first sequencing read products (dashed arrows), wherein the first sequencing read product includes a portion of the target barcode sequence and the insert sequence. The plurality of first sequencing read products are removed from the concatemer, and sequencing is repeated, wherein no more than 30 sequencing cycles are performed to generate another plurality of first sequencing read products (dashed arrows), wherein the first sequencing read product includes a portion of the target barcode sequence and the insert sequence. The plurality of first sequencing read products are removed from the concatemer, and sequencing is repeated again, wherein no more than 30 sequencing cycles are performed to generate another plurality of first sequencing read products (dashed arrows), wherein the first sequencing read product includes a portion of the target barcode sequence and the insert sequence. In some embodiments, sequencing can be performed iteratively up to 50 times. The sequences of all first sequencing reads can be determined and aligned with a first reference sequence (e.g., a reference barcode sequence and an insert sequence corresponding to the target RNA) to confirm the presence of the first target RNA molecule in the cell sample.

[0029] Figure 4 Is displayed for Figure 1 Schematic diagram of a non-limiting example workflow for sequencing concatemers generated within cells as shown in . Figure 4The concatemer depicted in s includes a tandem repeat unit, wherein each unit includes: (i) a universal sequencing primer binding site (Seq), (ii) a universal compaction oligonucleotide binding site (CO) and (iii) an insertion sequence corresponding to a given target cDNA. In some embodiments, a universal sequencing primer (solid arrow) hybridizes with the universal sequencing primer binding site, and performs no more than 30 sequencing cycles to generate a plurality of first sequencing reads products (dotted arrows), wherein the first sequencing reads the product including the portion of the insertion sequence. Multiple first sequencing reads are removed from the concatemer, and sequencing is repeated, wherein no more than 30 sequencing cycles are performed to generate another plurality of first sequencing reads products (dotted arrows), wherein the first sequencing reads the product including the portion of the insertion sequence. Multiple first sequencing reads are removed from the concatemer, and sequencing is repeated again, wherein no more than 30 sequencing cycles are performed to generate another plurality of first sequencing reads products (dotted arrows), wherein the first sequencing reads the product including the portion of the insertion sequence. In some embodiments, repeated sequencing can be performed up to 50 times. The sequences of all first sequencing reads can be determined and aligned to a first reference sequence (eg, corresponding to an insert sequence of the target RNA) to confirm the presence of the first target RNA molecule in the cell sample.

[0030] Figure 5 Is displayed for Figure 1 Schematic diagram of a non-limiting example workflow for sequencing concatemers generated within cells as shown in . Figure 5 The concatemer depicted in includes tandem repeat units, wherein each unit includes: (i) universal sequencing primer binding site (Seq) and (ii) an insertion sequence corresponding to a given target cDNA. In some embodiments, a universal sequencing primer (solid arrow) hybridizes with the universal sequencing primer binding site, and performs no more than 30 sequencing cycles to generate a plurality of first sequencing reads products (dotted arrows), wherein the first sequencing reads a portion of a product including an insertion sequence. Multiple first sequencing reads products are removed from the concatemer, and sequencing is repeated, wherein no more than 30 sequencing cycles are performed to generate another plurality of first sequencing reads products (dotted arrows), wherein the first sequencing reads a portion of a product including an insertion sequence. Multiple first sequencing reads products are removed from the concatemer, and sequencing is repeated again, wherein no more than 30 sequencing cycles are performed to generate another plurality of first sequencing reads products (dotted arrows), wherein the first sequencing reads a portion of a product including an insertion sequence. In some embodiments, repeated sequencing can be performed up to 50 times. The sequences of all first sequencing reads can be determined, and compared with a first reference sequence (e.g., corresponding to an insertion sequence of a target RNA) to confirm that there is a first target RNA molecule in the cell sample.

[0031] Figure 6An image showing fluorescent sequencing signals emitted by HEK 293 cells cultured on a polylysine-coated flow cell is shown. The cultured cells were fixed, permeabilized, subjected to reverse transcription, and subjected to rolling circle amplification on the flow cell, all under conditions suitable for retaining cellular nucleic acids within the cell. The coated flow cell lacked surface capture primers. The nucleic acids retained in the cells were sequenced using a two-stage sequencing method that employed detectably labeled multivalent molecules and unlabeled nucleotide analogs. Thirty cycles of the two-stage sequencing reaction were performed using multivalent molecules labeled with one of four fluorophores and unlabeled nucleotide analogs. Figure 6 Results from a second in situ sequencing experiment are shown (see Example 1). Figure 6 The fluorescence signal from cycles 20-25 is shown, where the sequence reads CCTCCT. A total of 4620 fluorescent spots were detected, with significant signal at all cycles. More than 90% of the fluorescent spots were detected with the sample index sequence.

[0032] Figure 7 Schematic diagrams of various non-limiting example configurations of multivalent molecules. Left (Class I): Schematic diagram of a multivalent molecule with a "starburst" or "helter skelter" configuration. Center (Class II): Schematic diagram of a multivalent molecule with a dendrimer configuration. Right (Class III): Schematic diagram of various multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. The nucleotide unit is designated "N," biotin is designated 13', and streptavidin is designated "SA."

[0033] Figure 8 is a schematic diagram of a non-limiting example multivalent molecule comprising a universal core attached to multiple nucleotide arms.

[0034] Figure 9 is a schematic diagram of a non-limiting example multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms.

[0035] Figure 10 A schematic diagram of a non-limiting example multivalent molecule comprising a core attached to a plurality of nucleotide arms, wherein the nucleotide arms comprise biotin, a spacer, a linker, and nucleotide units is shown.

[0036] Figure 11 is a schematic diagram of a non-limiting exemplary nucleotide arm comprising a core attachment portion, a spacer, a linker, and a nucleotide unit.

[0037] Figure 12 Shown are the chemical structures of non-limiting example spacers (top) and various non-limiting example linkers, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker (bottom).

[0038] Figure 13 Shown are the chemical structures of various non-limiting example linkers, including Linkers 1-9.

[0039] Figure 14 Shown are the chemical structures of various non-limiting example linkers joined / attached to the nucleotide units.

[0040] Figure 15 Shown are the chemical structures of various non-limiting example linkers joined / attached to the nucleotide units.

[0041] Figure 16 Shown are the chemical structures of various non-limiting example linkers joined / attached to the nucleotide units.

[0042] Figure 17 The chemical structure of a non-limiting example biotinylated nucleotide arm is shown. In this example, the nucleotide unit is linked to the linker via propargylamine attachment at the 5-position of the pyrimidine base or the 7-position of the purine base.

[0043] Figure 18 is a schematic diagram of a guanine tetrad (eg, a G-tetrad).

[0044] Figure 19 is a schematic diagram of a non-limiting exemplary intramolecular G-quadruplex structure.

[0045] Figure 20 Schematic diagram showing a workflow for generating circularized padlock probes, the workflow comprising generating first and second cDNAs from first and second target RNA molecules (respectively), hybridizing the first and second padlock probes to the first and second cDNA molecules (respectively) to generate first and second circularized padlock probes (respectively). The first padlock probe comprises (i) a first target barcode sequence (target BC-1) that uniquely identifies the first target RNA, (ii) a first batch-specific sequencing primer binding site (batch Seq-1) (or its complement), (iii) a universal binding site (universal RCA) (or its complement) for an amplification primer, and (iv) a universal binding site (or its complement) for a compaction oligonucleotide. The second padlock probe comprises (i) a second target barcode sequence (target BC-2) that uniquely identifies the second target RNA, (ii) a second batch-specific sequencing primer binding site (batch Seq-2) (or its complement), (iii) a universal binding site (universal RCA) (or its complement) for an amplification primer, and (iv) a universal binding site (or its complement) for a compaction oligonucleotide.

[0046] Figure 21Schematic diagram showing rolling circle and sequencing workflow, the workflow includes generating the first and second concatemers by performing rolling circle amplification using the first and second covalently closed circular molecules (respectively). The first and second concatemers are subjected to a first sequencing workflow using a first batch of specific sequencing primers, sequencing polymerase, and a plurality of nucleotide reagents. The first concatemer undergoes repeated sequencing, but the second concatemer does not undergo. The second concatemer is subjected to a second sequencing workflow using a second batch of specific sequencing primers, sequencing polymerase, and a plurality of nucleotide reagents. The second concatemer undergoes repeated sequencing, but the first concatemer does not undergo.

[0047] Figure 22 Schematic diagram showing first and second antibody-oligonucleotide conjugates. The first antibody-oligonucleotide conjugate comprises a first antibody that selectively binds to a first target polypeptide. The first antibody is coupled to a first oligonucleotide, wherein the first oligonucleotide carries a first and a second tag sequence. The second antibody-oligonucleotide conjugate comprises a second antibody that selectively binds to a second target polypeptide. The second antibody is coupled to a second oligonucleotide, wherein the second oligonucleotide carries a third and a fourth tag sequence. The first, second, third, and fourth tag sequences are different from each other.

[0048] Figure 23 is a schematic diagram showing first and second antibody-oligonucleotide conjugates, each conjugate bound to its respective target polypeptide and its respective padlock probe.

[0049] The first padlock probe comprises (i) a sequence that can hybridize to the first tag sequence, (ii) a third target barcode sequence (target BC-3) that uniquely identifies the first antibody that selectively binds to the first target polypeptide, (iii) a first batch-specific sequencing primer binding site (batch Seq-1) (or its complement), (iv) a universal binding site for an amplification primer (universal RCA) (or its complement), (v) a universal binding site for a compaction oligonucleotide (or its complement), and (vi) a sequence that can hybridize to the second tag sequence. The second padlock probe comprises (i) a sequence that can hybridize to the third tag sequence, (ii) a fourth target barcode sequence (target BC-4) that uniquely identifies the second antibody that selectively binds to the second target polypeptide, (iii) a second batch-specific sequencing primer binding site (batch Seq-2) (or its complement), (iv) a universal binding site for an amplification primer (universal RCA) (or its complement), (v) a universal binding site for a compaction oligonucleotide (or its complement), and (vi) a sequence that can hybridize to the fourth tag sequence.

[0050] Figure 24Schematic diagram showing a rolling circle amplification and sequencing workflow for a first target RNA and a first target polypeptide. The first target cDNA molecule (upper left schematic diagram) is hybridized to a target-specific padlock probe for the first cDNA, which carries (i) first and second binding arms that hybridize to the first target cDNA, (ii) a first barcode sequence that uniquely identifies the first target cDNA (BC-1), (iii) a first batch-specific sequencing primer binding site (batch Seq-1), (iv) a universal binding site for an amplification primer, and (v) a universal binding site for a compacting oligonucleotide. The hybridized first padlock probe includes a gap or notch that is enzymatically closed to generate a first covalently closed circular molecule carrying the first cDNA sequence. The covalently closed circular molecule undergoes rolling circle amplification to generate a first cDNA concatemer molecule (top concatemer molecule). The first cDNA concatemer molecule undergoes sequencing using the first batch-specific sequencing primer. A first antibody-oligonucleotide conjugate (upper right schematic diagram) is bound to the first target polypeptide. The oligonucleotide in the first antibody-oligonucleotide conjugate binds to a target-specific padlock probe of a first target polypeptide-oligonucleotide conjugate, which carries (i) first and second binding arms that hybridize to the first and second tag sequences, (ii) a third barcode sequence (BC-3) that uniquely identifies the first antibody-oligonucleotide conjugate that selectively binds to the first target polypeptide, and (iii) a first batch-specific sequencing primer binding site (batch Seq-1), (iv) a universal binding site for an amplification primer, and (v) a universal binding site for a compaction oligonucleotide. The hybridized first padlock probe includes a gap or nick that is enzymatically closed to generate a third covalently closed circular molecule carrying the first and second tag sequences. The covalently closed circular molecule undergoes rolling circle amplification to generate a third oligonucleotide-tagged concatemer molecule (bottom concatemer molecule). The third oligonucleotide-tagged concatemer molecule undergoes sequencing using the first batch-specific sequencing primer.

[0051] Figure 25Schematic diagram showing a rolling circle amplification and sequencing workflow for a second target RNA and a second target polypeptide. The second target cDNA molecule (upper left schematic diagram) is hybridized to a target-specific padlock probe for the second cDNA, which carries (i) first and second binding arms that hybridize to the second target cDNA, (ii) a second barcode sequence that uniquely identifies the second target cDNA (BC-2), (iii) a second batch-specific sequencing primer binding site (Batch Seq-2), (iv) a universal binding site for an amplification primer, and (v) a universal binding site for a compacting oligonucleotide. The hybridized second padlock probe includes a gap or nick that is enzymatically closed to generate a second covalently closed circular molecule carrying the second cDNA sequence. The covalently closed circular molecule undergoes rolling circle amplification to generate a second cDNA concatemer molecule (top concatemer molecule). The second cDNA concatemer molecule undergoes sequencing using a second batch-specific sequencing primer. A second antibody-oligonucleotide conjugate (upper right schematic diagram) is bound to the second target polypeptide. The oligonucleotide in the second antibody-oligonucleotide conjugate binds to a target-specific padlock probe of a second target polypeptide-oligonucleotide conjugate, which carries (i) first and second binding arms that hybridize to the third and fourth tag sequences, (ii) a fourth barcode sequence (BC-4) that uniquely identifies the second antibody-oligonucleotide conjugate that selectively binds to the second target polypeptide, and (iii) a second batch-specific sequencing primer binding site (batch Seq-2), (iv) a universal binding site for an amplification primer, and (v) a universal binding site for a compaction oligonucleotide. The hybridized fourth padlock probe includes a gap or nick that is enzymatically closed to generate a fourth covalently closed circular molecule carrying the third and fourth tag sequences. The covalently closed circular molecule undergoes rolling circle amplification to generate a fourth oligonucleotide-tagged concatemer molecule (bottom concatemer molecule). The fourth oligonucleotide-tagged concatemer molecule undergoes sequencing using the second batch-specific sequencing primer.

[0052] Figure 26 A computer system is shown that is programmed or otherwise configured to implement the methods provided herein.

[0053] Figure 27 Described is a non-limiting example method of an antibody conjugated to multiple oligonucleotides via multiple linkers. As described herein, detection efficiency can be improved by adding linker moieties directly or via multivalent structures. This can reduce non-detection caused by rolling circle amplification.

[0054] Figure 28A non-limiting example method as disclosed herein is depicted. A first antibody-oligonucleotide conjugate comprising a first antibody and a first oligonucleotide tag and a second antibody-oligonucleotide conjugate comprising a second antibody and a second oligonucleotide tag can be used against the same target protein corresponding to a target RNA. The first oligonucleotide tag and the second oligonucleotide tag can be used to circularize a padlock probe as disclosed herein. This method can improve binding specificity.

[0055] Figure 29 A non-limiting example method as disclosed herein is depicted. A first antibody-oligonucleotide conjugate comprising a first antibody and a first oligonucleotide tag and a second antibody-oligonucleotide conjugate comprising a second antibody and a second oligonucleotide tag can be used against the same target protein corresponding to a target RNA. The first oligonucleotide tag and the second oligonucleotide tag can be used to circularize a padlock probe as disclosed herein. This method can improve binding specificity. DETAILED DESCRIPTION

[0056] definition

[0057] The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole.

[0058] Unless otherwise defined, the technology and scientific terms used herein have the meaning commonly understood by those skilled in the art, unless otherwise defined. In general, the terms related to molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production and hybridization technology described herein are well-known in the art and commonly used those. The technology and procedures described herein are generally carried out according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, referring to Sambrook et al., Molecular Cloning:A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000), which is incorporated by reference in its entirety. Also referring to Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), which is incorporated by reference in its entirety. The nomenclature utilized in relation to laboratory procedures and technology described herein is well-known in the art and commonly used those.

[0059] Unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular. The singular forms "a," "an," and "the," as well as any word used in the singular, include plural referents unless expressly and unequivocally limited to one referent.

[0060] It will be understood that use of an alternative term (eg, "or") means one or both of the alternatives, or any combination thereof.

[0061] As used herein, the term "and / or" is intended to refer to the specific disclosure of each specified feature or component, whether or not other features or components are present. For example, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include: "A and B"; "A or B"; "A" (only A) or "B" (only B). In a similar manner, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: "A, B and C"; "A, B or C"; "A or C"; "A or B"; "B or C"; "A and B"; "B and C"; "A and C"; "A" (only A); "B" (only B) or "C" (only C).

[0062] As used herein and in the appended claims, the terms "comprises," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be non-limiting, such that the listing of one or more items does not exclude other items that can be substituted or added to the listed items. It should be understood that wherever aspects are described herein with the language of "comprising," otherwise similar aspects described with the terms "consisting of" and / or "consisting essentially of are also provided.

[0063] As used herein, the terms "about" and "approximately" refer to values ​​or compositions within an acceptable error range of a particular value or composition as determined by those skilled in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" or "approximately" can mean within one or more than one standard deviation. Alternatively, depending on the limitations of the measurement system, "about" or "approximately" can mean a range of up to 10% (i.e., ± 10%) or more. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value. When a particular value or composition is provided in the present disclosure, unless otherwise stated, it should be assumed that the meaning of "about" or "approximately" is within an acceptable error range of the particular value or composition. In addition, where a range and / or subrange of a value is provided, the range and / or subrange can include the endpoints of the range and / or subrange.

[0064] As used herein, the term "polymerase" and variants thereof include enzymes comprising a domain in conjunction with nucleotides (or nucleosides), wherein the polymerase can form a complex with a template nucleic acid and a complementary nucleotide. Polymerase can have one or more activities, including but not limited to base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity and nucleotide binding and recognition. Polymerase can be any enzyme that can catalyze nucleotides (including analogs thereof) to polymerize into a nucleic acid chain. Typically but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, the polymerase comprises one or more active sites, at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, the polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand displacement activity. Polymerases can include but are not limited to naturally occurring polymerases and any subunits thereof and truncations, mutant polymerases, variant polymerases, recombinant, fused or other engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases include catalytically inert polymerases, catalytically active polymerases, reverse transcriptases and other enzymes comprising a nucleotide binding domain. In some embodiments, polymerases can be isolated from cells or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases can be expressed in prokaryotes, eukaryotes, viruses or phage organisms. In some embodiments, polymerases can be post-translationally modified proteins or fragments thereof. Polymerases can be derived from prokaryotes, eukaryotes, viruses or phages. Polymerases include DNA-guided DNA polymerases and RNA-guided DNA polymerases.

[0065] As used herein, the term "strand displacement" refers to the ability of a polymerase to locally separate a double-stranded nucleic acid chain and synthesize a new chain in a template-based manner. The strand displacement polymerase displaces the complementary strand from the template strand and catalyzes the synthesis of a new chain. The strand displacement polymerase includes mesophilic and thermophilic polymerases. The strand displacement polymerase includes wild-type enzymes and variants, including exonuclease-minus mutants, mutant versions, chimeric enzymes, and truncation enzymes. The example of a strand displacement polymerase includes the large fragment of phi29 DNA polymerase, Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), the Klenow fragment of Escherichia coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV virus reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (eg, MagniPhi from Expedeon) or a variant EquiPhi29 DNA polymerase (eg, from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (eg, from 4basebio).

[0066] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," and other related terms are used interchangeably and refer to polymers of nucleotides and are not limited to any specific length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, wherein the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids include naturally occurring internucleoside bonds, such as phosphodiester bonds. Nucleic acids can lack phosphate groups. Nucleic acids include non-natural internucleoside bonds, including phosphorothioate, phosphorothioate, or peptide nucleic acid (PNA) bonds. In some embodiments, the nucleic acid comprises a mixture of one type of polynucleotide or two or more different types of polynucleotides.

[0067] As used herein, the terms "operably coupled" and "operably joined" or related terms refer to the juxtaposition of components. The juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically linked together, wherein the linkage joining the two components together comprises a phosphodiester bond. The first and second nucleic acid components can be linked together, wherein the first nucleic acid component can confer functionality to the second nucleic acid component. For example, the linkage between the primer binding sequence and the sequence of interest forms a nucleic acid library molecule having a portion that can be bound to a primer. In another example, a transgenic (e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest) can be linked to a vector, wherein the linkage allows expression or functionalization of the transgenic sequence contained in the vector. In some embodiments, the transgenic is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects transgenic expression. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signal sequence, etc. In some embodiments, the host cell regulatory sequence controls expression levels, transgenic timing, and / or transgenic position.

[0068] The terms "link," "join," "attach," "attach," and variations thereof include any type of fusion, bonding, adhesion, or association between any combination of compounds or molecules that is sufficiently stable to withstand use in a particular procedure. Procedures may include, but are not limited to, nucleotide binding; nucleotide incorporation; deblocking (e.g., removal of a chain terminating portion); washing; removal; flow; detection; imaging and / or identification, or any combination thereof. Such linkages may include, for example, covalent bonds, ionic bonds, hydrogen bonds, dipole-dipole bonds, hydrophilic bonds, hydrophobic bonds, or affinity bonds, bonds or associations involving van der Waals forces, mechanical bonds, and the like, or any combination thereof. In some embodiments, such linkages occur within a molecule, such as joining the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such linkages may occur between a combination of different molecules or between molecules and non-molecules, including, but not limited to, linkages between a nucleic acid molecule and a solid surface; linkages between a protein and a detectable reporter portion; linkages between a nucleotide and a detectable reporter portion, and the like. Some examples of linkages can be found, for example, in Hermanson, G., “Bioconjugate Techniques”, 2nd ed. (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998), which are incorporated herein by reference in their entirety.

[0069] The term "primer" and related terms used herein refer to oligonucleotides that can hybridize with DNA and / or RNA polynucleotide templates to form duplex molecules. Primers include natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers can have any length, but generally range from 4 to 50 nucleotides. Typical primers include 5' ends and 3' ends. The 3' end of a primer can include a 3'OH portion, which serves as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of a primer can lack a 3'OH portion or can include an end 3' blocking group that suppresses nucleotide polymerization in a polymerase-catalyzed reaction. Any nucleotide or more than one nucleotide along the length of the primer can be labeled with a detectable reporter moiety. Primers can be in solution (e.g., soluble primers) or can be fixed to a support (e.g., surface capture primers).

[0070] The terms "template nucleic acid," "template polynucleotide," "target nucleic acid," "target polynucleotide," "template strand," and other variants refer to a nucleic acid strand that serves as a base nucleic acid molecule for any of the iterative sequencing methods described herein. The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from naturally occurring sources, recombinant forms, or chemical synthesis to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or in other forms. The template nucleic acid can include an insert having an insert sequence. The template nucleic acid can also include at least one adapter sequence. The insert can be isolated in any form, including chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, clones, amplifications, cDNA, RNA such as pre-mRNA or mRNA, oligonucleotides, whole genomic DNA, obtained from fresh frozen paraffin-embedded tissue, needle biopsy, circulating tumor cells, cell-free circulating DNA, or any type of nucleic acid library. Inserts can be isolated from any source, including organisms such as prokaryotes, eukaryotes (e.g., humans, plants, and animals), fungi, viruses, cells, tissues, normal or diseased cells or tissues, body fluids, including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid samples, sweat, semen, environmental samples, culture samples, or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. Inserts can be isolated from any organ, including the head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testis, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary; thymus, skin, heart, larynx, or other organs. Nucleic acid analysis, including sequencing and compositional analysis, can be performed on the template nucleic acid.

[0071] The term "adapter" and related terms refer to oligonucleotides that can be operably coupled to a target polynucleotide, wherein the adaptor confers a co-engaged adaptor-target molecule function. The adaptor can include DNA, RNA, chimeric DNA / RNA, or its analogs. The adaptor can include at least one ribonucleoside residue. The adaptor can be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. The adaptor can be configured as a linear, stem-loop, hairpin, or Y-shaped form. The adaptor can be of any length, including 4-100 nucleotides or longer. The adaptor can have a blunt end, an overhang, or a combination of the two. The overhang includes a 5' overhang and a 3' overhang. The 5' end of a chain of a single-stranded adaptor or a double-stranded adaptor can have a 5' phosphate group or lack a 5' phosphate group. The adaptor can include a 5' tail (e.g., a tail adaptor) that does not hybridize with the target polynucleotide, or the adaptor can be tailless. The adapter may include a sequence that is at least partially complementary to a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). The adapter may include a random sequence or a degenerate sequence. The adapter may include at least one inosine residue. The adapter may include at least one phosphorothioate, thiophosphate, and / or phosphoramidate bond. The adapter may include a barcode sequence that can be used to distinguish polynucleotides (e.g., insertion sequences) from different sample sources in a multiplex assay. The adapter may include a unique identification sequence (e.g., a unique molecular index (UMI) or a unique molecular tag) that can be used to uniquely identify the nucleic acid molecule to which the adapter is attached. In some embodiments, a unique identification sequence can be used to increase error correction and accuracy, reduce false positive variant call rates, and / or improve the sensitivity of variant detection. The adapter may include at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from type I, type II, type III, type IV, type Hs, or type IIB.

[0072] In some embodiments, the length of any amplification primer sequence, sequencing primer sequence, capture primer sequence, target capture sequence, circularization anchor sequence, sample barcode sequence, spatial barcode sequence, or anchor region sequence can be about 3-50 nucleotides or about 5-40 nucleotides, or about 5-25 nucleotides.

[0073] The term "universal sequence" and related terms refer to a sequence in a nucleic acid molecule that is shared among two or more polynucleotide molecules. For example, an adaptor with a universal sequence can be operably joined to multiple polynucleotides such that the co-joined population of molecules carries the same universal adaptor sequence. Examples of universal adaptor sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or immobilized capture primers).

[0074] When used to refer to nucleic acid molecules, the term "hybridize" or "hybridizing" or "hybridization" or other related terms refer to the hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization can also include hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule with a duplex region. Hybridization can include Watson-Crick or Hoogsteen binding to form a duplex double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. The two different regions of a double-stranded nucleic acid or a single nucleic acid can be fully complementary or partially complementary. The complementary nucleic acid chains do not need to hybridize to each other across their entire length. Complementary base pairing can be standard AT or CG base pairing or can be other forms of base pairing interactions. The duplex nucleic acid can include mismatched base pairing nucleotides.

[0075] When used in reference to nucleic acids, the terms "extend," "extending," "extension," and other variants refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides into the terminal 3' OH end of a nucleic acid chain, resulting in extension of the nucleic acid chain. Nucleotide incorporation can be performed using natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, including primer extension catalyzed by DNA polymerase or RNA polymerase.

[0076] The term "nucleotide" and related terms refer to molecules comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose) and at least one phosphate group. Standard or non-standard nucleotides are consistent with the use of the term. In some embodiments, phosphate includes monophosphate, diphosphate or triphosphate, or corresponding phosphate analogs. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar. Nucleotide and nucleoside can be unlabeled or labeled with a detectable reporter moiety.

[0077] Nucleotides (and nucleosides) typically contain heterocyclic bases, which include substituted or unsubstituted nitrogen-containing parent heteroaromatic rings commonly found in nucleic acids, including naturally occurring, substituted, modified or engineered variants, or analogs thereof. The bases of the nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with suitable complementary bases. Non-limiting example bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), vinyladenine, N6-Δ2-isopentenyladenine (6iA), N6-Δ2-isopentenyl-2-methylthioadenine (2ms6iA); N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine, and O6-methylguanine; 7-deazapurines such as 7-deazaadenine (7- pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; muscimol; inosine; hydroxymethylcytosine; 5-methylcytosine; bases (Y); and methylated, glycosylated and acylated base moieties, etc. Additional non-limiting exemplary bases can be found in Fasman, 1989, in "Practical Handbook of Biochemistry and Molecular Biology", pp. 385-394, CRC Press, Boca Raton, Fla., which is incorporated herein by reference in its entirety.

[0078] Nucleotides (and nucleosides) typically contain sugar moieties, such as carbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48, which are incorporated herein by reference in their entirety), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7:3013-3016, both of which are incorporated herein by reference in their entirety), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36:2627-2638; Kim, et al., 1993 J. Med. Chem. 36:30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Patent No. 5,558,991, all of which are incorporated herein by reference in their entirety). Sugar moieties include: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribosyl, carbocyclic; acyclic or other modified sugars.

[0079] In some embodiments, nucleotide comprises a chain of one, two or three phosphorus atoms, wherein the chain is generally attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramidite bond. In some embodiments, nucleotide is an analog with a phosphorus chain, in which a phosphorus atom is linked to an O, S, NH, methylene or ethylene group in the middle. In some embodiments, the phosphorus atom in the chain comprises a substituted side group, including O, S or BH3. In some embodiments, the chain comprises a phosphate group substituted with an analog, including phosphoramidate, phosphorothioate, phosphorodithioate and O-methylphosphoramidite groups.

[0080] The term "reporter moiety", "reporter moieties" or related terms refer to a compound that produces or causes a detectable signal. A reporter moiety is sometimes referred to as a "label". Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophore, radioisotope, electrochemistry, mass spectrometry, Raman, hapten, affinity tag, atom or enzyme. The reporter moiety produces a detectable signal caused by a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzyme activity or adjacent event). An adjacent event includes two reporter moieties that are close to each other, or associate with each other or bind to each other. In some embodiments, the reporter moiety can be selected so that each reporter moiety absorbs excitation radiation and / or emits fluorescence at a wavelength that is distinguishable from other reporter moieties, to allow monitoring of the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties with spectrally different emission curves or with minimal overlapping spectral emission curves can be selected. The reporter moiety can be linked (eg, operably linked) to a nucleotide, nucleoside, nucleic acid, enzyme (eg, a polymerase or reverse transcriptase), or support (eg, a surface).

[0081] The reporter moiety (or label) may include a fluorescent label or fluorophore. Non-limiting examples of fluorescent moieties that can be used as fluorescent labels or fluorophores include, but are not limited to, fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthylfluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetylaminofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine, fluorescein thiocyanate, fluorescein thiosemicarbazide, fluorescein thioacetyl-aminofluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine, fluorescein thiocyanate, fluorescein thioacetyl-aminofluorescein ... Rhodamine, Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY530 / 550C3, BODIPY-530 / 550C3-SE, BODIPY530 / 550C3 hydrazide, BODIPY-493 / 503C3 hydrazide, BODIPY-FL C3 hydrazide, BODIPY-FL-IA, BODIPY 530 / 551IA, Br-BODIPY493 / 503, Cascade Blue Blue) and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow (Lucifer Yellow) and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanines and derivatives such as indolium-based cyanine dyes, benzindolium-based cyanine dyes, pyridinium-based cyanine dyes, thiazolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy 3, Cy 5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon green dye, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near infrared dyes and others known in the art such as described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th edition;those described in Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press New York (1999) or Hermanson, Bioconjugate Techniques, 2nd ed. (all references are incorporated herein in their entirety);or its derivatives or any combination thereof. Cyanine dyes may exist in sulfonated or non-sulfonated form and consist of two indoline, benzindolium, pyridinium, thiazolium and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which may include 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5 -dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may include 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indoline -2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indolium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolinoline-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate) and Cy7 (which may include 1-(5-carboxypenta 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for "cyanine" and the first digit identifies the number of carbon atoms between the two indoline groups. Cy2, which is an oxazole derivative rather than an indoline, and benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are non-limiting examples of exceptions to this rule. ;

[0082] In some embodiments, the reporter moiety can be a FRET pair, allowing multiple classifications to be performed in a single excitation and imaging step. As used herein, FRET can include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0083] The terms "amplify," "amplifying," "amplification," and other related terms when used with reference to nucleic acids include the production of multiple copies of an original polynucleotide template molecule, wherein the copies include a sequence that is complementary to the template sequence or the copies include a sequence that is identical to the template sequence. In some embodiments, the copies include a sequence that is substantially identical to the template sequence or a sequence that is substantially identical to a sequence that is complementary to the template sequence.

[0084] As used herein, the term "support" refers to a substrate designed to deposit biomolecules or biological samples for measurement and / or analysis. Examples of biomolecules to be deposited on a support include nucleic acids (e.g., DNA, RNA), polypeptides, carbohydrates, lipids, single cells or multiple cells. Examples of biological samples include, but are not limited to, saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs.

[0085] In some embodiments, the support is solid, semi-solid, or a combination thereof. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the support can be substantially flat, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0086] In some embodiments, the surface of the support can be substantially smooth.In some embodiments, the support can be regularly or irregularly textured, including bumps, etchings, holes, three-dimensional scaffolds, or any combination thereof.

[0087] In some embodiments, the support comprises beads having any shape, including spheres, hemispheres, cylinders, barrels, rings, disks, rods, cones, triangles, cubes, polygons, tubes, or wires.

[0088] The support can be made of any material, including but not limited to glass, fused silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various combinations of glass and plastic substrates are contemplated.

[0089] The support can have multiple (for example, two or more) nucleic acid templates fixed thereon.Multiple immobilized nucleic acid templates can have identical sequence or have different sequences.In some embodiments, the single nucleic acid template molecule in multiple nucleic acid templates is fixed to the different sites on the support.In some embodiments, two or more independent nucleic acid template molecules in multiple nucleic acid templates are fixed to the site on the support.

[0090] The term "array" refers to a support comprising a plurality of sites at predetermined positions on a support to form an array of sites. The sites may be discrete and separated by interstitial regions. In some embodiments, the predetermined sites on the support may be arranged in rows or columns in one dimension, or in rows and columns in two dimensions. In some embodiments, a plurality of predetermined sites are arranged on the support in an organized manner. In some embodiments, a plurality of predetermined sites are arranged in any organized pattern, including a straight line, a hexagonal pattern, a grid pattern, a pattern with reflection symmetry, a pattern with rotational symmetry, and the like. The spacing between different pairs of sites may be the same or may vary. In some embodiments, upholder includes at least 102 sites, at least 103 sites, at least 104 sites, at least 105 sites, at least 106 sites, at least 107 sites, at least 108 sites, at least 109 sites, at least 1010 sites, at least 1011 sites, at least 1012 sites, at least 1013 sites, at least 1014 sites, at least 1015 sites or more, wherein site can be positioned at the predetermined position on upholder. In some embodiments, multiple predetermined sites (for example, 102-1015 sites or more) on upholder are fixed to form nucleic acid template array with nucleic acid template. In some embodiments, nucleic acid template is fixed on multiple predetermined sites by hybridizing with immobilized surface capture primer or nucleic acid template is covalently attached to surface capture primer. In some embodiments, nucleic acid template is fixed on multiple predetermined sites, for example, is fixed on 102-1015 or more sites. In some embodiments, immobilized nucleic acid template is through clonal amplification, to generate immobilized nucleic acid clusters at multiple predetermined sites. In some embodiments, a single immobilized nucleic acid cluster comprises a linear cluster or comprises a single-stranded or double-stranded concatemer.

[0091] In some embodiments, the support comprising a plurality of sites positioned at random position on the support is referred to herein as the support with the site of random positioning thereon.The position of the site of random positioning on the support is not predetermined.A plurality of randomly positioned sites are arranged on the support in disorder and / or unpredictable mode. In some embodiments, the support comprises at least 102 sites, at least 103 sites, at least 104 sites, at least 105 sites, at least 106 sites, at least 107 sites, at least 108 sites, at least 109 sites, at least 1010 sites, at least 1011 sites, at least 1012 sites, at least 1013 sites, at least 1014 sites, at least 1015 sites or more, wherein the site is randomly positioned on the support. In some embodiments, a plurality of randomly positioned sites (for example, 102-1015 sites or more) are fixed with nucleic acid template on the support, to form the support fixed with nucleic acid template. In some embodiments, the nucleic acid template is fixed at a plurality of randomly positioned sites by hybridization with an immobilized surface capture primer or the nucleic acid template is covalently attached to a surface capture primer. In some embodiments, the nucleic acid template is fixed at a plurality of randomly positioned sites, for example, fixed at 102-1015 or more sites. In some embodiments, the immobilized nucleic acid template is cloned and amplified to generate immobilized nucleic acid clusters at a plurality of randomly positioned sites. In some embodiments, a single immobilized nucleic acid cluster comprises a linear cluster or comprises a single-stranded or double-stranded concatemer.

[0092] In some embodiments, the plurality of immobilized surface capture primers on the support are in fluid communication with each other to allow a reagent solution (e.g., a nucleic acid template molecule, a soluble primer, an enzyme, a nucleotide, a divalent cation, a buffer, etc.) to flow onto the support so that the plurality of immobilized surface capture primers on the support can react with the reagent substantially simultaneously in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized surface capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) substantially simultaneously on the plurality of immobilized surface capture primers.

[0093] In some embodiments, a plurality of immobilized nucleic acid clusters on the support are in fluid communication with each other, to allow a reagent solution (e.g., enzyme, nucleotide, divalent cation, etc.) to flow onto the support so that a plurality of immobilized nucleic acid clusters on the support can react with a reagent substantially simultaneously in a large-scale parallel manner. In some embodiments, the fluid communication of a plurality of immobilized nucleic acid clusters can be used to substantially simultaneously carry out nucleotide binding assays and / or carry out nucleotide polymerization reactions (e.g., primer extension or sequencing) on ​​a plurality of immobilized nucleic acid clusters, and optionally carry out detection and imaging for large-scale parallel sequencing.

[0094] The term "immobilized" and related terms, when used in reference to an immobilized enzyme, refers to an enzyme that is attached to a support (e.g., a polymerase) by covalent bonds or non-covalent interactions, or to a coating on a support, or is embedded in a matrix formed by a coating on a support.

[0095] When used to refer to immobilized nucleic acids, the term "immobilized" and related terms refer to nucleic acid molecules attached to a support by covalent bonds or non-covalent interactions, or nucleic acid molecules attached to a coating on a support, or nucleic acid molecules embedded in a matrix formed by a coating on a support, wherein the nucleic acid molecules include surface capture primers, nucleic acid template molecules, and extension products of the capture primers. The extension products of the capture primers include nucleic acid concatemers (e.g., nucleic acid clusters).

[0096] In some embodiments, one or more nucleic acid templates are fixed on a support, for example, fixed on a site on a support. In some embodiments, one or more nucleic acid templates are through clonal amplification. In some embodiments, one or more nucleic acid templates are cloned and amplified from a support (for example, in a solution), and are then deposited on a support and fixed on a support. In some embodiments, the clonal amplification reaction of one or more nucleic acid templates is carried out on a support, resulting in fixation on a support. In some embodiments, one or more nucleic acid templates use nucleic acid amplification reactions to carry out clonal amplification (for example, in a solution or on a support), including any one or any combination of the following: polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), chain displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), ring to ring amplification (circle-to-circle amplification), helicase-dependent amplification, recombinase-dependent amplification and / or single-stranded binding (SSB) protein-dependent amplification.

[0097] The term "duration" and related terms refer to the length of time that the binding complex formed between the target nucleic acid, polymerase, conjugated or unconjugated nucleotides remains stable without any binding components dissociating from the binding complex. The duration indicates the stability of the binding complex and the intensity of the binding interaction. The duration can be measured by observing the start and / or duration of the binding complex, such as by observing the signal from the labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in the binding complex, thereby allowing the signal from the label to be detected during the duration of the binding complex. A non-limiting example of labeling is a fluorescent label.

[0098] introduction

[0099] The present disclosure provides methods, compositions, systems and kits for processing or analyzing biomolecules. Biomolecules can include, for example, nucleic acids, polypeptides or proteins, lipids or carbohydrates. Methods, compositions, systems and kits as described herein can be used to obtain information about the position of nucleic acid sequences (such as RNA or DNA) and proteins encoded by nucleic acid sequences. For example, target nucleic acid sequences can be identified and sequenced as disclosed herein. For example, target RNA or DNA molecules associated with a target protein or polypeptide can be identified by padlock probes as described herein and can be further amplified and / or sequenced. In this way, information about the position and / or sequence of target proteins, RNA sequences and DNA sequences can be collected.

[0100] In some embodiments, disclosed herein are methods, compositions, systems and kits for processing and / or analyzing nucleic acid sequences. Methods, systems, kits and compositions as described herein can be used in in situ repeated short read sequencing. Sequencing can be performed in one or more cells, tissues or tumors. The nucleic acid sequence can be, for example, a DNA sequence or an RNA sequence. Methods, systems, kits and compositions as described herein can be useful in utilizing massively parallel sequencing technology. Such methods, compositions, kits and systems can also be found in U.S. Publication No. US20210139884, which is incorporated herein by reference in its entirety.

[0101] method

[0102] The present disclosure provides a method for analyzing nucleic acid sequences. The method disclosed herein may include introducing one or more reagents into a biological sample under conditions sufficient to identify RNA-encoding DNA, protein-encoding RNA, or proteins in single nucleic acid sequencing. Identification may include running any method disclosed herein. In some embodiments, the method disclosed herein does not require the use of barcoded nucleic acids, but may utilize spatially encoded reagents. For example, primers spatially encoded on a solid surface can be identified by their relative position on the solid surface. The method described herein can be used in situ repeated short-read sequencing within a cell sample including a single cell, multiple cells, tissue, or tumor. In some embodiments, the method includes repeatedly performing a short number of sequencing cycles on the same region of the template molecule. By performing repeated short sequencing cycles, the RNA content of the cell sample can be found. The method described herein can be used in a transcriptomics workflow that utilizes massively parallel sequencing technology. Compared to the long-read sequencing workflow, the repeated short sequencing cycles described herein use a reduced amount of sequencing reagents, which reduces costs and saves time. The method for performing repeated short sequencing cycles has many uses, including but not limited to detecting specific RNAs of interest, mutant RNA sequences, splice variants, and their abundance levels.

[0103] A non-limiting example of the purpose of the method described herein is to use large-scale parallel sequencing to detect and image the spatial localization of RNA in a cell sample. The workflow may include the following general steps: the cell sample can be placed on a solid support, which can be positioned on a fluorescence microscope configured to detect fluorescent signals. The cells can be treated with a chemical fixation agent to retain the target RNA within the cell. The cell sample can also be permeabilized to allow manipulation of the target RNA within the cell. The target RNA can be converted into a first-strand cDNA, which can be selectively hybridized with a target-specific padlock probe to generate a circularized padlock probe corresponding to a specific cDNA. The padlock probe can carry at least one universal adapter for a sequencing primer binding site. The padlock probe can also carry a target barcode sequence, each sequence corresponding to a given target cDNA. The circularized padlock probe can be connected and / or filled with a reaction to form a covalently closed circular padlock probe, which can be amplified via rolling circle amplification within the cell sample to generate a single-stranded concatemer. Rolling circle amplification can be performed in the presence of one or more compacting oligonucleotides. Concatemers can carry tandem repeat units of the cDNA of interest, universal sequencing primer binding sites, and target barcode sequences. Concatemers can be sequenced within a cell sample, wherein a short number of sequencing cycles are performed for each round, and multiple rounds of short read sequencing are performed. The full length of the target barcode and cDNA regions may not be sequenced. Instead, at least a portion of the target barcode region may be sequenced repeatedly. In some embodiments, the cDNA region does not need to be sequenced. In some embodiments, portions of the target barcode and cDNA regions are sequenced repeatedly. The entire length of the cDNA region may not need to be sequenced. There may be no need to assemble sequencing reads or obtain the full-length sequence of the cDNA of interest. The redundant sequencing information obtained from the short sequencing reads can eliminate the need for sequencing the complementary strands of the concatemer. Therefore, paired sequencing may not be required.

[0104] The methods described herein can provide several advantages over other in situ transcriptomics workflows. For example, a cell sample can be placed on a solid support, such as a planar support including glass or plastic, which can be made into any shape and size. It is not necessary to assemble a hybridization chamber on the support. It is also not necessary to prepare chemically washed glass beads for cell adhesion. The support can be passivated with a coating that promotes cell adhesion to the support. The coating does not need to be formulated to include tethered capture primers. The support to which the cell sample is adhered can be easily adapted to accommodate existing flow cell supports / brackets that are fluidically connected to an automatic fluid distribution system and configured on a fluorescence microscope. Any combination of steps for performing in situ repeated short read sequencing can be performed in an automated mode using a fluid distribution system, including cell seeding, cell fixation, cell permeabilization, reverse transcription reaction, padlock probe hybridization, padlock probe ligation reaction, rolling circle amplification, and sequencing.

[0105] Another advantage of the method described herein is to form concatemers in cell samples. Single-stranded concatemers can be folded into compact DNA nanoballs, wherein each nanoball carries numerous serial copies of polynucleotide units along its length, wherein the polynucleotide units include cDNA sequences of interest and at least one universal sequencing primer binding site. Each polynucleotide unit can combine sequencing primers, sequencing polymerase and detectably labeled nucleotide reagents (e.g., detectably labeled multivalent molecules) to form detectable sequencing complexes (e.g., detectable ternary complexes). Each nanoball carries numerous detectable sequencing complexes. Therefore, the compactness of nanoballs can increase the local concentration of the detectably labeled nucleotide reagents used during the sequencing workflow, which can thereby increase the signal intensity emitted by nanoballs. This can provide discrete detectable signals, which can be imaged as fluorescent spots in cell samples. Each spot corresponds to concatemers, and each concatemer corresponds to the target RNA molecule in the cell sample. Multiple spots in the imaging cell sample can be detected simultaneously.

[0106] In addition, can be from identical starting position to the short part resequencing of cDNA region in concatemer at least once (for example, sequencing repeatedly), to generate can with the overlapping sequencing read of reference sequence comparison.For example, the identical part of concatemer molecule can be sequenced at least twice, three times, four times, five times or 50 times at the most.The initial sequencing site can be any position of concatemer, and can be determined by the sequencing primer that is designed to anneal to the selected position in concatemer.Repeated short sequencing read can increase the redundancy of the sequencing information of single base in cDNA region.Repeated sequencing of a chain of concatemer template molecule can provide enough base coverage, to disclose the existence of target RNA in cell sample, make without the complementary chain being carried out paired sequencing.

[0107] Concatemer template molecules can include multiple sequencing primer binding sites along the same concatemer molecule, which can be used to generate multiple usable sequencing reads to increase sequencing depth. Compared to sequencing a copy of the template molecule, repeatedly sequencing one strand of the concatemer template together can increase sequencing base coverage and sequencing depth.

[0108] The methods described herein can be performed in singleplex or multiplex mode. Using different reverse transcription primers, different target-specific padlock probes, and universal sequencing primers, two or more different target RNAs can be detected and imaged simultaneously within a cell sample. For example, any of the iterative short-read sequencing methods described herein can be used to simultaneously detect and image the presence of a housekeeping RNA and at least one target RNA in a cell sample.

[0109] In the methods described herein, RNA can be extracted from cell samples and can not need to track sequencing information and map it back to the image of the cell sample.On the contrary, RNA can be retained in the cell sample to allow the spatial position of the target RNA in the cell to be directly imaged.In addition, the RNA in the cell sample can be without fragmentation and can be without the need to enrich the target RNA.Use target-specific and / or random sequence reverse transcription primers to enable detection of poly-A and non-poly-A RNA in single or multiple modes.

[0110] The method described here offers several advantages over other in situ transcriptomics workflows, including a simpler workflow, fewer reagents, lower cost, less time, milder conditions for cell samples, and no need for specialized equipment.

[0111] Methods for performing in situ repetitive short-read sequencing

[0112] The present disclosure provides a method for performing in situ sequencing in a biological sample. One or more nucleic acids can be analyzed in a biological sample (e.g., a cell). The nucleic acid can include, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Sequencing can be repeated so that the nucleic acid is subjected to multiple sequencing cycles.

[0113] The present disclosure provides a method for in situ detection of at least two nucleic acid sequences in a biological sample. The present disclosure provides a method for in situ detection of at least two target RNA molecules in a biological sample. The at least two target nucleic acid sequences may include a first target nucleic acid sequence and a second target nucleic acid sequence. The biological sample may be a cell sample. The method may include performing a sequencing reaction within the cell sample, wherein the cDNA amplicon may be a concatemer molecule. The biological sample may include a first nucleic acid molecule, a second nucleic acid molecule, or a combination thereof. The first nucleic acid molecule may include a first target nucleic acid sequence or a portion thereof, or a reverse complement thereof or a portion thereof. The second nucleic acid molecule may include a second target nucleic acid sequence or a portion thereof, or a reverse complement thereof or a portion thereof. The method may include determining the sequence of the first nucleic acid molecule or a portion thereof in the biological sample in situ to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first nucleic acid molecule or a portion thereof. The method may include determining the sequence of the second nucleic acid molecule or a portion thereof in situ to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second nucleic acid molecule or a portion thereof. The full sequence of the first target nucleic acid sequence and the full sequence of the second target nucleic acid sequence may differ by at least one nucleotide. The method may further include removing the first sequencing product nucleic acid molecule from the first nucleic acid molecule and removing the second sequencing product nucleic acid molecule from the second nucleic acid molecule. After removal, the first nucleic acid molecule and the second nucleic acid molecule can be located in the biological sample. The method can further include repeating the sequence of the first and second nucleic acids in situ. In some embodiments, repeating includes repeating at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times or at least 50 times. The method can further include repeating to remove the first sequencing product nucleic acid molecule from the first nucleic acid molecule and to remove the second sequencing product nucleic acid molecule from the second nucleic acid molecule. In some embodiments, repeating includes repeating at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times or at least 50 times.

[0114] The present disclosure provides a method for detecting at least two different target nucleic acid molecules in a biological sample in situ. The present disclosure provides a method for detecting at least two different target RNA molecules in a cell sample in situ, comprising step (a): providing a cell sample deposited on a solid support, wherein the cell sample carries at least a first plurality of DNA amplicons corresponding to a first target RNA molecule, and the cell sample carries a second plurality of DNA amplicons corresponding to a second target RNA molecule. In some embodiments, the cell sample may include a first target RNA molecule. In some embodiments, the cell sample may include at least a first plurality of DNA amplicons, which may correspond to a first target RNA molecule. In some embodiments, the cell sample may include a second target RNA molecule. In some embodiments, the cell sample may include at least a second plurality of DNA amplicons, which may correspond to a second target RNA. In some embodiments, the cell sample may be deposited on a solid support.

[0115] In some embodiments, the cell sample carries 2-25 different target RNA molecules, or carries 25-50 different target RNA molecules, or carries 50-75 different target RNA molecules, or carries 75-100 different target RNA molecules. In some embodiments, the cell sample carries more than 100 different target RNA molecules, or more than 250 different target RNA molecules, or more than 500 different target RNA molecules, or more than 1000 different target RNA molecules, or more. In some embodiments, the cell sample carries more than 10,000 different target RNA molecules. In some embodiments, the cell sample comprises a whole cell, a plurality of whole cells, a complete tissue, or a complete tumor. In some embodiments, the cell sample comprises a fresh cell sample, a fresh frozen cell sample, a sliced ​​cell sample, or a FFPE cell sample. In some embodiments, the cell sample is deposited on a solid support. In some embodiments, the cell sample is deposited on a solid support, and the solid support is passivated with a coating that promotes cell adhesion. In some embodiments, the cell sample is deposited on a support lacking immobilized capture oligonucleotides. In some embodiments, the cell sample comprises an expanded cell sample that has been cultured in a simple or complex cell culture medium.

[0116] In some embodiments, the first plurality of DNA amplicons comprises a first plurality of concatemers. In some embodiments, the second plurality of DNA amplicons comprises a second plurality of concatemers.

[0117] In some embodiments, the first plurality of DNA amplicons comprise a first target DNA sequence corresponding to a first target RNA molecule. In some embodiments, the first plurality of DNA amplicons comprise a first target DNA sequence corresponding to a first target RNA molecule and a first target barcode sequence corresponding to the first target RNA molecule. In some embodiments, the first plurality of DNA amplicons comprise a first target DNA sequence corresponding to a first target RNA molecule and at least one universal adapter sequence, such as a universal sequencing primer binding site (or its complement). In some embodiments, the first plurality of DNA amplicons comprise a first target DNA sequence corresponding to a first target RNA molecule and a universal primer binding site (or its complement) for a rolling circle amplification primer. In some embodiments, the first plurality of DNA amplicons comprise a first target DNA sequence corresponding to a first target RNA molecule and a universal compaction oligonucleotide binding site (or its complement).

[0118] In some embodiments, the second plurality of DNA amplicons comprise a second target DNA sequence corresponding to a second target RNA molecule. In some embodiments, the second plurality of DNA amplicons comprise a second target DNA sequence corresponding to a second target RNA molecule and a second target barcode sequence corresponding to the second target RNA molecule. In some embodiments, the second plurality of DNA amplicons comprise a second target DNA sequence corresponding to a second target RNA molecule and at least one universal adapter sequence, such as a universal sequencing primer binding site (or its complement). In some embodiments, the second plurality of DNA amplicons comprise a second target DNA sequence corresponding to a second target RNA molecule and a universal primer binding site (or its complement) for a rolling circle amplification primer. In some embodiments, the second plurality of DNA amplicons comprise a second target DNA sequence corresponding to a second target RNA molecule and a universal compaction oligonucleotide binding site (or its complement).

[0119] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cell sample further comprises step (b): sequencing a first plurality of DNA amplicons within the cell sample. Sequencing may include performing no more than 2-30 sequencing cycles to generate a plurality of first sequencing read products, and sequencing a second plurality of DNA amplicons within the cell sample, which includes performing no more than 2-30 sequencing cycles to generate a plurality of second sequencing read products. In some embodiments, (b) may include sequencing a first plurality of DNA amplicons within the cell sample, which may include performing no more than 2-30 sequencing cycles. In some embodiments, 2-30 sequencing cycles may generate a plurality of first sequencing read products. In some embodiments, (b) may include sequencing a second plurality of DNA amplicons within the cell sample, which may include performing no more than 2-30 sequencing cycles. In some embodiments, 2-30 sequencing cycles may generate a plurality of second sequencing read products. In some embodiments, the sequence of the first sequencing read product may be compared with the first target reference sequence to confirm the presence of the first target RNA in the cell product. In some embodiments, the sequence of the second sequencing read product can be aligned with the second target reference sequence to confirm the presence of the second target RNA in the cell sample. In some embodiments, the sequence of the first sequencing read product is aligned with the first target reference sequence to confirm the presence of the first target RNA in the cell sample, and the sequence of the second sequencing read product is aligned with the second target reference sequence to confirm the presence of the second target RNA in the cell sample.

[0120] In some embodiments, the first target reference sequence comprises a first target barcode sequence. In some embodiments, the first target reference sequence comprises a first target barcode sequence and at least a portion of a first target RNA sequence. In some embodiments, the first target reference sequence comprises at least a portion of a first target RNA sequence.

[0121] In some embodiments, the second target reference sequence comprises a second target barcode sequence. In some embodiments, the second target reference sequence comprises a second target barcode sequence and at least a portion of a second target RNA sequence. In some embodiments, the second target reference sequence comprises at least a portion of a second target RNA sequence.

[0122] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cell sample may further include (c): removing a plurality of first sequencing read products from the first DNA amplicon and retaining the first DNA amplicon within the cell sample, and removing a plurality of second sequencing read products from the second DNA amplicon and retaining the second DNA amplicon within the cell sample. In some embodiments, (c) may include removing a plurality of first sequencing read products from the first DNA amplicon. In some embodiments, (c) may include retaining the first DNA amplicon within the cell sample. In some embodiments, (c) may include removing a plurality of second sequencing read products from the second DNA amplicon. In some embodiments, (c) may include retaining the second DNA amplicon within the cell sample.

[0123] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cell sample further includes step (c): removing a plurality of first sequencing read products from the first DNA amplicon and retaining the first DNA amplicon within the cell sample, and removing a plurality of second sequencing read products from the first DNA amplicon and retaining the second DNA amplicon within the cell sample.

[0124] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cell sample may further comprise (d): iteratively sequencing the first and second pluralities of DNA amplicons by repeating (b) and (c) at least once. In some embodiments, repeating comprises repeating at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or at least 50 times. In some embodiments, (d) may comprise iteratively sequencing the first pluralities of DNA amplicons by repeating (b) and (c) at least once. In some embodiments, (d) may comprise iteratively sequencing the second pluralities of DNA amplicons by repeating (b) and (c) at least once. In some embodiments, (b) and (c) may be repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, (b) and (c) can be repeated up to 10 times, up to 20 times, up to 30 times, up to 40 times, or up to 50 times.

[0125] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cell sample further comprises step (d): iteratively sequencing the first and second pluralities of DNA amplicons by repeating steps (b) and (c) at least once. In some embodiments, steps (b) and (c) can be repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, steps (b) and (c) can be repeated at most 10 times, at most 20 times, at most 30 times, at most 40 times, or at most 50 times.

[0126] In some embodiments, the sequences of the first and second sequencing read products can be aligned after each round of generating first and second sequencing read products that are no longer than 30 bases (e.g., after step (b)) or after generating a set of repeated sequencing read products in which the first and second sequencing read products are no longer than 30 bases (e.g., after step (d)).

[0127] The present disclosure provides a method for in situ detection of at least two different target RNA molecules in a cell sample, comprising step (a): providing a cell sample with multiple RNAs, the multiple RNAs including at least a first target RNA molecule and a second target RNA molecule, wherein the cell sample is fixed and permeabilized. In some embodiments, the cell sample carries 2-25 different target RNA molecules, or carries 25-50 different target RNA molecules, or carries 50-75 different target RNA molecules, or carries 75-100 different target RNA molecules. In some embodiments, the cell sample carries more than 100 different target RNA molecules, or more than 250 different target RNA molecules, or more than 500 different target RNA molecules, or more than 1000 different target RNA molecules, or more. In some embodiments, the cell sample carries more than 10,000 different target RNA molecules. In some embodiments, the cell sample comprises whole cells, multiple whole cells, intact tissue, or complete tumor. In some embodiments, the cell sample comprises a fresh cell sample, a fresh frozen cell sample, a sliced ​​cell sample, or a FFPE cell sample. In some embodiments, the cell sample is deposited on a solid support. In some embodiments, the cell sample is deposited on a solid support, and the solid support is passivated with a coating that promotes cell adhesion. In some embodiments, the cell sample is deposited on a support that lacks immobilized capture oligonucleotide. In some embodiments, a plurality of RNAs may include at least a first target RNA molecule. In some embodiments, a plurality of RNAs may include at least a second target RNA molecule. In some embodiments, the cell sample is cultivated before carrying out step (b) described below.

[0128] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample further comprises step (b): generating a plurality of cDNA molecules within the cell sample, comprising a first target cDNA molecule corresponding to at least the first target RNA molecule, and the plurality of cDNA molecules comprising a second target cDNA molecule corresponding to the second target RNA molecule. The cDNA molecules in the plurality of cDNA molecules can be generated by reverse transcription of a messenger RNA (mRNA) molecule within the biological sample, wherein the cDNA molecule or the mRNA molecule comprises a target nucleic acid sequence or a reverse complement of a target nucleic acid sequence. The plurality of cDNA molecules can be generated by reverse transcription of one or more messenger RNA (mRNA) molecules within the biological sample, wherein the plurality of cDNA molecules or the one or more mRNA molecules comprise a target nucleic acid sequence or a reverse complement of a target nucleic acid sequence. The first target cDNA molecule or the second target cDNA molecule can be generated by reverse transcription of one or more messenger RNA (mRNA) molecules within the biological sample, wherein the plurality of cDNA molecules or the one or more mRNA molecules comprise a target nucleic acid sequence or a reverse complement of a target nucleic acid sequence. In some embodiments, the method includes generating at least 2-10,000 different target cDNA molecules corresponding to 2-10,000 different target RNA molecules. In some embodiments, the generating in step (b) includes contacting the plurality of RNAs in the cell sample with: (i) a plurality of reverse transcription primers, (ii) a plurality of reverse transcriptases, and (iii) a plurality of nucleotides under conditions suitable for performing a reverse transcription reaction to generate a plurality of cDNA molecules (e.g., a plurality of first-strand cDNA molecules) in the cell sample (e.g., Figure 1 In some embodiments, the plurality of reverse transcription primers includes a first subset of target-specific reverse transcription primers that selectively hybridize to a first target RNA, and includes a second subset of target-specific reverse transcription primers that selectively hybridize to a second target RNA. In some embodiments, the plurality of reverse transcription primers includes a first subset of random sequence reverse transcription primers that hybridize to a first target RNA, and includes a second subset of random sequence reverse transcription primers that hybridize to a second target RNA.

[0129] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample may further include step (b): generating multiple cDNA molecules in the cell sample, which may include at least a first target cDNA molecule that may correspond to a first target RNA molecule. Multiple cDNA molecules may include a second target cDNA molecule that may correspond to a second target RNA molecule. In some embodiments, multiple cDNA molecules may include at least a first target cDNA molecule. In some embodiments, the first target cDNA molecule may correspond to the first target RNA molecule. In some embodiments, multiple cDNA molecules may include at least a second target cDNA molecule. In some embodiments, the second target cDNA molecule may correspond to the second target RNA molecule. In some embodiments, the method may include generating at least 2-10,000 different target cDNA molecules, which may correspond to 2-10,000 different target RNA molecules. In some embodiments, the generation in step (b) may include contacting the multiple RNAs in the cell sample with: (i) multiple reverse transcription primers, (ii) multiple reverse transcriptases, and (iii) multiple nucleotides. In some embodiments, the generating in step (b) can be performed under conditions suitable for performing a reverse transcription reaction to generate a plurality of cDNA molecules (e.g., a plurality of first-strand cDNA molecules) in the cell sample (e.g., Figure 1). In some embodiments, the generation in step (b) may include contacting a plurality of RNAs in the cell sample with a plurality of reverse transcription primers. In some embodiments, the generation in step (b) may include contacting a plurality of RNAs in the cell sample with a plurality of reverse transcriptases. In some embodiments, the generation in step (b) may include contacting a plurality of RNAs in the cell sample with a plurality of nucleotides. In some embodiments, a plurality of reverse transcription primers may include a first subgroup of target-specific reverse transcription primers that can selectively hybridize with a first target RNA. In some embodiments, a plurality of reverse transcription primers may include a second subgroup of target-specific reverse transcription primers that can selectively hybridize with a second target RNA. In some embodiments, a plurality of reverse transcription primers may include a first subgroup of target-specific reverse transcription primers. In some embodiments, the first subgroup of target-specific reverse transcription primers can selectively hybridize with a first target RNA. In some embodiments, a plurality of reverse transcription primers may include a second subgroup of target-specific reverse transcription primers. In some embodiments, the second subgroup of target-specific reverse transcription primers can selectively hybridize with a second target RNA. In some embodiments, a plurality of reverse transcription primers may include a first subgroup of random sequence reverse transcription primers that can hybridize with a first target RNA, and may include a second subgroup of random sequence reverse transcription primers that can hybridize with a second target RNA. In some embodiments, a plurality of reverse transcription primers may include a first subgroup of random sequence reverse transcription primers. In some embodiments, the first subgroup of random sequence reverse transcription primers can hybridize with a first target RNA. In some embodiments, a plurality of reverse transcription primers may include a second subgroup of random sequence reverse transcription primers. In some embodiments, the second subgroup of random sequence reverse transcription primers can hybridize with a second target RNA.

[0130] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample further comprises step (c): contacting a plurality of cDNA molecules in the cell sample with a plurality of target-specific padlock probes, the plurality of target-specific padlock probes comprising at least a first plurality of target-specific padlock probes and a second plurality of target-specific padlock probes. In some embodiments, the plurality of target-specific padlock probes may comprise at least a first plurality of target-specific padlock probes. In some embodiments, the plurality of target-specific padlock probes may comprise at least a second plurality of target-specific padlock probes. In some embodiments, the method comprises contacting a plurality of cDNA molecules in the cell sample with at least 2-10,000 different target-specific padlock probes.

[0131] In some embodiments, a single padlock probe in the plurality of first target-specific padlock probes comprises a first and a second end (e.g., a first and a second padlock binding arm), wherein the first end selectively hybridizes to a first region of the first target cDNA molecule and the second end selectively hybridizes to a second region of the first target cDNA molecule. In some embodiments, the contacting in step (c) comprises: hybridizing the first and second ends of the first target-specific padlock probe to a proximal position on the first target cDNA molecule to form a circularized first target-specific padlock probe (e.g., a gap or notch) having a gap between the hybridized first and second ends. Figure 1 ). In some embodiments, the first target-specific padlock probe comprises a first target barcode sequence corresponding to a first target cDNA sequence. In some embodiments, the first target-specific padlock probe comprises a first target barcode sequence located adjacent to a region in the first target-specific padlock probe that selectively hybridizes to the first target cDNA molecule. In some embodiments, the first target-specific padlock probe comprises at least one universal adapter sequence, such as a universal sequencing primer binding site (or its complement). In some embodiments, the first target-specific padlock probe comprises a universal primer binding site (or its complement) for a rolling circle amplification primer. In some embodiments, the first target-specific padlock probe comprises a universal compaction oligonucleotide binding site (or its complement).

[0132] In some embodiments, the method includes: contacting a first target nucleic acid sequence or its reverse complement in situ with a first oligonucleotide comprising a first end and a second end. The first end and the second end of the first oligonucleotide may be complementary. The first end and the second end of the first oligonucleotide may bind to two adjacent segments of the first target nucleic acid sequence or its reverse complement, such that the first oligonucleotide forms a ring structure with a gap between the first end and the second end. In some embodiments, the method further includes: contacting a second target nucleic acid sequence or its reverse complement in situ with a second oligonucleotide comprising a first end and a second end. In some embodiments, the first end and the second end of the second oligonucleotide are complementary. In some embodiments, the first end and the second end bind to two adjacent segments of the second target nucleic acid sequence or its reverse complement, such that the second oligonucleotide forms a ring structure with a gap between the first end and the second end. In some embodiments, the first target nucleic acid sequence comprises a first cDNA molecule or a first mRNA molecule. In some embodiments, the second target nucleic acid sequence comprises a second cDNA molecule or a second mRNA molecule. In some embodiments, the gap in the first oligonucleotide or the second oligonucleotide is one nucleotide in size. In some embodiments, the gap in the first oligonucleotide and the second oligonucleotide is one nucleotide in size. In some embodiments, the gap of the first oligonucleotide has a size of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more nucleotides. In some embodiments, the gap of the second oligonucleotide has a size of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more nucleotides. In some embodiments, the gap of the first oligonucleotide or the second oligonucleotide has a size of at least two nucleotides. In some embodiments, the gap of the first oligonucleotide and the second oligonucleotide has a size of at least two nucleotides. In some embodiments, the first oligonucleotide further includes a first identification sequence for identifying the first target nucleic acid sequence. In some embodiments, the second oligonucleotide further includes a second identification sequence for identifying the second target nucleic acid sequence. In some embodiments, the first oligonucleotide further includes a nucleic acid sequence that is a complementary sequence to the nucleic acid sequence of a sequencing primer. In some embodiments, the second oligonucleotide further includes a nucleic acid sequence that is a complementary sequence to the nucleic acid sequence of a sequencing primer. In some embodiments, the first oligonucleotide further includes a nucleic acid sequence that is a complementary sequence to the nucleic acid sequence of a primer for nucleic acid amplification. In some embodiments, the second oligonucleotide further includes a nucleic acid sequence that is a complementary sequence to the nucleic acid sequence of a primer for nucleic acid amplification. In some embodiments, the primer for nucleic acid amplification is a primer for rolling circle amplification (RCA). In some embodiments, the primer for RCA produces concatemers.In some embodiments, the concatemer includes at least two repeats of the target nucleic acid sequence in at least two target nucleic acid sequences or a portion thereof or its reverse complement. In some embodiments, the first oligonucleotide further includes the reverse complement of the compaction oligonucleotide. In some embodiments, the second oligonucleotide further includes the reverse complement of the compaction oligonucleotide. In some embodiments, the first section of the compaction oligonucleotide is complementary to and binds to the first portion of the concatemer. In some embodiments, the second section of the compaction oligonucleotide is complementary to and binds to the second portion of the concatemer. In some embodiments, the combination with the first and / or second portion of the concatemer causes the size of the concatemer to decrease or the shape to change. In some embodiments, the method further includes in situ joining the first end and the second end of the first oligonucleotide to produce a first circular oligonucleotide in the biological sample. In some embodiments, the method further includes in situ joining the first end and the second end of the second oligonucleotide to produce a second circular oligonucleotide in the biological sample. In some embodiments, joining the first end and the second end of the first oligonucleotide includes joining the first end and the second end of the first oligonucleotide by a first nucleic acid enzyme. In some embodiments, joining the first end and the second end of the second oligonucleotide includes joining the first end and the second end of the second oligonucleotide by a second nucleic acid enzyme. In some embodiments, the first nucleic acid enzyme and the second nucleic acid enzyme are enzymes of the same type. In some embodiments, the first end and the second end of the second oligonucleotide are joined together by the second nucleic acid enzyme. The first nucleic acid enzyme and the second nucleic acid enzyme are enzymes of different types. In some embodiments, the first nucleic acid enzyme includes nucleic acid ligase, nucleic acid ligase, nucleic acid polymerase, nucleic acid polymerase or a combination thereof. In some embodiments, the second nucleic acid enzyme includes nucleic acid ligase, nucleic acid ligase, nucleic acid polymerase, nucleic acid polymerase or a combination thereof. In some embodiments, the method further includes amplifying the first circular oligonucleotide in situ to produce the first nucleic acid molecule. In some embodiments, the method further includes amplifying the second circular oligonucleotide in situ to produce the second nucleic acid molecule. In some embodiments, amplification includes rolling circle amplification (RCA), wherein the first nucleic acid molecule includes the first concatemer, and the second nucleic acid molecule includes the second concatemer. In some embodiments, the first concatemer includes at least two repetitions of the first unit nucleic acid sequence, and the first unit nucleic acid sequence includes the first target nucleic acid sequence or a portion thereof, or the reverse complementary sequence of the first target nucleic acid sequence or a portion thereof. In some embodiments, the second concatemer includes at least two repeats of a second unit nucleic acid sequence that includes the second target nucleic acid sequence or a portion thereof, or the reverse complement of the second target nucleic acid sequence or a portion thereof.In some embodiments, the first concatemer further includes a first identification sequence or a sequencing primer or a reverse complement sequence thereof for identifying the first target nucleic acid sequence, wherein the second concatemer further includes a second identification sequence or a sequencing primer or a reverse complement sequence thereof for identifying the second target nucleic acid sequence. In some embodiments, the first concatemer further includes a first compaction oligonucleotide. In some embodiments, the first section of the first compaction oligonucleotide is complementary to and binds to the first portion of the first concatemer. In some embodiments, the second section of the first compaction oligonucleotide is complementary to and binds to the second portion of the first concatemer, so as to cause the first concatemer to be reduced in size or changed in shape. In some embodiments, the second concatemer further includes a second compaction oligonucleotide. In some embodiments, the first section of the second compaction oligonucleotide is complementary to and binds to the first portion of the second concatemer. In some embodiments, the second section of the second compaction oligonucleotide is complementary to and binds to the second portion of the second concatemer, so as to cause the second concatemer to be reduced in size or changed in shape.

[0133] In some embodiments, a single padlock probe in the plurality of second target-specific padlock probes comprises a first and a second end, wherein the first end selectively hybridizes to a first region of the second target cDNA molecule and the second end selectively hybridizes to a second region of the second target cDNA molecule. In some embodiments, the contacting in step (c) comprises hybridizing the first and second ends of the second target-specific padlock probe to a proximal position on the second target cDNA molecule to form a circularized second target-specific padlock probe having a gap or notch between the hybridized first and second ends. In some embodiments, the second target-specific padlock probe comprises a second target barcode sequence corresponding to the second target cDNA sequence. In some embodiments, the second target-specific padlock probe comprises a second target barcode sequence located adjacent to a region in the second target-specific padlock probe that selectively hybridizes to the second target cDNA molecule. In some embodiments, the second target-specific padlock probe comprises at least one universal adapter sequence, for example, comprising at least one universal sequencing primer binding site (or its complement). In some embodiments, the second target-specific padlock probe comprises a universal primer binding site (or its complement) for a rolling circle amplification primer. In some embodiments, the second target-specific padlock probe comprises a universal compacted oligonucleotide binding site (or its complement).

[0134] In some embodiments, the target-specific padlock probe includes a universal sequencing primer binding site and a target barcode sequence adjacent to each other, so that the target barcode region of the concatemer is sequenced first. The target barcode sequence can be of any length, for example, 3-15 bases, or 15-25 bases, or 25-40 bases, or longer.

[0135] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample may further include step (d): closing the gap or gap in at least the first and second circularized target-specific padlock probes by performing an enzymatic reaction, thereby generating at least a first covalently closed circular padlock probe and a second covalently closed circular padlock probe within the cell sample. In some embodiments, step (d) may include closing the gap or gap in at least the first circularized target-specific padlock probe by performing an enzymatic reaction, thereby generating at least a first covalently closed circular padlock probe. In some embodiments, step (d) may include closing the gap or gap in at least the second circularized target-specific padlock probe by performing an enzymatic reaction, thereby generating at least a second covalently closed circular padlock probe. In some embodiments, closing the gap may include performing an enzymatic ligation reaction. In some embodiments, closing the gap may include performing a polymerase-catalyzed fill-in reaction using the first or second target cDNA molecule as a template, and performing an enzymatic ligation reaction. In some embodiments, closing the gap may include performing a polymerase-catalyzed fill-in reaction using the first target cDNA molecule as a template. In some embodiments, closing the gap can include performing a polymerase-catalyzed fill-in reaction using a second target cDNA molecule as a template. In some embodiments, the method can include closing the gap or nick in at least 2-10,000 circularized target-specific padlock probes by performing an enzymatic reaction, thereby generating at least 2-10,000 covalently closed circular padlock probes within the cell sample.

[0136] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample may further include step (e): performing a rolling circle amplification reaction in the cell sample using the first and second covalently closed circular padlock probes as template molecules to generate a plurality of concatemer molecules, which include at least a first concatemer molecule corresponding to the first target RNA molecule, and the plurality of concatemer molecules may include at least a second concatemer molecule corresponding to the second target RNA molecule. In some embodiments, step (e) may include performing a rolling circle amplification reaction in the cell sample using the first covalently closed circular padlock probe as a template molecule. In some embodiments, the rolling circle amplification of the first covalently closed circular padlock probe may generate a plurality of concatemer molecules. In some embodiments, the plurality of concatemer molecules may include the first concatemer molecule. In some embodiments, the first concatemer molecule may correspond to the first target RNA molecule. In some embodiments, step (e) may include performing a rolling circle amplification reaction in the cell sample using the second covalently closed circular padlock probe as a template molecule. In some embodiments, the rolling circle amplification of the second covalently closed circular padlock probe may generate a plurality of concatemer molecules. In some embodiments, the plurality of concatemer molecules may include the second concatemer molecule. In some embodiments, the second concatemer molecule can correspond to the second target RNA molecule. In some embodiments, the first concatemer molecule can include a tandem repeat unit, wherein the unit can include the sequence of the first target cDNA and a universal sequencing primer binding site (or its complementary sequence). In some embodiments, the second concatemer molecule can include a tandem repeat unit, wherein the unit can include the sequence of the second target cDNA and a universal sequencing primer binding site (or its complementary sequence). In some embodiments, the method can include using at least 2-10,000 covalently closed circular padlock probes as template molecules to carry out rolling circle amplification reactions in a cell sample, thereby generating at least 2-10,000 concatemer molecules corresponding to at least 2-10,000 target RNA molecules. In some embodiments, rolling circle amplification can be carried out in the presence of multiple compaction oligonucleotides. In some embodiments, each compaction oligonucleotide can include a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide can have a first region at one end and a second region at the other end. In some embodiments, the first region can hybridize with a portion of the concatemer molecule, and the second region can hybridize with another portion of the concatemer molecule. In some embodiments, the hybridization of the first region and the second region of the compacting oligonucleotide can compress the concatemer molecule. In some embodiments, the compacting oligonucleotide can compress the size of the concatemer molecule. In some embodiments, the compacting oligonucleotide can compress the shape of the concatemer molecule. In some embodiments, the compacting oligonucleotide can compress the size and shape of the concatemer molecule. In some embodiments, the compacting oligonucleotide can compress the concatemer molecule to form a compact nanosphere.

[0137] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample may further include step (f): sequencing a plurality of concatemer molecules within the cell sample, which may include sequencing a first concatemer molecule by performing no more than 2-30 sequencing cycles to generate a plurality of first sequencing read products, and sequencing a second concatemer molecule by performing no more than 2-30 sequencing cycles to generate a plurality of second sequencing read products. In some embodiments, step (f) may include sequencing a plurality of concatemer molecules within the cell sample. In some embodiments, sequencing a plurality of concatemer molecules within the cell sample may include sequencing a first concatemer molecule by performing no more than 2-30 sequencing cycles to generate a plurality of first sequencing read products. In some embodiments, step (f) may include sequencing a plurality of concatemer molecules within the cell sample. In some embodiments, sequencing a plurality of concatemer molecules within the cell sample may include sequencing a second concatemer molecule by performing no more than 2-30 sequencing cycles to generate a plurality of second sequencing read products. In some embodiments, the sequencing in step (f) may include base sequencing of no more than 2-30 first concatemer molecules to generate a plurality of first sequencing read products, and it may include base sequencing of no more than 2-30 second concatemer molecules to generate a plurality of second sequencing read products. In some embodiments, the sequencing in step (f) may include base sequencing of no more than 2-30 first concatemer molecules to generate a plurality of first sequencing read products. In some embodiments, the sequencing in step (f) may include base sequencing of no more than 2-30 second concatemer molecules to generate a plurality of second sequencing read products. In some embodiments, the method may include sequencing at least 2-10,000 concatemer molecules within a cell sample, which may include performing no more than 2-30 sequencing cycles on the 2-10,000 concatemer molecules to generate a plurality of sequencing read products. In some embodiments, only the first target barcode region of the first concatemer molecule is sequenced (e.g., Figure 2 In some embodiments, at least a portion or the entire length of the first target barcode in the first concatemer molecule is sequenced (e.g., Figure 2 In some embodiments, the first target barcode is sequenced and a portion of the first cDNA region of the first concatemer molecule is sequenced (e.g., Figure 3 In some embodiments, at least a portion of the first cDNA region in the first concatemer molecule is sequenced (e.g., Figure 4 or Figure 5 In some embodiments, only the second target barcode region of the second concatemer molecule is sequenced (e.g., Figure 2In some embodiments, at least a portion or the entire length of the second target barcode in the second concatemer molecule is sequenced (e.g., Figure 2 In some embodiments, the second target barcode is sequenced and a portion of the second cDNA region of the second concatemer molecule is sequenced (e.g., Figure 3 In some embodiments, at least a portion of the second cDNA region of the second concatemer molecule is sequenced (e.g., Figure 4 or Figure 5 ).

[0138] In some embodiments, the sequencing in step (f) comprises using a sequence comprising greater than 1.0 mm 2 The optical imaging system sequences at least a portion of the first and second nucleic acid concatemers using a field of view (FOV).

[0139] In some embodiments, in the sequencing in step (f), a plurality of first and second sequencing reads can be detectable by imaging, and wherein sequencing can include decoding a plurality of first and second sequencing reads from an image obtained during no more than 2-30 sequencing cycles. In some embodiments, in the sequencing in step (f), a plurality of first sequencing reads can be detectable by imaging. In some embodiments, in the sequencing in step (f), a plurality of first sequencing reads can be detectable by imaging, and wherein sequencing can include decoding a plurality of first sequencing reads from an image obtained during no more than 2-30 sequencing cycles. In some embodiments, in the sequencing in step (f), a plurality of second sequencing reads can be detectable by imaging. In some embodiments, in the sequencing in step (f), a plurality of second sequencing reads can be detectable by imaging, and wherein sequencing can include decoding a plurality of second sequencing reads from an image obtained during no more than 2-30 sequencing cycles.

[0140] In some embodiments, in the sequencing in step (f), a plurality of first and second sequencing reads can be detectable by imaging, and wherein sequencing can include decoding a plurality of first and second sequencing reads from an image obtained during no more than 2-30 sequencing cycles. In some embodiments, in the sequencing in step (f), a plurality of first sequencing reads can be detectable by imaging. In some embodiments, in the sequencing in step (f), a plurality of first sequencing reads can be detectable by imaging, and wherein sequencing can include decoding a plurality of first sequencing reads from an image obtained during no more than 2-30 sequencing cycles. In some embodiments, in the sequencing in step (f), a plurality of second sequencing reads can be detectable by imaging. In some embodiments, in the sequencing in step (f), a plurality of second sequencing reads can be detectable by imaging, and wherein sequencing can include decoding a plurality of second sequencing reads from an image obtained during no more than 2-30 sequencing cycles.

[0141] In some embodiments, the sequence of the first sequencing read product can be aligned with the first target reference sequence to confirm the presence of the first target RNA in the cell product. In some embodiments, the sequence of the second sequencing read product can be aligned with the second target reference sequence to confirm the presence of the second target RNA in the cell sample.

[0142] In some embodiments, the sequencing in step (f) may include (1) contacting the plurality of concatemer molecules within the cell sample with (i) a plurality of universal sequencing primers, (ii) a plurality of sequencing polymerases, and (iii) a plurality of nucleotide reagents under conditions suitable for hybridizing the plurality of universal sequencing primers to their respective universal sequencing primer binding sites on the concatemer. In some embodiments, the sequencing in step (f) may include (1) contacting the plurality of concatemer molecules within the cell sample with a plurality of universal sequencing primers. In some embodiments, the sequencing in step (f) may include (1) contacting the plurality of concatemer molecules within the cell sample with a plurality of sequencing polymerases. In some embodiments, the sequencing in step (f) may include (1) contacting the plurality of concatemer molecules within the cell sample with a plurality of nucleotide reagents. In some embodiments, the sequencing may further include (2) performing no more than 2-30 sequencing cycles to generate at least a first plurality of sequencing read products. In some embodiments, the sequencing may further include (2) performing no more than 2-30 sequencing cycles to generate at least a first plurality of sequencing read products and a second plurality of sequencing read products. In some embodiments, sequencing may further include (3) removing a first plurality of sequencing read products from the concatemer and retaining the plurality of concatemers within the cell sample. In some embodiments, sequencing may further include (3) removing a first plurality of sequencing read products from the first concatemer molecule. In some embodiments, sequencing in (3) may further include retaining the first concatemer molecule within the cell sample. In some embodiments, sequencing may further include (3) removing a first plurality of sequencing read products from the concatemer and retaining the first concatemer molecule within the cell sample, and removing a second plurality of sequencing read products from the second concatemer molecule and retaining the second concatemer molecule within the cell sample. In some embodiments, sequencing may further include (3) removing a second plurality of sequencing read products from the second concatemer molecule. In some embodiments, sequencing in (3) may further include retaining the second concatemer molecule within the cell sample. In some embodiments, sequencing may further include (4) repeating (1)–(3) at least once. In some embodiments, (4) can include repeating (1)-(3) at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, (4) can include repeating (1)-(3) at most 10 times, at most 20 times, at most 30 times, at most 40 times, or at most 50 times.

[0143] In some embodiments, iterative sequencing can be performed using a combination of sequencing procedures, labeled and / or unlabeled chain-terminating nucleotides, or multivalent molecules. Descriptions of these three sequencing methods are described below.

[0144] In some embodiments, multiple universal sequencing primers can be hybridized to concatemer template molecules using a hybridization reagent comprising SSC buffer (e.g., 2X saline-sodium citrate) buffer and formamide (e.g., 10%-20% formamide). Hybridization conditions include a temperature of about 20-30° C. for about 10-60 minutes.

[0145] In some embodiments, multiple sequencing read products can be removed from concatemers using a dehybridization reagent comprising an SSC buffer (e.g., saline-sodium citrate) buffer with or without formamide at a temperature that promotes nucleic acid denaturation, e.g., 30-90°C, and the multiple concatemers can remain within the cell sample.

[0146] In some embodiments, the multiple nucleotide reagents in step (f) include detectable labels or unlabeled multiple nucleotides. In some embodiments, a single nucleotide is coupled to a detectable reporting portion. In some embodiments, the detectable reporting portion includes a fluorophore. In some embodiments, the nucleotide analogs of a plurality of detectable labels include a plurality of chain-terminating nucleotides, wherein the chain-terminating portion is coupled to a 3' nucleotide sugar position to form a nucleotide analog of 3' blocking. In some embodiments, the chain-terminating portion can be removed to convert the nucleotide analog of 3' blocking into an extendable nucleotide with a 3' OH group on the sugar. In some embodiments, labeled nucleotide analogs are coupled to different fluorophores corresponding to core bases adenine, cytosine, guanine, thymine or uracil, wherein different fluorophores emit fluorescent signals during the sequencing in step (f). In some embodiments, the sequencing cycles include (1) contacting the concatemer / sequencing primer duplex with a sequencing polymerase and a detectably labeled chain-terminating nucleotide under conditions suitable for polymerase-catalyzed incorporation of the detectably labeled chain-terminating nucleotide into the end of the sequencing primer, (2) detecting and imaging the fluorescent signal and color emitted by the incorporated chain-terminating nucleotide, and (3) removing the chain-terminating portion (e.g., unblocking) and retaining the concatemer / sequencing primer duplex. In some embodiments, no more than 2-30 sequencing cycles can be performed on multiple concatemers within a cell sample to generate multiple first sequencing reads and multiple second sequencing reads. In some embodiments, no more than 2-30 sequencing cycles can be performed on multiple concatemers within a cell sample to generate multiple first sequencing reads. In some embodiments, no more than 2-30 sequencing cycles can be performed on multiple concatemers within a cell sample to generate multiple second sequencing reads. In some embodiments, the sequence of the first sequencing read product can be determined and compared to a first reference sequence to confirm the presence of the first target RNA molecule within the cell sample. In some embodiments, the sequence of the second sequencing read product can be determined and compared to the second reference sequence to confirm the presence of the first target polypeptide in the cell sample. In some embodiments, after each round of generating the first and second sequencing read products, the sequences of the first and second sequencing read products can be compared. In some embodiments, the length of the first and second sequencing read products can be no more than 30 bases. In some embodiments, after generating a set of iterative sequencing read products, the sequences of the first and second sequencing read products can be compared. In some embodiments, wherein the length of the first and second sequencing read products can be no more than 30 bases. In some embodiments, after each round of generating the first sequencing read product, the sequence of the first sequencing read product can be compared. In some embodiments, the length of the first sequencing read product can be no more than 30 bases. In some embodiments, after generating a set of iterative sequencing read products, the sequence of the first sequencing read product can be compared.In some embodiments, the length of the first sequencing read product may be no more than 30 bases. In some embodiments, after each round of generating the second sequencing read product, the sequence of the second sequencing read product can be compared. In some embodiments, the length of the second sequencing read product may be no more than 30 bases. In some embodiments, after generating repeated sequencing read product sets, the sequence of the second sequencing read product can be compared. In some embodiments, the length of the second sequencing read product may be no more than 30 bases. In some embodiments, sequencing reaction can be carried out on a sequencing device with a detector for capturing fluorescent signals from the sequencing reaction in a cell sample. Sequencing device can be configured to relay the fluorescent signal data captured by the detector to a computer system, which is programmed to display the image of different fluorescent spots co-located in a cell sample, wherein a single fluorescent spot corresponds to different target RNA molecules or different target polypeptides. In some embodiments, when sequencing is carried out using different fluorescent-labeled nucleotide reagents corresponding to different core bases (e.g., A, G, C, T / U), the image can have different color fluorescent spots co-located in the same cell sample in different sequencing cycles.

[0147] In some embodiments, during the synchronous sequencing reaction on the clonal amplification template amplicon, asynchronous phasing and / or pre-phasing events can occur, wherein the sequencing reaction includes a polymerase-catalyzed sequencing reaction using a detectably labeled chain terminator nucleotide. In some embodiments, the sequencing reaction on one template molecule in the clonal amplification template molecule is ahead of (e.g., pre-phasing) or behind (e.g., phasing) the sequencing of other template molecules in the clonal amplification template molecule. During sequencing, a fluorescent signal is typically detected corresponding to the incorporation of labeled chain terminator nucleotides. Therefore, phasing and pre-phasing events can be detected and monitored using the incorporation of labeled chain terminator nucleotides.

[0148] In some embodiments, the multiple nucleotide reagents in step (f) include multiple multivalent molecules, and each multivalent molecule includes the core attached to multiple nucleotide arms, and wherein the nucleotide arms are attached to nucleotide units. In some embodiments, single multivalent molecule can detect reporter moiety labeling. In some embodiments, detectable reporter moiety includes fluorophore. In some embodiments, the core fluorophore labeling of multivalent molecule, and the fluorophore wherein attached to the given core of multivalent molecule corresponds to the nucleotide base (for example, adenine, guanine, cytosine, thymine or uracil) of nucleotide arms. In some embodiments, at least one nucleotide arm of multivalent molecule includes joints and / or nucleotide bases attached to fluorophore, and the fluorophore wherein attached to given nucleotide base corresponds to the nucleotide base (for example, adenine, guanine, cytosine, thymine or uracil) of nucleotide arms. In some embodiments, the sequencing cycle includes (1) contacting the concatemer / sequencing primer duplex with a first sequencing polymerase to form a composite polymerase, (2) contacting the composite polymerase with a detectably labeled multivalent molecule under conditions suitable for binding complementary nucleotide units of the multivalent molecule to the composite polymerase, thereby forming a multivalent binding complex, and the conditions are suitable for inhibiting incorporation of the complementary nucleotide units into the termini of the sequencing primer, (3) detecting and imaging the fluorescent signal and color emitted by the bound detectably labeled multivalent molecule, (4) removing the first sequencing polymerase and the bound detectably labeled multivalent molecule and retaining the concatemer / sequencing primer duplex, (5) contacting the retained concatemer / sequencing primer duplex with a second sequencing polymerase and an unlabeled chain-terminating nucleotide under conditions suitable for polymerase-catalyzed incorporation of an unlabeled chain-terminating nucleotide into the termini of the sequencing primer, and (6) removing the chain-terminating moiety (e.g., unblocking) and retaining the concatemer / sequencing primer duplex. In some embodiments, the sequencing cycle may include contacting the concatemer / sequencing primer duplex with a first sequencing polymerase to form a composite polymerase. In some embodiments, the sequencing cycle may include contacting the composite polymerase with a detectably labeled multivalent molecule under conditions suitable for binding the complementary nucleotide units of the multivalent molecule to the composite polymerase, thereby forming a multivalent binding complex, and the conditions are suitable for inhibiting the incorporation of the complementary nucleotide units into the ends of the sequencing primer. In some embodiments, the sequencing cycle may include detecting and imaging the fluorescent signal and color emitted by the bound detectably labeled multivalent molecule. In some embodiments, the sequencing cycle may include removing the first sequencing polymerase and the bound detectably labeled multivalent molecule and retaining the concatemer / sequencing primer duplex. In some embodiments, the sequencing cycle may include contacting the retained concatemer / sequencing primer duplex with a second sequencing polymerase and an unlabeled chain-terminating nucleotide under conditions suitable for polymerase-catalyzed incorporation of unlabeled chain-terminating nucleotides into the ends of the sequencing primer.In some embodiments, the sequencing cycle can include removing the chain termination portion (e.g., unblocking) and retaining the concatemer / sequencing primer duplex. In some embodiments, a single cycle time can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) can exceed 1 mm. 2 And used to scan large areas (>10mm 2) can have a cycle time of less than 5 minutes. In some embodiments, no more than 2-30 sequencing cycles can be performed on multiple concatemers in a cell sample to generate multiple first sequencing read products and multiple second sequencing read products. In some embodiments, no more than 2-30 sequencing cycles can be performed on multiple concatemers in a cell sample to generate multiple first sequencing read products. In some embodiments, no more than 2-30 sequencing cycles can be performed on multiple concatemers in a cell sample to generate multiple second sequencing read products. In some embodiments, the sequence of the first sequencing read product can be determined and compared with the first reference sequence to confirm that the first target RNA molecule exists in the cell sample. In some embodiments, the sequence of the second sequencing read product can be determined and compared with the second reference sequence to confirm that the second target RNA molecule exists in the cell sample. In some embodiments, after each round of generating the first and second sequencing read products having a length of no more than 30 bases or after generating a set of repeated sequencing read products (wherein the length of the first and second sequencing read products is no more than 30 bases), the sequences of the first and second sequencing read products can be compared. In some embodiments, after each round of generating the first and second sequencing read products, the sequences of the first and second sequencing read products can be aligned. In some embodiments, the length of the first and second sequencing read products can be no more than 30 bases. In some embodiments, after generating a set of iterative sequencing read products, the sequences of the first and second sequencing read products can be aligned. In some embodiments, wherein the length of the first and second sequencing read products can be no more than 30 bases. In some embodiments, after each round of generating the first sequencing read product, the sequence of the first sequencing read product can be aligned. In some embodiments, the length of the first sequencing read product can be no more than 30 bases. In some embodiments, after generating a set of iterative sequencing read products, the sequence of the first sequencing read product can be aligned. In some embodiments, wherein the length of the first sequencing read product can be no more than 30 bases. In some embodiments, after each round of generating the second sequencing read product, the sequence of the second sequencing read product can be aligned. In some embodiments, the length of the second sequencing read product can be no more than 30 bases. In some embodiments, after generating a set of iterative sequencing read products, the sequence of the second sequencing read product can be aligned. In some embodiments, the second sequencing read product may be no longer than 30 bases.In some embodiments, the sequencing reaction may be performed on a sequencing device having a detector that can capture fluorescent signals from the sequencing reaction within the cell sample.The sequencing device can be configured to relay the fluorescence signal data captured by the detector to a computer system, which can be programmed to display an image of different fluorescent spots that can co-localize in the cell sample, where a single fluorescent spot can correspond to a different target RNA molecule (e.g., a first target RNA molecule, a second target RNA molecule, or a combination of the first RNA molecule and the second RNA molecule).

[0149] In some embodiments, when sequencing with a detectably labeled multivalent molecule, a step (2) in which a multivalent binding complex is formed, and a step (3) in which the detected labeled multivalent molecule is imaged and detected, the conditions are mild compared to a sequencing workflow using a detectably labeled chain-terminating nucleotide. For example, steps (2) and (3) can be performed at a mild temperature of about 35-45°C or about 39-42°C. Steps (2) and (3) can be performed at a mild temperature, which helps to maintain the compact size and shape of the DNA nanoballs during multiple sequencing cycles (e.g., up to 30 cycles), which can improve the FWHM (full width at half maximum) of the DNA nanoball spot image within the cell sample. In some embodiments, the DNA nanoballs do not spread out during multiple sequencing cycles. In some embodiments, the spot image of the DNA nanoballs does not amplify during multiple sequencing cycles. In some embodiments, the spot image of the DNA nanoballs remains as discrete spots during multiple sequencing cycles. The spot image can be represented as a Gaussian spot, and the size can be measured as the FWHM. Smaller spot sizes, as indicated by smaller FWHM, generally correlate with improved spot images. In some embodiments, the FWHM of the nanosphere spots can be about 10 um or less.

[0150] In some embodiments, during the sequencing reaction using the synchronous polymerase catalysis of the multivalent molecule of detectable label, asynchronous phasing and / or pre-phasing events can occur. During sequencing, a fluorescent signal can be detected, which corresponds to the combination of the complementary nucleotide unit of the multivalent molecule and the composite polymerase, thereby forming a multivalent binding complex. Therefore, phasing and pre-phasing events can be detected and monitored using the combination of the labeled multivalent molecule. In some embodiments, when performing up to 30 sequencing cycles with the multivalent molecule of detectable label, phasing and / or pre-phasing rates can be less than about 5%, or less than about 1%, or less than about 0.01% or less than about 0.001%. In contrast, using the labeled chain terminator nucleotides to perform up to 30 sequencing cycles of phasing and / or pre-phasing rates can be about 5%.

[0151] In some embodiments, the sequencing in (f) can include determining the sequence of a first nucleic acid molecule or a portion thereof, wherein the first nucleic acid molecule or a portion thereof is composed of 2-30 nucleotides. In some embodiments, the sequence of (f) can include determining the sequence of a second nucleic acid molecule or a portion thereof, wherein the second nucleic acid molecule or a portion thereof is composed of 2-30 nucleotides. In some embodiments, the 2-30 nucleotides of the first nucleic acid molecule or a portion thereof include a first identification sequence, and the 2-30 nucleotides of the second nucleic acid molecule or a portion thereof include a second identification sequence. In some embodiments, the 2-30 nucleotides of the first nucleic acid molecule or a portion thereof further include at least a portion of the sequence of a first cDNA molecule or a first mRNA molecule, and the 2-30 nucleotides of the second nucleic acid molecule or a portion thereof further include at least a portion of the sequence of a second cDNA molecule or a second mRNA molecule.

[0152] In some embodiments, determining comprises contacting the first concatemer with a polymerase, a plurality of nucleotides, and a primer sequence complementary to a portion of the first concatemer under conditions sufficient to form a binding complex, the binding complex comprising a polymerase, a nucleotide from the plurality of nucleotides that are complementary to and bound to the nucleotide units of the first concatemer, and the first concatemer hybridized to the primer sequence, wherein the nucleotide comprises a fluorescent label and a removable blocking group at the 3' carbon position of the sugar portion. In some embodiments, the method further comprises incorporating the nucleotide into the 3' end of the primer sequence. In some embodiments, the method further comprises identifying the nucleobase of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide.

[0153] In some embodiments, determining includes contacting the second concatemer with a polymerase, a plurality of nucleotides, and a primer sequence that is complementary to a portion of the second concatemer under conditions sufficient to form a binding complex, the binding complex comprising a polymerase, a nucleotide from a plurality of nucleotides that are complementary to and bound to the nucleotide units of the second concatemer, and a second concatemer hybridized to the primer sequence, wherein the nucleotide comprises a fluorescent label and a removable blocking group at the 3' carbon position of the sugar portion. In some embodiments, the method further comprises incorporating the nucleotide into the 3' end of the primer sequence. In some embodiments, the method further comprises identifying the nucleobase of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide. In some embodiments, determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar portion. In some embodiments, the blocking group includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, a silyl group, or any combination thereof. In some embodiments, a plurality of nucleotides are composed of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group comprising dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the fluorescent label emitted light of the nucleotides of another type in the group. In some embodiments, a plurality of nucleotides include at least two types of nucleotides, wherein at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the fluorescent label emitted light of the nucleotides of another type in the group.

[0154] In some embodiments, determining comprises: contacting the two first concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to portions of the first concatemers under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two first concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide moieties, wherein each of the at least two nucleotide moieties is complementary to and binds to a nucleotide in each of the two first concatemers. In some embodiments, determining comprises detecting the multivalent binding complex by the label of the nucleotide conjugate; and identifying the nucleobases of the nucleotides in the two first concatemers, each of which is complementary to and binds to each of the two of the at least two nucleotide moieties of the nucleotide conjugate. In some embodiments, the conditions inhibit incorporation of at least two nucleotide moieties of the nucleotide conjugate into the two first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide moiety is attached to one of the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, detecting comprises imaging the fluorescent label. In some embodiments, determining further includes: removing the two polymerases and the nucleotide conjugate from the two first concatemers; contacting each of the two first concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, the binding complex comprising each of the two second polymerases, each of the two first concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides; and incorporating each of two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two first concatemers, wherein an unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

[0155] In some embodiments, determining comprises: contacting the two second concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to portions of the second concatemers under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate in the plurality of nucleotide conjugates, and each of the two second concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide moieties, wherein each of the at least two nucleotide moieties is complementary to and binds to a nucleotide in each of the two second concatemers; detecting the multivalent binding complex by the label of the nucleotide conjugate; and identifying the nucleobases of the nucleotides in the two second concatemers, each of which is complementary to and binds to each of the two at least two nucleotide moieties of the nucleotide conjugate. In some embodiments, the conditions inhibit the incorporation of at least two nucleotide moieties of the nucleotide conjugate into the two first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide moiety is attached to one nucleotide arm of the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, detecting comprises imaging the fluorescent label. In some embodiments, determining further includes: removing the two polymerases and the nucleotide conjugate from the two first concatemers; contacting each of the two first concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, the binding complex comprising each of the two second polymerases, each of the two first concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides; and incorporating each of two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two first concatemers, wherein an unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

[0156] In some embodiments, determining further comprises: removing two polymerases and nucleotide conjugates from the two second concatemers; contacting each of the two second concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions sufficient to form a binding complex, the binding complex comprising each of the two second polymerases, each of the two second concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides, and incorporating each of the two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to the nucleotide of each of the two second concatemers, wherein the unlabeled nucleotides in the plurality of unlabeled nucleotides are included in a removable blocking group at the 3' carbon of the sugar portion. In some embodiments, the conditions inhibit the incorporation of at least two nucleotide moieties of the nucleotide conjugate into the two first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide moiety is attached to one nucleotide arm in the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, detection comprises imaging the fluorescent label. In some embodiments, determining further includes: removing the two polymerases and the nucleotide conjugate from the two first concatemers; contacting each of the two first concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, the binding complex comprising each of the two second polymerases, each of the two first concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides; and incorporating each of two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two first concatemers, wherein an unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

[0157] In some embodiments, determining comprises: contacting two first concatemers with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to the first portion of the first concatemer, and a second primer sequence complementary to the second portion of the first concatemer under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate in the plurality of nucleotide conjugates, a first portion of the first concatemer hybridized to the first primer sequence, and a second portion of the first concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein each of the at least two nucleotide portions is complementary to and binds to the nucleotides of the first and second portions of the first concatemer; detecting the multivalent binding complex by the label of the nucleotide conjugate; and identifying the nucleobases of the nucleotides in the first and second portions of the first concatemer, each of which is complementary to and binds to each of the two of the at least two nucleotide portions of the nucleotide conjugate. In some embodiments, the conditions inhibit the incorporation of at least two nucleotide portions of the nucleotide conjugate into the two first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide portion is attached to one of the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, detecting comprises imaging the fluorescent label. In some embodiments, determining further comprises: removing the two polymerases and the nucleotide conjugate from the two first concatemers; contacting each of the two first concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, the binding complex comprising each of the two second polymerases, each of the two first concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides; and incorporating each of the two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two first concatemers, wherein the unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

[0158] In some embodiments, determining comprises: contacting the two second concatemers with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to the first portion of the second concatemer, and a second primer sequence complementary to the second portion of the second concatemer under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate in the plurality of nucleotide conjugates, a first portion of the second concatemer hybridized to the first primer sequence, and a second portion of the second concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein each of the at least two nucleotide portions is complementary to and binds to the nucleotides of the first and second portions of the second concatemer; detecting the multivalent binding complex by the label of the nucleotide conjugate; and identifying the nucleobases of the nucleotides in the first and second portions of the second concatemer, each of which is complementary to and binds to each of the two of the at least two nucleotide portions of the nucleotide conjugate. In some embodiments, the conditions inhibit the incorporation of at least two nucleotide portions of the nucleotide conjugate into the two first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide portion is attached to one of the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, detection includes imaging of fluorescent labels. In some embodiments, a plurality of nucleotides are composed of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group including dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the light emitted by the fluorescent label of the nucleotides of another type in the group. In some embodiments, a plurality of nucleotides include at least two types of nucleotides, wherein at least two types of nucleotides are selected from the group including dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the light emitted by the fluorescent label of the nucleotides of another type in the group. In some embodiments, determining further includes contacting the nucleotides with a reagent to remove the blocking group from the nucleotides and generate a 3'OH group on the sugar portion. In some embodiments, the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0159] In the methods disclosed herein, the first target nucleic acid sequence or the second target nucleic acid sequence may correspond to at least a portion of a messenger RNA (mRNA) molecule. In the methods disclosed herein, the first target nucleic acid sequence and the second target nucleic acid sequence may correspond to at least a portion of a messenger RNA (mRNA) molecule. In the methods disclosed herein, the first target nucleic acid sequence or the second target nucleic acid sequence may correspond to at least a portion of a complementary DNA (cDNA) molecule. In the methods disclosed herein, the first target nucleic acid sequence and the second target nucleic acid sequence may correspond to at least a portion of a complementary DNA (cDNA) molecule. In some embodiments, the first target nucleic acid sequence and / or the second target nucleic acid sequence may correspond to two separate portions of the same mRNA or cDNA molecule. In some embodiments, the first target nucleic acid sequence and the second target nucleic acid sequence may correspond to two different mRNA or cDNA molecules. In some embodiments, determining comprises detecting the first or second sequencing product nucleic acid molecule in a biological sample in situ by imaging. In some embodiments, determining comprises detecting the first and second sequencing product nucleic acid molecules in a biological sample simultaneously in situ by imaging. In some embodiments, imaging comprises fluorescence imaging. In some embodiments, the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, a complete tissue, a tumor, a complete tumor, an organ, an organism, a protozoa, an algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal. In some embodiments, the biological sample comprises a fresh sample, a processed sample, a fresh frozen sample, a sliced ​​sample, or a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the biological sample comprises a fresh cell sample, a fresh frozen cell sample, a sliced ​​cell sample, or a FFPE cell sample. In some embodiments, at least two target nucleic acid sequences comprise a target DNA sequence. In some embodiments, at least two target nucleic acid sequences comprise a target RNA sequence. In some embodiments, at least two target nucleic acid sequences comprise a first target RNA sequence and a second target RNA sequence. In some embodiments, the first target RNA sequence comprises coding RNA, non-coding RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small interfering RNA (siRNA), antisense RNA, mature microRNA, or immature microRNA. In some embodiments, the second target RNA sequence comprises a coding RNA, a non-coding RNA, an mRNA, a tRNA, an rRNA, a miRNA, a gRNA, a snRNA, a siRNA, an antisense RNA, a mature microRNA, or an immature microRNA. In some embodiments, the at least two target nucleic acid sequences comprise a first target DNA sequence and a second target DNA sequence.In some embodiments, the first target DNA sequence comprises complementary DNA (cDNA), genomic DNA (gDNA), non-coding DNA or coding DNA. In some embodiments, the second target DNA sequence comprises cDNA, gDNA, non-coding DNA or coding DNA. In some embodiments, the at least two target nucleic acid sequences comprise a target RNA sequence and a target DNA sequence. In some embodiments, the target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, the target DNA sequence comprises cDNA, gDNA, non-coding DNA or coding DNA.

[0160] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample may further include step (g): removing a plurality of first sequencing read products from the first concatemer molecule and retaining the first concatemer molecule in the cell sample, and removing a plurality of second sequencing read products from the second concatemer molecule and retaining the second concatemer molecule in the cell sample. In some embodiments, (g) may include removing a plurality of first sequence read products from the first concatemer molecule. In some embodiments, step (g) may further include retaining the first concatemer molecule in the cell sample. In some embodiments, step (g) may include removing a plurality of second sequence read products from the second concatemer molecule. In some embodiments, step (g) may further include retaining the second concatemer molecule in the cell sample.

[0161] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample further comprises step (h): iteratively sequencing the plurality of concatemers by repeating steps (f) and (g) at least once, wherein the sequences of the plurality of first sequencing read products confirm the presence of the first target RNA molecule in the cell sample, and wherein the sequences of the plurality of second sequencing read products confirm the presence of the second target RNA molecule in the cell sample. In some embodiments, repeating comprises repeating at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or at least 50 times. In some embodiments, step (h) may comprise iteratively sequencing the plurality of concatemers by repeating steps (f) and (g) at least once. In some embodiments, repeating comprises repeating at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or at least 50 times. In some embodiments, the sequences of the plurality of first sequencing read products can confirm the presence of the first target RNA molecule in the cell sample. In some embodiments, the sequences of the plurality of second sequencing read products can confirm the presence of the second target RNA molecule in the cell sample. In some embodiments, the sequences of the plurality of first sequencing read products can confirm the presence of the first target RNA molecule in the cell sample and the sequences of the plurality of second sequencing read products can confirm the presence of the second target RNA molecule in the cell sample.

[0162] The present disclosure provides a method for in situ detection of at least two target nucleic acid molecules and at least two polypeptides in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, wherein the at least two target polypeptides include a first target polypeptide encoded by the first target nucleic acid sequence or its reverse complement and a second target polypeptide encoded by the second target nucleic acid sequence or its reverse complement. In some embodiments, the method includes step (a) providing a biological sample. In some embodiments, the biological sample comprises a first nucleic acid molecule, which comprises a first target nucleic acid sequence or a portion thereof, or a reverse complement sequence or a portion thereof of the first target nucleic acid sequence. In some embodiments, the biological sample comprises a second nucleic acid molecule, which comprises a second target nucleic acid sequence or a portion thereof, or a reverse complement sequence or a portion thereof of the second target nucleic acid sequence. In some embodiments, the biological sample comprises a third nucleic acid molecule, which comprises a third target nucleic acid sequence or a portion thereof, or a reverse complement sequence or a portion thereof of the third target nucleic acid sequence, wherein the presence of the third target nucleic acid sequence or its reverse complement identifies the presence of the first target polypeptide in the biological sample. In some embodiments, the biological sample comprises a fourth nucleic acid molecule comprising a fourth target nucleic acid sequence or a portion thereof, or a reverse complement sequence or a portion thereof of the fourth target nucleic acid sequence, wherein the presence of the fourth target nucleic acid sequence or its reverse complement sequence identifies the presence of a second target polypeptide in the biological sample. In some embodiments, the method further comprises step (b) determining the sequence of the first nucleic acid molecule or a portion thereof in the biological sample in situ to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first nucleic acid molecule or a portion thereof. In some embodiments, step (b) further comprises determining the sequence of the third nucleic acid molecule or a portion thereof in the biological sample in situ to generate a third sequencing product nucleic acid molecule that is complementary to and binds to the third nucleic acid molecule or a portion thereof. In some embodiments, the method further comprises step (c) identifying the sequence of the following in situ: the second nucleic acid molecule or a portion thereof in the biological sample to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second nucleic acid molecule or a portion thereof. In some embodiments, step (c) further comprises identifying the sequence of the fourth nucleic acid molecule or a portion thereof in the biological sample in situ to generate a fourth sequencing product nucleic acid molecule that is complementary to and binds to the fourth nucleic acid molecule or a portion thereof. In some embodiments, the full sequence of the first target nucleic acid sequence and the full sequence of the second target nucleic acid sequence have a difference of at least one nucleotide. In some embodiments, the full sequence of the first target polypeptide and the full sequence of the second target polypeptide differ by at least one amino acid. In some embodiments, step (b) is performed under conditions that prevent step (c) from being performed.

[0163] The present disclosure provides a method for in situ detection of at least two target nucleic acid molecules and at least two polypeptides in a biological sample. In some embodiments, the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence. In some embodiments, the at least two target polypeptides include a first target polypeptide encoded by a first target nucleic acid sequence or its reverse complementary sequence. In some embodiments, the at least two target polypeptides include a second target polypeptide encoded by a second target nucleic acid sequence or its reverse complementary sequence. In some embodiments, the method includes (a) providing a biological sample, which includes: (i) a first nucleic acid molecule, which includes a first target nucleic acid sequence or a portion thereof, or a reverse complementary sequence or a portion thereof of the first target nucleic acid sequence. In some embodiments, the biological sample includes (ii) a second nucleic acid molecule, which includes a second target nucleic acid sequence or a portion thereof, or a reverse complementary sequence or a portion thereof of the second target nucleic acid sequence. In some embodiments, the biological sample includes (iii) a third nucleic acid molecule, which includes a third target nucleic acid sequence or a portion thereof, or a reverse complementary sequence or a portion thereof of the third target nucleic acid sequence. In some embodiments, the presence of the third target nucleic acid sequence or its reverse complementary sequence identifies the presence of the first target polypeptide in the biological sample. In some embodiments, the biological sample further includes (iv) a fourth nucleic acid molecule comprising a fourth target nucleic acid sequence or a portion thereof, or a reverse complement sequence of the fourth target nucleic acid sequence or a portion thereof, wherein the presence of the fourth target nucleic acid sequence or its reverse complement sequence identifies the presence of a second target polypeptide in the biological sample. In some embodiments, the method further includes step (b) determining the sequence of the following in situ: (i) a first nucleic acid molecule or a portion thereof in the biological sample to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first nucleic acid molecule or a portion thereof, wherein the first nucleic acid molecule or a portion thereof consists of 2-30 nucleotides. In some embodiments, step (b) further includes determining the sequence of the following in situ: (ii) a third nucleic acid molecule or a portion thereof in the biological sample to generate a third sequencing product nucleic acid molecule that is complementary to and binds to the third nucleic acid molecule or a portion thereof, wherein the third nucleic acid molecule or a portion thereof consists of 2-30 nucleotides. In some embodiments, the method further includes (c) removing the first sequencing product nucleic acid molecule from the first nucleic acid molecule, and removing the third sequencing product nucleic acid molecule from the third nucleic acid molecule. In some embodiments, after removal, the first nucleic acid molecule and the third nucleic acid molecule are located in the biological sample. In some embodiments, the method further comprises step (d): repeating (b) and (c). In some embodiments, the method further comprises step (e): identifying in situ the sequence of: (i) a second nucleic acid molecule or a portion thereof in the biological sample to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second nucleic acid molecule or the portion thereof, wherein the second nucleic acid molecule or the portion thereof consists of 2-30 nucleotides.In some embodiments, step (e) further includes in situ identification of the sequence of the following items: (ii) a fourth nucleic acid molecule or a portion thereof in the biological sample to generate a fourth sequencing product nucleic acid molecule that is complementary to and bound to the fourth nucleic acid molecule or a portion thereof. In some embodiments, the fourth nucleic acid molecule or a portion thereof consists of 2-30 nucleotides. In some embodiments, the method further includes step (f): removing the second sequencing product nucleic acid molecule from the second nucleic acid molecule, and removing the fourth sequencing product nucleic acid molecule from the fourth nucleic acid molecule, wherein after removal, the second nucleic acid molecule and the fourth nucleic acid molecule are located in the biological sample. In some embodiments, the method further includes step (g): repeating steps (e) and (f). In some embodiments, the full sequence of the first target nucleic acid sequence and the full sequence of the second target nucleic acid sequence have a difference of at least one nucleotide. In some embodiments, the full sequence of the first target polypeptide and the full sequence of the second target polypeptide have a difference of at least one amino acid. In some embodiments, step (b) is performed under conditions that prevent step (e) from being performed.

[0164] The present disclosure provides a method for in situ detection of at least two target RNA sequences and at least two target polypeptides in a biological sample. In some embodiments, the method includes step (a) providing a biological sample fixed on a surface, permeabilized and fixed. In some embodiments, the biological sample comprises (i) a first target RNA sequence in at least two target RNA sequences and a first target polypeptide in at least two polypeptides, wherein the first target polypeptide is encoded by the first target RNA sequence or its reverse complementary sequence. In some embodiments, the biological sample comprises (ii) a second target RNA sequence in at least two target RNA sequences and a second target polypeptide in at least two polypeptides, wherein the second target polypeptide is encoded by the second target RNA sequence or its reverse complementary sequence. In some embodiments, the method further includes step (b) producing a first target cDNA sequence by reverse transcription of the first target RNA sequence, and producing a second target cDNA sequence by reverse transcription of the second target RNA sequence. In some embodiments, the method further includes the step of (c) contacting the first target cDNA sequence with a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target cDNA sequence, so that the first oligonucleotide forms a ring structure with a gap between the first end and the second end, wherein the first oligonucleotide includes a first identification sequence for identifying the first target RNA sequence, a first sequencing primer, and a nucleic acid amplification primer. In some embodiments, step (c) further includes contacting the second target cDNA sequence with a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target cDNA sequence, so that the second oligonucleotide forms a ring structure with a gap between the first end and the second end. In some embodiments, the second oligonucleotide includes a second identification sequence for identifying the second target RNA sequence, a second sequencing primer, and a nucleic acid amplification primer. In some embodiments, the first sequencing primer and the second sequencing primer have a difference of at least one nucleotide. In some embodiments, step (c) comprises contacting the first target polypeptide with a first oligonucleotide conjugate comprising a first short nucleic acid and a first binding portion that specifically binds to the first target polypeptide, wherein the first short nucleic acid comprises a first tag sequence, a second tag sequence, wherein the first oligonucleotide conjugate specifically binds to the first target polypeptide through the first binding portion to form a first binding complex, wherein the first and second tag sequences identify the first binding portion. In some embodiments, step (c) comprises contacting the second target polypeptide with a second oligonucleotide conjugate comprising a second short nucleic acid and a second binding portion that specifically binds to the second target polypeptide, wherein the second short nucleic acid comprises a third tag sequence, a fourth tag sequence, wherein the second oligonucleotide conjugate specifically binds to the second target polypeptide through the second binding portion to form a second binding complex, wherein the third and fourth tag sequences identify the second binding portion.In some embodiments, the method further comprises the step of (d) contacting the first binding complex with a third oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the third oligonucleotide are complementary to and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, such that the third oligonucleotide forms a ring structure having a gap between the first end and the second end. In some embodiments, step (d) comprises contacting the second binding complex with a fourth oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the fourth oligonucleotide are complementary to and bind to the third tag sequence and the fourth tag sequence, such that the fourth oligonucleotide forms a ring structure having a gap between the first end and the second end. In some embodiments, the method further comprises step (e) joining the first end and the second end of the first oligonucleotide to produce a first circular oligonucleotide, joining the first end and the second end of the second oligonucleotide to produce a second circular oligonucleotide, joining the first end and the second end of the third oligonucleotide to produce a third circular oligonucleotide and joining the first end and the second end of the fourth oligonucleotide to produce a fourth circular oligonucleotide, wherein the first circular oligonucleotide and the third circular oligonucleotide include a first sequencing primer or its reverse complementary sequence, wherein the second circular oligonucleotide and the fourth circular oligonucleotide include a second sequencing primer or its reverse complementary sequence, wherein the sequence of the first and second sequencing primers has a difference of at least one nucleotide. In some embodiments, the method further comprises step (f) amplifying the first circular oligonucleotide by rolling circle amplification to produce a first concatemer comprising a plurality of first circular oligonucleotides. In some embodiments, step (f) further comprises amplifying the second circular oligonucleotide by rolling circle amplification to produce a second concatemer comprising a plurality of second circular oligonucleotides. In some embodiments, step (f) further comprises amplifying the third circular oligonucleotide by rolling circle amplification to produce a third concatemer comprising a plurality of third circular oligonucleotides. In some embodiments, step (f) further comprises amplifying a fourth circular oligonucleotide by rolling circle amplification to produce a fourth concatemer comprising a plurality of fourth circular oligonucleotides. In some embodiments, the method further comprises the step of determining the sequence of the first concatemer or a portion thereof in situ to generate a first sequencing product nucleic acid molecule that is complementary to and bound to the first concatemer, wherein the sequence of the first concatemer or a portion thereof is composed of 2-30 nucleotides. In some embodiments, step (g) further comprises determining the sequence of the third concatemer or a portion thereof in situ to generate a third sequencing product nucleic acid molecule that is complementary to and bound to the third concatemer, wherein the sequence of the third concatemer or a portion thereof is composed of 2-30 nucleotides. In some embodiments, step (g) is performed under conditions that prevent step (j) from being performed.In some embodiments, the method further comprises the step of (h) removing the first sequencing product nucleic acid molecule from the first concatemer and removing the third sequencing product nucleic acid molecule from the third concatemer, wherein after removal, the first concatemer and the third concatemer are located in the biological sample. In some embodiments, the method further comprises step (i) repeating (g) and (h) at least once. In some embodiments, the method further comprises the step of (j) determining the sequence of the second concatemer or a portion thereof in situ to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second concatemer, wherein the sequence of the second concatemer or a portion thereof consists of 2-30 nucleotides. In some embodiments, step (j) further comprises determining the sequence of the fourth concatemer or a portion thereof in situ to generate a fourth sequencing product nucleic acid molecule that is complementary to and binds to the fourth concatemer, wherein the sequence of the fourth concatemer or a portion thereof consists of 2-30 nucleotides. In some embodiments, step (j) is performed under conditions that prevent step (g) from being performed. In some embodiments, the full sequence of the first target RNA sequence and the full sequence of the second target RNA sequence have a difference of at least one nucleotide. In some embodiments, the full sequence of the first target polypeptide and the full sequence of the second target polypeptide have a difference of at least one amino acid. In some embodiments, the method further comprises step (k) removing the second sequencing product nucleic acid molecule from the second concatemer and removing the fourth sequencing product nucleic acid molecule from the fourth concatemer. In some embodiments, after removal, the second concatemer and the fourth concatemer are localized in the biological sample. In some embodiments, the method further comprises step (i) repeating (j) and (k) at least once.

[0165] In some embodiments, determining comprises imaging the first or third sequencing product nucleic acid molecule, wherein identifying comprises imaging the second or fourth sequencing product nucleic acid molecule and the fourth sequencing product nucleic acid molecule. In some embodiments, the method further comprises simultaneously imaging the first sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule to analyze the spatial distribution of the first and second sequencing product nucleic acid molecules within the biological sample. In some embodiments, the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, a complete tissue, a tumor, a complete tumor, an organ, an organism, a protozoa, an algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal. In some embodiments, the biological sample comprises a fresh sample, a processed sample, a fresh frozen sample, a sectioned sample, or a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the first target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA, and / or wherein the first target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, the second target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA, and / or wherein the second target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA. In some embodiments, the biological sample comprises a human sample, a monkey sample, an ape sample, a dog sample, a cat sample, a cow sample, a horse sample, a mouse sample, a pig sample, a goat sample, a lupin sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a virus sample, a protozoan sample, or a fungal sample. In some embodiments, the first, second, third, or fourth concatemer further comprises a compaction oligonucleotide, wherein: the first segment of the compaction oligonucleotide is complementary to and binds to the first portion of the first, second, third, or fourth concatemer. In some embodiments, the second segment of the compaction oligonucleotide is complementary to and binds to the second portion of the first, second, third, or fourth concatemer to cause the second concatemer to decrease in size or change in shape. In some embodiments, the first sequencing product nucleic acid molecule comprises a first identification sequence that identifies the first target RNA sequence, and the second sequencing product nucleic acid molecule comprises a second identification sequence that identifies the second target RNA sequence.In some embodiments, the first sequencing product nucleic acid molecule comprises a first identification sequence that identifies a first target RNA sequence and a portion of the first target RNA sequence, wherein the second sequencing product nucleic acid molecule comprises a second identification sequence that identifies a second target RNA sequence and a portion of the second target RNA sequence. In some embodiments, determining comprises contacting the first, second, third, or fourth concatemer with a polymerase, a plurality of nucleotides, and a primer sequence that is complementary to a portion of the first, second, third, or fourth concatemer under conditions sufficient to form a binding complex, the binding complex comprising a polymerase, a plurality of nucleotides that are complementary to and bound to the nucleotide units in the first, second, third, or fourth concatemer, and the first, second, third, or fourth concatemer that hybridizes to the primer sequence. In some embodiments, the nucleotide comprises a fluorescent label and a removable blocking group at the 3' carbon position of the sugar portion. In some embodiments, determining further comprises incorporating the nucleotide into the 3' end of the primer sequence. In some embodiments, determining further comprises identifying the nucleobase of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide. In some embodiments, determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar portion. In some embodiments, the blocking group includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, a plurality of nucleotides include a type of nucleotide selected from dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, a plurality of nucleotides include a mixture of any combination of two or more types of nucleotides selected from dATP, dGTP, dCTP, dTTP and / or dUTP.

[0166] In some embodiments, determining comprises contacting two first, second, third, or fourth concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to portions of the first, second, third, or fourth concatemers under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate in the plurality of nucleotide conjugates, and each of the two first, second, third, or fourth concatemers hybridized to each of the two primer sequences. In some embodiments, the nucleotide conjugate comprises a label and at least two nucleotide portions. In some embodiments, two of the at least two nucleotide portions are each complementary to and bound to a nucleotide of each of the two first concatemers. In some embodiments, determining comprises detecting the multivalent binding complex by the label of the nucleotide conjugate. In some embodiments, determining further comprises identifying the nucleobases of the nucleotides in the two first concatemers, each of which is complementary to and bound to two of the at least two nucleotide portions of the nucleotide conjugates.

[0167] In some embodiments, determining further comprises: removing two polymerases and nucleotide conjugates from the two first, second, third or fourth concatemers. In some embodiments, determining further comprises contacting each of the two first, second, third or fourth concatemers that hybridize to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, the binding complex comprising each of the two second polymerases, each of the first, second, third or fourth concatemers that hybridize to each of the two primer sequences, and two of the plurality of unlabeled nucleotides, and incorporating each of the two of the plurality of unlabeled nucleotides into each of the two primer sequences. In some embodiments, each of the two of the plurality of unlabeled nucleotides is complementary to and binds to the nucleotide of each of the two first, second, third or fourth concatemers. In some embodiments, the unlabeled nucleotides in the plurality of unlabeled nucleotides include a removable blocking group at the 3' carbon of the sugar portion.

[0168] In some embodiments, determining comprises contacting two first, second, third, or fourth concatemers with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to the first portion of the first, second, third, or fourth concatemer, and a second primer sequence complementary to the second portion of the first, second, third, or fourth concatemer under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, the first portion of the first, second, third, or fourth concatemer hybridized to the first primer sequence, and the second portion of the first, second, third, or fourth concatemer hybridized to the second primer sequence. In some embodiments, the nucleotide conjugate comprises a label and at least two nucleotide portions. In some embodiments, two of the at least two nucleotide portions are each complementary to and bind to a nucleotide in the first portion and the second portion of the first, second, third, or fourth concatemer. In some embodiments, determining further comprises detecting the multivalent binding complex via the label of the nucleotide conjugate. In some embodiments, determining further comprises identifying the nucleobases of the nucleotides in the first and second portions of the first, second, third, or fourth concatemer, each of which is complementary to and binds to each of two of the at least two nucleotide portions in the nucleotide conjugate. In some embodiments, the condition inhibits at least two nucleotide moieties of the nucleotide conjugate from being incorporated into two first, second, third or fourth concatemers. In some embodiments, the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each nucleotide moiety is attached to a nucleotide arm in the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, detection comprises imaging the fluorescent label. In some embodiments, a plurality of nucleotides are composed of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the light emitted by the fluorescent label of the nucleotides of another type in the group. In some embodiments, a plurality of nucleotides include at least two types of nucleotides, wherein at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the light emitted by the fluorescent label of the nucleotides of another type in the group. In some embodiments, determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar moiety.In some embodiments, the blocking group includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group. In some embodiments, a plurality of nucleotides are composed of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group including dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the fluorescent label emitted light of the nucleotides of another type in the group. In some embodiments, a plurality of nucleotides include at least two types of nucleotides, wherein at least two types of nucleotides are selected from the group including dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of the fluorescent label emitted light of the nucleotides of one type in the group is different from the wavelength of the fluorescent label emitted light of the nucleotides of another type in the group. In some embodiments, the first target RNA sequence comprises coding RNA, non-coding RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small interfering RNA (siRNA), antisense RNA, mature microRNA or immature microRNA. In some embodiments, the second target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA.

[0169] In some embodiments, the biological sample comprises a human sample, a monkey sample, ape sample, dog sample, cat sample, bovine sample, horse sample, mouse sample, porcine sample, goat sample, lupine sample, frog sample, fish sample, plant sample, insect sample, bacterial sample, algae sample, viral sample, protozoan sample, or fungal sample. In some embodiments, the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, an intact tissue, a tumor, an intact tumor, an organ, or an organism. In some embodiments, the biological sample is immobilized on a surface. In some embodiments, the surface comprises the interior surface of a flow cell.

[0170] In situ batch sequencing of RNA and peptides

[0171] The present disclosure provides methods for in situ multiplex and multi-omics detection and identification using coded padlock probes. Padlock probes are designed to selectively detect target RNAs or polypeptides. Padlock probes can contain one or more batch-specific primer binding sites that are specific for primers. Primers can be spatially positioned, and therefore padlock probes may not require barcode sequences. However, in some embodiments, padlock probes may contain barcode sequences.

[0172] RNA-specific padlock probes selectively hybridize to cDNA corresponding to the target RNA. The RNA-specific probes carry a barcode that uniquely identifies the cDNA. The RNA-specific padlock probes also carry a batch-specific sequencing primer binding site. The padlock probes can contain one or more batch-specific primer binding sites specific for the primers. The primers can be spatially positioned, and therefore the padlock probes may not require a barcode sequence. However, in some embodiments, the padlock probes may contain a barcode sequence.

[0173] Target polypeptides are detected using antibody-oligonucleotide conjugates and polypeptide-specific padlock probes that selectively hybridize to the oligonucleotide conjugated to the antibody. The polypeptide-specific padlock probes carry a barcode that uniquely identifies the antibody that selectively binds to the target polypeptide. The polypeptide-specific padlock probes also carry batch-specific sequencing primers, identical to those carried by the corresponding RNA-specific padlock probes, enabling simultaneous sequencing and detection of the target RNA and the polypeptide encoded by it. Thus, the padlock probes enable simultaneous detection and identification of RNA and its encoded peptide.

[0174] Both types of padlock probes are used to generate concatemers with multiple copies of batch-specific sequencing binding sites and barcodes. The concatemers can be folded into DNA nanoballs with a compact shape and size, which produces increased signal intensity and color differentiation during sequencing.

[0175] For in situ sequencing, the limitation of optical resolution hinders the ability to perform highly multiplexed sequencing. Batch-specific sequencing primer binding sites on padlock probes enable the sequencing of desired concatemer subsets (e.g., batches) using selected batch-specific sequencing primers to reduce overcrowded signals and images. Batch-specific sequencing primers produce intense and distinguishable optical images. By performing multiple rounds of sequencing on the same cell sample using different batch-specific sequencing primers, multiplexed and multi-omics sequencing can reveal numerous target RNAs and their encoded polypeptides.

[0176] The batch-specific sequencing methods described herein have many uses. For example, the number of spots imaged and associated with sequencing can be counted. The counted spots can be used to measure RNA and polypeptide levels in cell samples.

[0177] For example, a paired sequencing kit that can be used to perform 150 forward sequencing cycles and 150 reverse sequencing cycles (e.g., 300 sequencing cycles per kit) can be used to reveal more than 300,000 molecules. A non-limiting example cell has approximately 30 copies of 5,000 different transcripts, totaling approximately 150,000 transcripts, and the call has approximately 15,000 polypeptides encoded by the transcripts, totaling 300,000 total RNA and polypeptide molecules. The paired kit is used to perform 300 sequencing cycles (not in paired mode) to reveal 300,000 different RNA and polypeptide molecules in the cell sample. See Table 1. Table 1 lists the estimated total sequencing cycles and the total time for decoding the RNA and polypeptide in the cell sample.

[0178] Table 1: Estimated total sequencing cycles and total time to decode RNA and peptides from cell samples

[0179]

[0180] Column A: Resolution (um)

[0181] Column B: Cell volume (um 2 )

[0182] Column C: resolved polymerase communities / cells (4 colors)

[0183] Column D: Transcripts

[0184] Column E: Copy number / transcript

[0185] Column F: Protein

[0186] Column G: copy number / protein

[0187] Column H: Total numerator

[0188] Column I: Decoding loop

[0189] Column J: Total cycles of decoding all molecules

[0190] Column K: Total time for decoding (hours)

[0191] The present disclosure provides a method for in situ detection of at least two different target RNA molecules and two different polypeptides encoded by the at least two different target RNA molecules, comprising the step (a): providing a cell sample deposited on a solid support, wherein the cell sample carries (i) a first plurality of DNA amplicons corresponding to the first target RNA molecule, (ii) a second plurality of DNA amplicons corresponding to the second target RNA molecule, (iii) a third plurality of DNA amplicons corresponding to the first polypeptide encoded by the first target RNA molecule, and (iv) a fourth plurality of DNA amplicons corresponding to the second polypeptide encoded by the second target RNA molecule.

[0192] In some embodiments, the method further includes step (b): sequencing the first and third multiple DNA amplicons within the cell sample under conditions that inhibit sequencing of the second and fourth multiple DNA amplicons, wherein sequencing the first multiple DNA amplicons within the cell sample includes generating multiple first sequencing read products, wherein the sequences of the first sequencing read products are aligned with the first target reference sequence to confirm the presence of the first target RNA in the cell sample, and wherein sequencing the third multiple DNA amplicons within the cell sample includes generating multiple second sequencing read products, wherein the sequences of the second sequencing read products are aligned with the second target reference sequence to confirm the presence of the first target polypeptide in the cell sample.

[0193] In some embodiments, the method further includes step (c): sequencing the second and fourth multiple DNA amplicons within the cell sample under conditions that inhibit sequencing of the first and third multiple DNA amplicons, wherein sequencing the second multiple DNA amplicons within the cell sample includes generating multiple third sequencing read products, wherein the sequences of the third sequencing read products are aligned with the third target reference sequence to confirm the presence of the second target RNA in the cell sample, and wherein sequencing the fourth multiple DNA amplicons within the cell sample includes generating multiple fourth sequencing read products, wherein the sequences of the fourth sequencing read products are aligned with the fourth target reference sequence to confirm the presence of the second target polypeptide in the cell sample.

[0194] The present disclosure provides a method for in situ detection of at least two different target RNA molecules and two different polypeptides encoded by the at least two different target RNA molecules, comprising the step (a): providing a cell sample deposited on a solid support, wherein the cell sample carries (i) a first plurality of DNA amplicons corresponding to the first target RNA molecule, (ii) a second plurality of DNA amplicons corresponding to the second target RNA molecule, (iii) a third plurality of DNA amplicons corresponding to the first polypeptide encoded by the first target RNA molecule, and (iv) a fourth plurality of DNA amplicons corresponding to the second polypeptide encoded by the second target RNA molecule.

[0195] In some embodiments, the method further comprises step (b): sequencing the first and third multiple DNA amplicons within the cell sample under conditions that inhibit sequencing of the second and fourth multiple DNA amplicons. In some embodiments, step (b) comprises sequencing the first multiple DNA amplicons within the cell sample, comprising performing no more than 2-30 sequencing cycles to generate multiple first sequencing read products, wherein the sequence of the first sequencing read product is aligned with the first target reference sequence to confirm the presence of the first target RNA in the cell sample. In some embodiments, step (b) comprises sequencing the first multiple DNA amplicons within the cell sample, comprising performing 1-250 sequencing cycles to generate multiple first sequencing read products, wherein the sequence of the first sequencing read product is aligned with the first target reference sequence to confirm the presence of the first target RNA in the cell sample. In some embodiments, step (b) comprises sequencing the third multiple DNA amplicons within the cell sample, comprising performing no more than 2-30 sequencing cycles to generate multiple second sequencing read products, wherein the sequence of the second sequencing read product is aligned with the second target reference sequence to confirm the presence of the first target polypeptide in the cell sample. In some embodiments, step (b) includes sequencing a third plurality of DNA amplicons within the cell sample, comprising performing 1-250 sequencing cycles to generate a plurality of second sequencing read products, wherein the sequences of the second sequencing read products are aligned with the second target reference sequence to confirm the presence of the first target polypeptide in the cell sample.

[0196] In some embodiments, the method further comprises step (c): removing a plurality of first sequencing read products from the first DNA amplicon and retaining the first DNA amplicon within the cell sample, and removing a plurality of second sequencing read products from the third DNA amplicon and retaining the third DNA amplicon within the cell sample. In some embodiments, a 3' blocking portion can be added to the first and second sequencing read products to inhibit further sequencing reactions. For example, a nucleotide analog can be incorporated, wherein the nucleotide analog inhibits the incorporation of subsequent nucleotides. Non-limiting example blocking nucleotide analogs include dideoxynucleotides or nucleotides with 2' or 3' chain termination portions.

[0197] In some embodiments, the method further comprises step (d): iteratively sequencing the first and third plurality of DNA amplicons by repeating steps (b) and (c) at least once. In some embodiments, repeating comprises repeating at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or at least 50 times.

[0198] In some embodiments, the method further comprises step (e): sequencing the second and fourth multiple DNA amplicons within the cell sample under conditions that inhibit sequencing of the first and third multiple DNA amplicons. In some embodiments, step (e) comprises sequencing the second multiple DNA amplicons within the cell sample, comprising performing no more than 2-30 sequencing cycles to generate multiple third sequencing read products, wherein the sequence of the third sequencing read product is aligned with the third target reference sequence to confirm the presence of a second target RNA in the cell sample. In some embodiments, step (e) comprises sequencing the second multiple DNA amplicons within the cell sample, comprising performing 1-250 sequencing cycles to generate multiple third sequencing read products, wherein the sequence of the third sequencing read product is aligned with the third target reference sequence to confirm the presence of a second target RNA in the cell sample. In some embodiments, step (e) comprises sequencing the fourth multiple DNA amplicons within the cell sample, comprising performing no more than 2-30 sequencing cycles to generate multiple fourth sequencing read products, wherein the sequence of the fourth sequencing read product is aligned with the fourth target reference sequence to confirm the presence of a second target polypeptide in the cell sample. In some embodiments, step (e) includes sequencing a fourth plurality of DNA amplicons within the cell sample, comprising performing 1-250 sequencing cycles to generate a plurality of fourth sequencing read products, wherein the sequences of the fourth sequencing read products are aligned with a fourth target reference sequence to confirm the presence of the second target polypeptide in the cell sample.

[0199] In some embodiments, the method includes step (f): removing a plurality of third sequencing read products from the second DNA amplicon and retaining the second DNA amplicon within the cell sample, and removing a plurality of fourth sequencing read products from the fourth DNA amplicon and retaining the fourth DNA amplicon within the cell sample. In some embodiments, a 3' blocking portion can be added to the third and fourth sequencing read products to inhibit further sequencing reactions. For example, a nucleotide analog can be incorporated, wherein the nucleotide analog inhibits the incorporation of subsequent nucleotides. Non-limiting example blocking nucleotide analogs include dideoxynucleotides or nucleotides with 2' or 3' chain termination portions.

[0200] In some embodiments, the method comprises step (g): iteratively sequencing the second and fourth pluralities of DNA amplicons by repeating steps (e) and (f) at least once. In some embodiments, repeating comprises repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times.

[0201] The present disclosure provides a method for in situ detection of at least two different target RNA molecules and two different polypeptides encoded by the at least two different target RNA molecules, comprising step (a): providing a cell sample deposited on a solid support, wherein the cell sample is fixed and permeabilized, wherein the cell sample carries a first plurality of target RNAs and a first plurality of target polypeptides encoded by the first plurality of target RNAs, and the cell sample carries a second plurality of target RNAs and a second plurality of target polypeptides encoded by the second plurality of target RNAs.

[0202] In some embodiments, the cell sample carries 2-25 different target RNA molecules, or carries 25-50 different target RNA molecules, or carries 50-75 different target RNA molecules, or carries 75-100 different target RNA molecules. In some embodiments, the cell sample carries more than 100 different target RNA molecules, or more than 250 different target RNA molecules, or more than 500 different target molecules, or more than 1000 different target RNA molecules, or more. In some embodiments, the cell sample carries more than 10,000 different target RNA molecules. In some embodiments, the cell sample comprises a whole cell, a plurality of whole cells, a complete tissue, or a complete tumor. In some embodiments, the cell sample comprises a fresh cell sample, a fresh frozen cell sample, a sliced ​​cell sample, or a FFPE cell sample. In some embodiments, the cell sample is deposited on a solid support. In some embodiments, the cell sample is deposited on a solid support, and the solid support is passivated with a coating that promotes cell adhesion. In some embodiments, the cell sample is deposited on a support lacking immobilized capture oligonucleotides. In some embodiments, the cell sample is cultured prior to performing step (b) described below.

[0203] In some embodiments, the cell sample contains 2-25 different target polypeptide molecules, or 25-50 different target polypeptide molecules, or 50-75 different target polypeptide molecules, or 75-100 different target polypeptide molecules. In some embodiments, the cell sample contains more than 100 different target polypeptide molecules, or more than 250 different target polypeptide molecules, or more than 500 different target molecules, or more than 1000 different target polypeptide molecules, or more. In some embodiments, the cell sample contains more than 10,000 different target polypeptide molecules. The target polypeptide molecules are encoded by target RNA molecules.

[0204] In some embodiments, the method includes step (b): generating a plurality of cDNAs within the cell sample by: (i) generating at least a first plurality of target cDNAs from a first plurality of target RNAs, and (ii) generating at least a second plurality of target cDNAs from a second plurality of target RNAs (e.g., Figure 20). In some embodiments, the first target cDNA corresponds to a first target RNA molecule. In some embodiments, the second target cDNA corresponds to a second target RNA molecule. In some embodiments, the method includes generating at least 2-10,000 different target cDNA molecules, which correspond to 2-10,000 different target RNA molecules. In some embodiments, the generation in step (b) includes contacting a plurality of RNAs in a cell sample with (i) a plurality of reverse transcription primers, (ii) a plurality of reverse transcriptases, and (iii) a plurality of nucleotides under conditions suitable for performing a reverse transcription reaction to generate a plurality of cDNA molecules (e.g., a plurality of first-chain cDNA molecules) in the cell sample. In some embodiments, a plurality of reverse transcription primers include a first subset of target-specific reverse transcription primers that selectively hybridize with the first target RNA, and include a second subset of target-specific reverse transcription primers that selectively hybridize with the second target RNA. In some embodiments, a plurality of reverse transcription primers include a first subset of random sequence reverse transcription primers that hybridize with the first target RNA, and include a second subset of random sequence reverse transcription primers that hybridize with the second target RNA.

[0205] In some embodiments, the method includes step (c): generating a plurality of DNA concatemers corresponding to a first and a second plurality of target RNA molecules within a cell sample, comprising: (1) generating a first plurality of covalently closed circular padlock probes by contacting a first plurality of target cDNAs with a first plurality of padlock probes, wherein the contacting is performed under conditions suitable for hybridizing the first and second binding arms of the first padlock probes to...

Claims

1. A method for in situ detection of at least two target nucleic acid sequences in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, the method comprising: (a) providing the biological sample, which comprises: (i) a first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or a reverse complementary sequence thereof or a portion thereof; as well as (ii) a second nucleic acid molecule comprising the second target nucleic acid sequence or a portion thereof, or a reverse complement sequence thereof or a portion thereof; (b) determining in situ the sequence of: (i) the first nucleic acid molecule or a portion thereof in the biological sample to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first nucleic acid molecule or a portion thereof; as well as (ii) the second nucleic acid molecule or a portion thereof in the biological sample, to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second nucleic acid molecule or a portion thereof, wherein the full sequence of the first target nucleic acid sequence and the full sequence of the second target nucleic acid sequence differ by at least one nucleotide; (c) removing the first sequencing product nucleic acid molecule from the first nucleic acid molecule, and removing the second sequencing product nucleic acid molecule from the second nucleic acid molecule, wherein after the removal, the first nucleic acid molecule and the second nucleic acid molecule are localized within the biological sample; and (d) repeating (b) and (c) to detect the at least two target nucleic acid sequences in the biological sample in situ.

2. The method of claim 1, wherein the biological sample is fixed and permeabilized.

3. The method according to claim 1 or 2, further comprising: a. generating in situ a first complementary DNA (cDNA) molecule by reverse transcription of a first messenger RNA (mRNA) molecule in the biological sample, wherein the first cDNA molecule or the first mRNA molecule comprises the first target nucleic acid sequence or the reverse complement of the first target nucleic acid sequence; and / or b. generating a second cDNA molecule in situ by reverse transcription of a second mRNA molecule in the biological sample, wherein the second cDNA molecule or the second mRNA molecule comprises the second target nucleic acid sequence or the reverse complementary sequence of the second target nucleic acid sequence.

4. The method of claim 3, further comprising: a. contacting the first target nucleic acid sequence or its reverse complementary sequence in situ with a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target nucleic acid sequence or its reverse complementary sequence, so that the first oligonucleotide forms a ring structure having a gap between the first end and the second end; and / or b. contacting the second target nucleic acid sequence or its reverse complementary sequence in situ with a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target nucleic acid sequence or its reverse complementary sequence, so that the second oligonucleotide forms a circular structure with a gap between the first end and the second end.

5. The method of claim 4, wherein: a. the first target nucleic acid sequence comprises the first cDNA molecule or the first mRNA molecule; and / or b. The second target nucleic acid sequence includes the second cDNA molecule or the second mRNA molecule. The method of claim 4 , wherein the gap of the first oligonucleotide or the second oligonucleotide has a size of one nucleotide.

7. The method of claim 4 or 5, wherein the gap of the first oligonucleotide or the second oligonucleotide has a size of at least two nucleotides.

8. The method of any one of claims 4-7, wherein the first oligonucleotide further comprises a first identification sequence that identifies the first target nucleic acid sequence, and the second oligonucleotide further comprises a second identification sequence that identifies the second target nucleic acid sequence.

9. The method of any one of claims 4 to 8, wherein the first oligonucleotide and the second oligonucleotide further comprise a nucleic acid sequence that is complementary to a nucleic acid sequence of a sequencing primer.

10. The method according to any one of claims 4 to 9, wherein the first oligonucleotide and the second oligonucleotide further comprise a nucleic acid sequence that is complementary to a nucleic acid sequence of a primer for nucleic acid amplification.

11. The method of claim 10, wherein the primers for nucleic acid amplification are primers for rolling circle amplification (RCA) to produce concatemers, wherein the concatemers comprise at least two repeats of a target nucleic acid sequence in the at least two target nucleic acid sequences or parts thereof or their reverse complement sequences.

12. The method of claim 11, wherein the first oligonucleotide and the second oligonucleotide further comprise a reverse complement sequence of a compaction oligonucleotide, wherein: a. the first segment of the compacted oligonucleotide is complementary to and binds to the first portion of the concatemer; and b. the second segment of the compacted oligonucleotide is complementary to and binds to the second portion of the concatemer, This may result in a size reduction or a shape change of the concatemer.

13. The method of any one of claims 4 to 12, further comprising: (a) joining the first end and the second end of the first oligonucleotide in situ to generate a first circular oligonucleotide within the biological sample; as well as (b) joining the first end and the second end of the second oligonucleotide in situ to generate a second circular oligonucleotide within the biological sample.

14. The method of claim 13, wherein: (a) joining the first end and the second end of the first oligonucleotide comprises joining the first end and the second end of the first oligonucleotide by a first nuclease; and (b) joining the first end and the second end of the second oligonucleotide comprises joining the first end and the second end of the second oligonucleotide by a second nuclease, wherein the first nuclease and the second nuclease are the same type of enzyme.

15. The method of claim 14, wherein the first and second nucleases comprise nucleic acid ligases, nucleic acid ligases, nucleic acid polymerases, nucleic acid polymerases, or a combination thereof.

16. The method of any one of claims 13-15, further comprising in situ amplification: (a) the first circular oligonucleotide to produce the first nucleic acid molecule; and / or (b) the second circular oligonucleotide to produce the second nucleic acid molecule.

17. The method of claim 16, wherein the amplification comprises rolling circle amplification (RCA), wherein the first nucleic acid molecule comprises a first concatemer and the second nucleic acid molecule comprises a second concatemer.

18. The method of claim 17, wherein: a. the first concatemer comprises at least two repeats of a first unit nucleic acid sequence, wherein the first unit nucleic acid sequence comprises the first target nucleic acid sequence or a portion thereof, or a reverse complementary sequence of the first target nucleic acid sequence or a portion thereof; and / or b. The second concatemer comprises at least two repeats of a second unit nucleic acid sequence, wherein the second unit nucleic acid sequence comprises the second target nucleic acid sequence or a portion thereof, or a reverse complementary sequence of the second target nucleic acid sequence or a portion thereof.

19. The method of claim 18, wherein the first concatemer further comprises the first identification sequence or a sequencing primer or a reverse complementary sequence thereof for identifying the first target nucleic acid sequence, wherein the second concatemer further comprises the second identification sequence or a sequencing primer or a reverse complementary sequence thereof for identifying the second target nucleic acid sequence.

20. The method of claim 19, wherein the first concatemer further comprises a first compacted oligonucleotide, wherein: a. the first segment of the first compacted oligonucleotide is complementary to and binds to the first portion of the first concatemer; and b. the second segment of the first compacted oligonucleotide is complementary to and binds to the second portion of the first concatemer, This causes the first concatemer to reduce in size or change in shape.

21. The method of claim 20, wherein the second concatemer further comprises a second compacted oligonucleotide, wherein: a. the first segment of the second compacted oligonucleotide is complementary to and binds to the first portion of the second concatemer; and b. the second segment of the second compacted oligonucleotide is complementary to and binds to the second portion of the second concatemer, This causes the second series to decrease in size or change in shape.

22. The method of any one of claims 1 to 21, wherein the determining comprises: a. determining the sequence of the first nucleic acid molecule or a portion thereof, wherein the first nucleic acid molecule or a portion thereof consists of 2-30 nucleotides; and / or b. Determining the sequence of the second nucleic acid molecule or a portion thereof, wherein the second nucleic acid molecule or a portion thereof consists of 2-30 nucleotides.

23. The method of claim 22, wherein the 2-30 nucleotides of the first nucleic acid molecule or portion thereof comprise the first identification sequence, and the 2-30 nucleotides of the second nucleic acid molecule or portion thereof comprise the second identification sequence.

24. A method as claimed in claim 23, wherein the 2-30 nucleotides of the first nucleic acid molecule or a portion thereof further include at least a portion of the sequence of the first cDNA molecule or the first mRNA molecule, and the 2-30 nucleotides of the second nucleic acid molecule or a portion thereof further include at least a portion of the sequence of the second cDNA molecule or the second mRNA molecule.

25. The method of claim 22, wherein the determining comprises: (a) contacting the first concatemer with a polymerase, a plurality of nucleotides, and a primer sequence complementary to a portion of the first concatemer under conditions sufficient to form a binding complex, the binding complex comprising the polymerase, a nucleotide of the plurality of nucleotides that is complementary to and bound to a nucleotide unit of the first concatemer, and the first concatemer hybridized to the primer sequence, wherein the nucleotide comprises a fluorescent label and a removable blocking group at the 3' carbon position of the sugar moiety; (b) incorporating the nucleotide into the 3' end of the primer sequence; as well as (c) identifying the nucleobase of said incorporated nucleotide by imaging said fluorescent label of said incorporated nucleotide.

26. The method of claim 22, wherein the determining comprises: (a) contacting the second concatemer with a polymerase, a plurality of nucleotides, and a primer sequence complementary to a portion of the second concatemer under conditions sufficient to form a binding complex, the binding complex comprising the polymerase, a nucleotide of the plurality of nucleotides that is complementary to and bound to a nucleotide unit of the second concatemer, and the second concatemer hybridized to the primer sequence, wherein the nucleotide comprises a fluorescent label and a removable blocking group at the 3' carbon position of the sugar moiety; (b) incorporating the nucleotide into the 3' end of the primer sequence; as well as (c) identifying the nucleobase of said incorporated nucleotide by imaging said fluorescent label of said incorporated nucleotide.

27. The method of claim 25 or 26, wherein the determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar moiety.

28. The method of any one of claims 25-27, wherein the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

29. The method of claim 28, wherein the plurality of nucleotides consists of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

30. The method of claim 28, wherein the plurality of nucleotides comprises at least two types of nucleotides, wherein the at least two types of nucleotides are selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

31. The method of claim 22, wherein the determining comprises: (a) contacting two of the first concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to portions of the first concatemers under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two first concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein two of the at least two nucleotide portions are each complementary to and bind to a nucleotide of each of the two first concatemers; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; (c) identifying nucleobases of the nucleotides in the two first concatemers that are each complementary to and bound to each of the two of the at least two nucleotide portions of the nucleotide conjugate.

32. The method of claim 22, wherein the determining comprises: (a) contacting two of the second concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to portions of the second concatemers under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two second concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein two of the at least two nucleotide portions are each complementary to and bind to a nucleotide of each of the two second concatemers; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; (c) identifying nucleobases of the nucleotides in the two second concatemers that are each complementary to and bound to each of the two of the at least two nucleotide portions of the nucleotide conjugate.

33. The method of claim 31 or 32, wherein the conditions inhibit incorporation of the at least two nucleotide moieties of the nucleotide conjugate into the two first or second concatemers.

34. The method of any one of claims 31-33, wherein the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each of the nucleotide moieties is attached to one of the plurality of nucleotide arms.

35. The method of any one of claims 31-34, wherein the detectable label comprises a fluorescent label.

36. The method of claim 35, wherein said detecting comprises imaging said fluorescent label.

37. The method of claim 31, wherein the determining further comprises: (a) removing the two polymerases and the nucleotide conjugates from the two first concatemers; (b) contacting each of the two first concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, wherein the binding complex comprises each of the two second polymerases, each of the two first concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides; as well as (c) incorporating each of the two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two first concatemers, wherein an unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

38. The method of claim 32, wherein the determining further comprises: (a) removing the two polymerases and the nucleotide conjugates from the two second concatemers; (b) contacting each of the two second concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions sufficient to form a binding complex, wherein the binding complex comprises each of the two second polymerases, each of the two second concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides; and incorporating each of the two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two second concatemers, wherein an unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

39. The method of claim 22, wherein the determining comprises: (a) contacting two of the first concatemers with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to a first portion of the first concatemer, and a second primer sequence complementary to a second portion of the first concatemer under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, a first portion of the first concatemer hybridized to the first primer, and a second portion of the first concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein two of the at least two nucleotide portions are each complementary to and bind to nucleotides in the first portion and the second portion of the first concatemer; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; (c) identifying nucleobases of the nucleotides in the first and second portions of the first concatemer that are each complementary to and bound to each of the two of the at least two nucleotide portions of the nucleotide conjugate.

40. The method of claim 22, wherein the determining comprises: (a) contacting two of the second concatemers with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to a first portion of the second concatemer, and a second primer sequence complementary to a second portion of the second concatemer under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, a first portion of the second concatemer hybridized to the first primer sequence, and a second portion of the second concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein two of the at least two nucleotide portions are each complementary to and bind to nucleotides in the first portion and the second portion of the second concatemer; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; (c) identifying nucleobases of the nucleotides in the first and second portions of the second concatemer that are each complementary to and bound to each of the two of the at least two nucleotide portions of the nucleotide conjugate.

41. The method of claim 39 or 40, wherein the conditions inhibit incorporation of the at least two nucleotide moieties of the nucleotide conjugate into the two first or second concatemers.

42. The method of any one of claims 39-41, wherein the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each of the nucleotide moieties is attached to one of the plurality of nucleotide arms.

43. The method of any one of claims 39-42, wherein the detectable label comprises a fluorescent label.

44. The method of claim 43, wherein said detecting comprises imaging said fluorescent label.

45. The method of claim 39 or 40, wherein the plurality of nucleotides consists of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

46. ​​A method as claimed in claim 39 or 40, wherein the plurality of nucleotides comprises at least two types of nucleotides, wherein the at least two types of nucleotides are selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

47. The method of claim 39 or 40, wherein the determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3'OH group on the sugar moiety.

48. The method of any one of claims 39-47, wherein the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

49. The method of claim 1, wherein the first target nucleic acid sequence and the second target nucleic acid sequence correspond to two separate portions of the same mRNA or cDNA molecule.

50. The method of claim 1, wherein the first target nucleic acid sequence and the second target nucleic acid sequence correspond to two different mRNA or cDNA molecules.

51. The method of any one of claims 1-50, wherein the repeating comprises repeating at least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or at least 50 times.

52. The method of any one of claims 1-51, wherein the determining comprises in situ detection of the first or second sequencing product nucleic acid molecule within the biological sample by imaging.

53. The method of claim 22, wherein the determining comprises simultaneously detecting in situ the first and second sequencing product nucleic acid molecules within the biological sample by imaging.

54. The method of claim 52 or 53, wherein the imaging comprises fluorescence imaging.

55. The method of any one of claims 1-54, wherein the biological sample comprises an organelle, a cell, a whole cell, a whole group of cells, a tissue, an intact tissue, a tumor, an intact tumor, an organ, an organism, a protozoa, an algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal.

56. The method of any one of claims 1-55, wherein the biological sample comprises a fresh sample, a processed sample, a fresh frozen sample, a sectioned sample, or a formalin-fixed paraffin-embedded (FFPE) sample.

57. The method of claim 56, wherein the biological sample comprises a fresh cell sample, a fresh frozen cell sample, a sectioned cell sample, or a FFPE cell sample.

58. The method of any one of claims 1-57, wherein the at least two target nucleic acid sequences comprise a target DNA sequence.

59. The method of any one of claims 1-57, wherein the at least two target nucleic acid sequences comprise a target RNA sequence.

60. The method of claim 59, wherein the at least two target nucleic acid sequences comprise a first target RNA sequence and a second target RNA sequence.

61. The method of claim 60, wherein the first target RNA sequence comprises a coding RNA, a noncoding RNA, a messenger RNA (mRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a micro RNA (miRNA), a guide RNA (gRNA), a small nuclear RNA (snRNA), a small interfering RNA (siRNA), an antisense RNA, a mature micro RNA, or an immature micro RNA.

62. The method of claim 60, wherein the second target RNA sequence comprises a coding RNA, a non-coding RNA, an mRNA, a tRNA, an rRNA, a miRNA, a gRNA, a snRNA, a siRNA, an antisense RNA, a mature microRNA, or an immature microRNA.

63. The method of claim 58, wherein the at least two target nucleic acid sequences include a first target DNA sequence and a second target DNA sequence.

64. The method of claim 58, wherein the first target DNA sequence comprises complementary DNA (cDNA), genomic DNA (gDNA), non-coding DNA, or coding DNA.

65. The method of claim 58, wherein the second target DNA sequence comprises cDNA, gDNA, non-coding DNA, or coding DNA.

66. The method of claim 58 or 59, wherein the at least two target nucleic acid sequences comprise a target RNA sequence and a target DNA sequence.

67. The method of claim 66, wherein the target RNA sequence comprises a coding RNA, a noncoding RNA, an mRNA, a tRNA, an rRNA, a miRNA, a gRNA, a snRNA, a siRNA, an antisense RNA, a mature microRNA, or an immature microRNA.

68. The method of claim 66, wherein the target DNA sequence comprises cDNA, gDNA, non-coding DNA, or coding DNA.

69. The method of claim 1, wherein the biological sample comprises a human sample, a simian sample, an ape sample, a canine sample, a feline sample, a bovine sample, a equine sample, a murine sample, a porcine sample, a goat sample, a lupin sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a viral sample, a protozoan sample, or a fungal sample.

70. The method of claim 69, wherein the biological sample comprises an organelle, a cell, a whole cell, a whole group of cells, a tissue, an intact tissue, a tumor, a whole tumor, an organ, or an organism.

71. The method of any one of claims 1-70, wherein the biological sample is immobilized on a surface.

72. The method of claim 71, wherein the surface comprises an interior surface of a flow cell.

73. A method for in situ detection of at least two target nucleic acid molecules and at least two target polypeptides in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, wherein the at least two target polypeptides include a first target polypeptide encoded by the first target nucleic acid sequence or its reverse complement sequence and a second target polypeptide encoded by the second target nucleic acid sequence or its reverse complement sequence, the method comprising: (a) providing the biological sample, which comprises: (i) a first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or a reverse complementary sequence of the first target nucleic acid sequence or a portion thereof; (ii) a second nucleic acid molecule comprising the second target nucleic acid sequence or a portion thereof, or a reverse complementary sequence of the second target nucleic acid sequence or a portion thereof; (iii) a third nucleic acid molecule comprising a third target nucleic acid sequence or a portion thereof, or the reverse complement of the third target nucleic acid sequence or a portion thereof, wherein the presence of the third target nucleic acid sequence or its reverse complement identifies the presence of the first target polypeptide in the biological sample; and (iv) a fourth nucleic acid molecule comprising a fourth target nucleic acid sequence or a portion thereof, or the reverse complement of the fourth target nucleic acid sequence or a portion thereof, wherein the presence of the fourth target nucleic acid sequence or its reverse complement identifies the presence of the second target polypeptide in the biological sample; (b) determining in situ the sequence of: (i) the first nucleic acid molecule or a portion thereof in the biological sample to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first nucleic acid molecule or a portion thereof; and (ii) the third nucleic acid molecule or a portion thereof in the biological sample, to generate a third sequencing product nucleic acid molecule that is complementary to and binds to the third nucleic acid molecule or a portion thereof; and (c) identifying in situ the sequence of: (i) the second nucleic acid molecule or a portion thereof in the biological sample to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second nucleic acid molecule or a portion thereof; as well as (ii) the fourth nucleic acid molecule or a portion thereof in the biological sample, to generate a fourth sequencing product nucleic acid molecule that is complementary to and binds to the fourth nucleic acid molecule or a portion thereof, The full sequence of the first target nucleic acid sequence and the full sequence of the second target nucleic acid sequence differ by at least one nucleotide, the full sequence of the first target polypeptide and the full sequence of the second target polypeptide differ by at least one amino acid, and step (b) is performed under conditions that prevent step (c) from being performed.

74. A method for in situ detection of at least two target nucleic acid sequences and at least two target polypeptides in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, wherein the at least two target polypeptides include a first target polypeptide encoded by the first target nucleic acid sequence or its reverse complement sequence and a second target polypeptide encoded by the second target nucleic acid sequence or its reverse complement sequence, the method comprising: (a) providing the biological sample, which comprises: (i) a first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or a reverse complementary sequence of the first target nucleic acid sequence or a portion thereof; (ii) a second nucleic acid molecule comprising the second target nucleic acid sequence or a portion thereof, or a reverse complementary sequence of the second target nucleic acid sequence or a portion thereof; (iii) a third nucleic acid molecule comprising a third target nucleic acid sequence or a portion thereof, or a reverse complement of the third target nucleic acid sequence or a portion thereof, wherein the presence of the third target nucleic acid sequence or its reverse complement identifies the presence of the first target polypeptide in the biological sample; and (iv) a fourth nucleic acid molecule comprising a fourth target nucleic acid sequence or a portion thereof, or a reverse complement of the fourth target nucleic acid sequence or a portion thereof, wherein the presence of the fourth target nucleic acid sequence or its reverse complement identifies the presence of the second target polypeptide in the biological sample; (b) determining in situ the sequence of: (i) the first nucleic acid molecule or a portion thereof in the biological sample to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first nucleic acid molecule or a portion thereof, wherein the first nucleic acid molecule or a portion thereof consists of 2-30 nucleotides; as well as (ii) the third nucleic acid molecule or a portion thereof in the biological sample, to generate a third sequencing product nucleic acid molecule that is complementary to and binds to the third nucleic acid molecule or a portion thereof, wherein the third nucleic acid molecule or a portion thereof consists of 2-30 nucleotides; as well as (c) removing the first sequencing product nucleic acid molecule from the first nucleic acid molecule, and removing the third sequencing product nucleic acid molecule from the third nucleic acid molecule, wherein after the removal, the first nucleic acid molecule and the third nucleic acid molecule are localized in the biological sample; (d) Repeat (b) and (c); (e) identifying in situ the sequence of: (i) the second nucleic acid molecule or a portion thereof in the biological sample to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second nucleic acid molecule or a portion thereof, wherein the second nucleic acid molecule or a portion thereof consists of 2-30 nucleotides; as well as (ii) the fourth nucleic acid molecule or a portion thereof in the biological sample, to generate a fourth sequencing product nucleic acid molecule that is complementary to and binds to the fourth nucleic acid molecule or a portion thereof, wherein the fourth nucleic acid molecule or a portion thereof consists of 2-30 nucleotides; (f) removing the second sequencing product nucleic acid molecule from the second nucleic acid molecule, and removing the fourth sequencing product nucleic acid molecule from the fourth nucleic acid molecule, wherein after the removal, the second nucleic acid molecule and the fourth nucleic acid molecule are located in the biological sample; and (g) repeat (e) and (f), The full sequence of the first target nucleic acid sequence and the full sequence of the second target nucleic acid sequence differ by at least one nucleotide, the full sequence of the first target polypeptide and the full sequence of the second target polypeptide differ by at least one amino acid, and step (b) is performed under conditions that prevent step (e) from being performed.

75. A method for in situ detection of at least two target RNA sequences and at least two target polypeptides in a biological sample, the method comprising: (a) providing the biological sample immobilized on a surface, permeabilized and fixed, wherein the biological sample comprises: (i) a first target RNA sequence among at least two target RNA sequences and a first target polypeptide among the at least two polypeptides, wherein the first target polypeptide is encoded by the first target RNA sequence or its reverse complement sequence; and (ii) a second target RNA sequence among the at least two target RNA sequences and a second target polypeptide among the at least two polypeptides, wherein the second target polypeptide is encoded by the second target RNA sequence or its reverse complement sequence; (b) generating a first target cDNA sequence by reverse transcription of the first target RNA sequence, and generating a second target cDNA sequence by reverse transcription of the second target RNA sequence; (c) cause: (iii) contacting the first target cDNA sequence with a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target cDNA sequence, so that the first oligonucleotide forms a circular structure having a gap between the first end and the second end, wherein the first oligonucleotide comprises a first identification sequence for identifying the first target RNA sequence, a first sequencing primer, and a nucleic acid amplification primer; (iv) contacting the second target cDNA sequence with a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target cDNA sequence, so that the second oligonucleotide forms a circular structure having a gap between the first end and the second end, wherein the second oligonucleotide comprises a second identification sequence for identifying the second target RNA sequence, a second sequencing primer, and a nucleic acid amplification primer, wherein the first sequencing primer and the second sequencing primer have a difference of at least one nucleotide; (v) contacting the first target polypeptide with a first oligonucleotide conjugate comprising a first short nucleic acid and a first binding moiety that specifically binds to the first target polypeptide, wherein the first short nucleic acid comprises a first tag sequence and a second tag sequence, wherein the first oligonucleotide conjugate specifically binds to the first target polypeptide through the first binding moiety to form a first binding complex, wherein the first and second tag sequences identify the first binding moiety; as well as (vi) contacting the second target polypeptide with a second oligonucleotide conjugate comprising a second short nucleic acid and a second binding moiety that specifically binds to the second target polypeptide, wherein the second short nucleic acid comprises a third tag sequence, a fourth tag sequence, wherein the second oligonucleotide conjugate specifically binds to the second target polypeptide through the second binding moiety to form a second binding complex, wherein the third and fourth tag sequences identify the second binding moiety; (d) cause: (vii) contacting the first binding complex with a third oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the third oligonucleotide are complementary to and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, so that the third oligonucleotide forms a ring structure having a gap between the first end and the second end; and (viii) contacting the second binding complex with a fourth oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the fourth oligonucleotide are complementary to and bind to the third tag sequence and the fourth tag sequence, so that the fourth oligonucleotide forms a ring structure having a gap between the first end and the second end; (e) joining the first end and the second end of the first oligonucleotide to produce a first circular oligonucleotide, joining the first end and the second end of the second oligonucleotide to produce a second circular oligonucleotide, joining the first end and the second end of the third oligonucleotide to produce a third circular oligonucleotide and joining the first end and the second end of the fourth oligonucleotide to produce a fourth circular oligonucleotide, wherein the first circular oligonucleotide and the third circular oligonucleotide comprise a first sequencing primer or a reverse complement thereof, wherein the second circular oligonucleotide and the fourth circular oligonucleotide comprise a second sequencing primer or a reverse complement thereof, wherein the sequences of the first and second sequencing primers differ by at least one nucleotide; (f) Amplification: (ix) the first circular oligonucleotide to produce a first concatemer comprising a plurality of the first circular oligonucleotides, wherein the amplification is by rolling circle amplification; (x) the second circular oligonucleotide to produce a second concatemer comprising a plurality of the second circular oligonucleotides, wherein the amplification is by rolling circle amplification; (xi) the third circular oligonucleotide to produce a third concatemer comprising a plurality of the third circular oligonucleotides, wherein the amplification is by rolling circle amplification; and (xii) the fourth circular oligonucleotide to produce a fourth concatemer comprising a plurality of the fourth circular oligonucleotides, wherein the amplification is by rolling circle amplification; (g) determining in situ the sequence of: (i) the first concatemer or a portion thereof, to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first concatemer, wherein the sequence of the first concatemer or a portion thereof consists of 2-30 nucleotides; as well as (ii) the third concatemer or a portion thereof, to generate a third sequencing product nucleic acid molecule that is complementary to and binds to the third concatemer, wherein the sequence of the third concatemer or a portion thereof consists of 2-30 nucleotides, wherein step (g) is performed under conditions that prevent step (j) from being performed; (h) removing the first sequencing product nucleic acid molecule from the first concatemer, and removing the third sequencing product nucleic acid molecule from the third concatemer, wherein after the removal, the first concatemer and the third concatemer are localized in the biological sample; (i) repeat (g) and (h) at least once; (j) determining in situ the sequence of: (xiii) the second concatemer or a portion thereof, to generate a second sequencing product nucleic acid molecule that is complementary to and binds to the second concatemer, wherein the sequence of the second concatemer or a portion thereof consists of 2 to 30 nucleotides; and (xiv) the fourth concatemer or a portion thereof, to generate a fourth sequencing product nucleic acid molecule that is complementary to and binds to the fourth concatemer, wherein the sequence of the fourth concatemer or a portion thereof consists of 2 to 30 nucleotides, wherein step (j) is performed under conditions that prevent step (g) from being performed, wherein the full sequence of the first target RNA sequence and the full sequence of the second target RNA sequence differ by at least one nucleotide, wherein the full sequence of the first target polypeptide and the full sequence of the second target polypeptide differ by at least one amino acid; (k) removing the second sequencing product nucleic acid molecule from the second concatemer, and removing the fourth sequencing product nucleic acid molecule from the fourth concatemer, wherein after the removal, the second concatemer and the fourth concatemer are located in the biological sample; (xv) Repeat (j) and (k) at least once.

76. The method of any one of claims 73-75, wherein the determining comprises imaging the first or third sequencing product nucleic acid molecule, wherein the identifying comprises imaging the second or fourth sequencing product nucleic acid molecule and the fourth sequencing product nucleic acid molecule.

77. The method of any one of claims 73-75, further comprising simultaneously imaging the first sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule to analyze the spatial distribution of the first and second sequencing product nucleic acid molecules within the biological sample.

78. The method of any one of claims 73-75, wherein the biological sample comprises an organelle, a cell, a whole cell, a whole group of cells, a tissue, an intact tissue, a tumor, a whole tumor, an organ, an organism, a protozoa, an algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal.

79. The method of any one of claims 73-78, wherein the biological sample comprises a fresh sample, a processed sample, a fresh frozen sample, a sectioned sample, or a formalin-fixed paraffin-embedded (FFPE) sample.

80. The method of any one of claims 73-79, wherein the first target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA, or immature microRNA, and / or wherein the first target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA, or immature microRNA.

81. The method of any one of claims 73-79, wherein the second target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA, or immature microRNA, and / or wherein the second target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA, or immature microRNA.

82. The method of any one of claims 73-81, wherein the biological sample comprises a human sample, a simian sample, an ape sample, a canine sample, a feline sample, a bovine sample, a equine sample, a murine sample, a porcine sample, a goat sample, a lupin sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a viral sample, a protozoan sample, or a fungal sample.

83. The method of claim 75, wherein the first, second, third or fourth concatemer further comprises a compacted oligonucleotide, wherein: (a) the first segment of the compacted oligonucleotide is complementary to and binds to the first portion of the first, second, third or fourth concatemer; and (b) the second segment of the compacted oligonucleotide is complementary to and binds to a second portion of the first, second, third or fourth concatemer, thereby causing a size reduction or a shape change of the second concatemer.

84. The method of claim 75, wherein the first sequencing product nucleic acid molecule comprises a first identification sequence that identifies the first target RNA sequence, and the second sequencing product nucleic acid molecule comprises a second identification sequence that identifies the second target RNA sequence.

85. The method of claim 75, wherein the first sequencing product nucleic acid molecule comprises a first identification sequence that identifies the first target RNA sequence and a portion of the first target RNA sequence, and wherein the second sequencing product nucleic acid molecule comprises a second identification sequence that identifies the second target RNA sequence and a portion of the second target RNA sequence.

86. The method of claim 75, wherein the determining comprises: (a) contacting the first, second, third or fourth concatemer with a polymerase, a plurality of nucleotides, and a primer sequence complementary to a portion of the first, second, third or fourth concatemer under conditions sufficient to form a binding complex, the binding complex comprising the polymerase, nucleotides of the plurality of nucleotides that are complementary to and bind to nucleotide units in the first, second, third or fourth concatemer, and the first, second, third or fourth concatemer hybridized to the primer sequence, wherein the nucleotides comprise a fluorescent label and a removable blocking group at the 3' carbon position of the sugar moiety; (b) incorporating the nucleotide into the 3' end of the primer sequence; as well as (c) identifying the nucleobase of said incorporated nucleotide by imaging said fluorescent label of said incorporated nucleotide.

87. The method of claim 86, wherein the determining further comprises contacting the nucleotide with a reagent to remove a blocking group from the nucleotide and generate a 3' OH group on the sugar moiety.

88. The method of claim 87, wherein the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

89. The method of claim 86, wherein the plurality of nucleotides comprises a type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

90. The method of claim 86, wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from dATP, dGTP, dCTP, dTTP and / or dUTP.

91. The method of claim 75, wherein the determining comprises: (a) contacting two of the first, second, third or fourth concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to portions of the first, second, third or fourth concatemers under conditions sufficient to form a multivalent binding complex, wherein the multivalent binding complex comprises each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two of the first, second, third or fourth concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein two of the at least two nucleotide portions are each complementary to and bind to a nucleotide of each of the two first concatemers; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; (c) identifying nucleobases of the nucleotides in the two first concatemers that are each complementary to and bound to each of the two of the at least two nucleotide portions of the nucleotide conjugate.

92. The method of claim 91, wherein the determining further comprises: (a) removing the two polymerases and the nucleotide conjugates from the two of the first, second, third or fourth concatemers; (b) contacting each of the two first, second, third or fourth concatemers hybridized to each of the two primer sequences with two second polymerases and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex, wherein the binding complex comprises each of the two second polymerases, each of the two first, second, third or fourth concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides, and incorporating each of the two of the plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and binds to a nucleotide of each of the two first, second, third or fourth concatemers, wherein an unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group at the 3' carbon of the sugar moiety.

93. The method of claim 72, wherein the determining comprises: (a) subjecting the two first, contacting a second, third, or fourth concatemer with two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to a first portion of the first, second, third, or fourth concatemer, and a second primer sequence complementary to a second portion of the first, second, third, or fourth concatemer, the multivalent binding complex comprising each of the two polymerases, a nucleotide conjugate from the plurality of nucleotide conjugates, a first portion of the first, second, third, or fourth concatemer hybridized to the first primer sequence, and a second portion of the first, second, third, or fourth concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, wherein two of the at least two nucleotide portions are each complementary to and bind to nucleotides in the first portion and the second portion of the first, second, third, or fourth concatemer; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; (c) identifying nucleobases of the nucleotides in the first and second parts of the first, second, third or fourth concatemer that are each complementary to and bound to each of the two of the at least two nucleotide parts in the nucleotide conjugate.

94. The method of claim 93, wherein the conditions inhibit incorporation of the at least two nucleotide moieties of the nucleotide conjugate into the two of the first, second, third or fourth concatemers.

95. The method of claim 93 or 94, wherein the nucleotide conjugate comprises a core coupled to a plurality of nucleotide arms, wherein each of the nucleotide moieties is attached to one of the plurality of nucleotide arms.

96. The method of any one of claims 93-95, wherein the detectable label comprises a fluorescent label.

97. The method of claim 93, wherein the detecting comprises imaging the fluorescent label.

98. A method as claimed in claim 93, wherein the plurality of nucleotides consists of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP, and wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

99. A method as claimed in claim 93, wherein the plurality of nucleotides comprises at least two types of nucleotides, wherein the at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

100. The method of claim 93, wherein the determining further comprises contacting the nucleotide with a reagent to remove the blocking group from the nucleotide and generate a 3' OH group on the sugar moiety.

101. The method of claim 100, wherein the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

102. A method as claimed in claim 86 or 87, wherein the plurality of nucleotides consists of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP, and wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

103. A method as claimed in claim 86 or 87, wherein the plurality of nucleotides comprises at least two types of nucleotides, wherein the at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, and wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

104. The method of claim 75, wherein the first target RNA sequence comprises a coding RNA, a noncoding RNA, a messenger RNA (mRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a micro RNA (miRNA), a guide RNA (gRNA), a small nuclear RNA (snRNA), a small interfering RNA (siRNA), an antisense RNA, a mature micro RNA, or an immature micro RNA.

105. The method of claim 75, wherein the second target RNA sequence comprises a coding RNA, a non-coding RNA, an mRNA, a tRNA, an rRNA, a miRNA, a gRNA, a snRNA, a siRNA, an antisense RNA, a mature microRNA, or an immature microRNA.

106. The method of any one of claims 73-75, wherein the biological sample comprises a human sample, a simian sample, an ape sample, a dog sample, a cat sample, a cow sample, a horse sample, a mouse sample, a pig sample, a goat sample, a lupin sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a viral sample, a protozoan sample, or a fungal sample.

107. The method of claim 106, wherein the biological sample comprises an organelle, a cell, a whole cell, a whole group of cells, a tissue, an intact tissue, a tumor, a whole tumor, an organ, or an organism.

108. The method of any one of claims 73-107, wherein the biological sample is immobilized on a surface.

109. The method of claim 108, wherein the surface comprises an interior surface of a flow cell.

110. A system for in situ detection of at least two target nucleic acid sequences in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, the system comprising: (a) a biological sample comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises the first target nucleic acid sequence or its reverse complement sequence or a portion thereof, and wherein the second nucleic acid molecule comprises the second target nucleic acid sequence or its reverse complement sequence or a portion thereof; (b) a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target nucleic acid sequence or its reverse complement sequence or a portion thereof, so that the first oligonucleotide forms a first circular oligonucleotide in the biological sample, wherein the first circular oligonucleotide comprises a gap between the first end and the second end; and (c) a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target nucleic acid sequence or its reverse complement sequence or a portion thereof, so that the second oligonucleotide forms a second circular oligonucleotide in the biological sample, wherein the second circular oligonucleotide comprises a gap between the first end and the second end.

111. The system of claim 110, wherein the first circular oligonucleotide further comprises a first nuclease configured to join the first end and the second end of the first oligonucleotide, and the second circular oligonucleotide further comprises a second nuclease configured to join the first end and the second end of the second oligonucleotide.

112. The system of claim 111, wherein the first and second nucleases comprise nucleic acid ligases, nucleic acid ligases, nucleic acid polymerases, nucleic acid polymerases, or a combination thereof.

113. The system of any one of claims 111-112, further comprising a first amplicon of the first circular oligonucleotide and a second amplicon of the second circular oligonucleotide.

114. The system of claim 113, wherein the first amplicon comprises a first concatemer and / or the second amplicon comprises a second concatemer.

115. The system of claim 114, wherein: (a) the first concatemer comprises at least two repeats of a first unit nucleic acid sequence, wherein the first unit nucleic acid sequence comprises the first target nucleic acid sequence or a portion thereof, or a reverse complementary sequence thereof or a portion thereof; and / or (b) The second concatemer comprises at least two repeats of a second unit nucleic acid sequence, wherein the second unit nucleic acid sequence comprises the second target nucleic acid sequence or a portion thereof, or a reverse complementary sequence thereof or a portion thereof.

116. A system as described in claim 115, wherein the first concatemer further comprises the first identification sequence or its sequencing primer or reverse complementary sequence for identifying the first target nucleic acid sequence, and wherein the second concatemer further comprises the second identification sequence or its sequencing primer or reverse complementary sequence for identifying the second target nucleic acid sequence.

117. The system of claim 116, wherein the first concatemer further comprises a first compacted oligonucleotide, wherein: (d) the first segment of the first compacted oligonucleotide is complementary to and binds to the first portion of the first concatemer; and (e) The second segment of the first compacted oligonucleotide is complementary to and binds to the second portion of the first concatemer, thereby causing the first concatemer to be reduced in size or changed in shape.

118. The system of claim 116 or 117, wherein the second concatemer further comprises a second compacted oligonucleotide, wherein: (f) the first segment of the second compacted oligonucleotide is complementary to and binds to the first portion of the second concatemer; and (g) the second segment of the second compacted oligonucleotide is complementary to and binds to the second portion of the second concatemer, thereby causing the second concatemer to be reduced in size or changed in shape.

119. The system of any one of claims 114-118, further comprising a polymerase, a plurality of nucleotides, and a primer sequence that is complementary to at least a portion of the first concatemer or the second concatemer under conditions sufficient to form a binding complex.

120. The system of claim 119, further comprising a reagent configured to remove a blocking group from a nucleotide in the plurality of nucleotides and generate a 3'OH group on a sugar moiety of the nucleotide.

121. A system as described in claim 120, wherein the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group.

122. The system of any one of claims 119-121, wherein the plurality of nucleotides comprises a fluorescent label.

123. The system of any one of claims 119-122, wherein the plurality of nucleotides consists of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

124. A system as described in any of claims 119-122, wherein the plurality of nucleotides comprises at least two types of nucleotides, wherein the at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, and wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

125. The system of any one of claims 110-124, further comprising a plurality of nucleotide conjugates, wherein the nucleotide conjugates in the plurality of nucleotide conjugates are configured to form a multivalent binding complex, the multivalent binding complex comprising two or more of the polymerases, the nucleotide conjugates and the at least two target nucleic acid sequences, wherein the nucleotide conjugate comprises a label and at least two nucleotide portions, each of the at least two nucleotide portions being complementary to and binding to a nucleotide of each of the at least two target nucleic acid sequences.

126. The system of any one of claims 110-125, further comprising a first oligonucleotide conjugate, the first oligonucleotide conjugate comprising a first short nucleic acid and a first binding portion that specifically binds to a first target polypeptide, wherein the first short nucleic acid comprises a first tag sequence and a second tag sequence, wherein the first oligonucleotide conjugate specifically binds to the first target polypeptide in the biological sample through the first binding portion to form a first binding complex, wherein the first and second tag sequences identify the first binding portion in a nucleic acid sequence reaction.

127. The system of any one of claims 110-126, further comprising a second oligonucleotide conjugate, the second oligonucleotide conjugate comprising a second short nucleic acid and a second binding portion that specifically binds to a second target polypeptide, wherein the second short nucleic acid comprises a third tag sequence and a fourth tag sequence, wherein the second oligonucleotide conjugate specifically binds to the second target polypeptide in the biological sample through the second binding portion to form a second binding complex, wherein the third and fourth tag sequences identify the second binding portion in a nucleic acid sequence reaction.

128. The system of claim 127, wherein the first or second binding moiety is an antibody or an antigen-binding fragment thereof.

129. The system of any one of claims 126-128, further comprising a third oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the third oligonucleotide are complementary to and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, so that the third oligonucleotide forms a circular structure having a gap between the first end and the second end.

130. The system of any one of claims 126-129, further comprising a fourth oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the fourth oligonucleotide are complementary to and bind to the third tag sequence and the fourth tag sequence, so that the fourth oligonucleotide forms a circular structure having a gap between the first end and the second end.

131. The system of any one of claims 126-130, further comprising a third circular oligonucleotide produced by joining the first end and the second end of the third oligonucleotide and a fourth circular oligonucleotide produced by joining the first end and the second end of the fourth oligonucleotide, wherein the first circular oligonucleotide and the third circular oligonucleotide comprise a first sequencing primer or its reverse complement, wherein the second circular oligonucleotide and the fourth circular oligonucleotide comprise a second sequencing primer or its reverse complement, wherein the sequences of the first and second sequencing primers differ by at least one nucleotide, and wherein the joining is performed by the first or second nuclease.

132. The system of any one of claims 126-131, further comprising a third amplicon of the third circular oligonucleotide and a fourth amplicon of the fourth circular oligonucleotide.

133. The system of claim 132, wherein the third amplicon comprises a third concatemer and / or the fourth amplicon comprises a fourth concatemer.

134. The system of claim 133, wherein: (h) the third concatemer comprises at least two repeats of a third unit nucleic acid sequence, wherein the third unit nucleic acid sequence comprises the third circular oligonucleotide or a portion thereof, or a reverse complementary sequence thereof or a portion thereof; and / or (i) The fourth concatemer comprises at least two repeats of the second unit nucleic acid sequence, and the second unit nucleic acid sequence comprises the fourth circular oligonucleotide or a portion thereof, or a reverse complementary sequence or a portion thereof.

135. The system of claim 134, wherein the third concatemer further comprises the first or second tag sequence or its reverse complement that identifies the first binding moiety, wherein the fourth concatemer further comprises the third or fourth tag sequence or its reverse complement that identifies the second binding moiety.

136. The system of claim 135, wherein the third concatemer further comprises a third compacted oligonucleotide, wherein: (j) the first segment of the third compacted oligonucleotide is complementary to and binds to the first portion of the third concatemer; and (k) The second segment of the third compacted oligonucleotide is complementary to and binds to the second portion of the third concatemer, thereby causing the third concatemer to be reduced in size or changed in shape.

137. The system of claim 136, wherein the fourth concatemer further comprises a fourth compacted oligonucleotide, wherein: (1) the first segment of the fourth compacted oligonucleotide is complementary to and binds to the first portion of the fourth concatemer; and (m) the second segment of the fourth compacted oligonucleotide is complementary to and binds to the second portion of the fourth concatemer, thereby causing the fourth concatemer to be reduced in size or changed in shape.

138. The system of any one of claims 126-137, further comprising a second polymerase, a second plurality of nucleotides, and a second primer sequence that is complementary to at least a portion of the third concatemer or the fourth concatemer under conditions sufficient to form a binding complex, wherein the binding complex comprises the third or fourth concatemer hybridized to the second primer sequence, the second polymerase, and a second nucleotide of the second plurality of nucleotides that is complementary to and bound to nucleotides of the third or fourth concatemer.

139. The system of claim 138, further comprising a second reagent configured to remove a second blocking group from a second nucleotide in the second plurality of nucleotides and generate a 3'OH group on the sugar moiety of the second nucleotide.

140. A system as described in claim 139, wherein the second blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group.

141. The system of any one of claims 138-140, wherein the second plurality of nucleotides comprises a second fluorescent label.

142. A system as described in claim 141, wherein the second plurality of nucleotides consists of at least two nucleotides of the same type, wherein the nucleotides of the same type are selected from the group including dATP, dGTP, dCTP, dTTP and dUTP, and wherein the wavelength of light emitted by the second fluorescent marker of one type of nucleotide in the group is different from the wavelength of light emitted by the second fluorescent marker of another type of nucleotide in the group.

143. A system as described in claim 141 or 142, wherein the second plurality of nucleotides includes at least two types of nucleotides, wherein the at least two types of nucleotides are selected from the group including dATP, dGTP, dCTP, dTTP and dUTP, and wherein the wavelength of light emitted by the second fluorescent marker of one type of nucleotide in the group is different from the wavelength of light emitted by the second fluorescent marker of another type of nucleotide in the group.

144. The system of any one of claims 126-143, further comprising a second plurality of nucleotide conjugates, wherein a second nucleotide conjugate in the second plurality of nucleotide conjugates is configured to form a multivalent binding complex, the multivalent binding complex comprising two or more of the second polymerases, the second nucleotide conjugate in the second plurality of nucleotide conjugates, and the at least two first short nucleic acids or the short nucleic acids or portions thereof, wherein the second nucleotide conjugate comprises a tag and at least two nucleotide portions, each of the at least two nucleotide portions being complementary to and binding to a nucleotide of each of the at least two first short nucleic acids or the short nucleic acids or portions thereof.

145. The system of any one of claims 110-144, further comprising a solid surface comprising the biological sample immobilized to the solid surface.

146. The system of claim 145, wherein the biological sample is permeabilized.

147. The system of claims 145 and 146, wherein the solid surface further comprises a hydrophilic polymer coating coupled thereto.

148. The system of claim 147, wherein the hydrophilic polymer coating has a water contact angle of less than 50 degrees.

149. The system of any one of claims 110-148, further comprising an optical imaging module configured to image the biological sample coupled to the solid surface to detect in situ the at least two target nucleic acid sequences and / or the at least two target polypeptides in the biological sample.

150. The system of any one of claims 126-149, wherein the first target polypeptide is encoded by the first target nucleic acid molecule or its reverse complement, and the second target polypeptide is encoded by the second target nucleic acid molecule or its reverse complement.

151. A computer-implemented system comprising a computing device including at least one processor, an operating system configured to execute executable instructions, a memory, and a computer program comprising instructions executable by the computing device, wherein the instructions include a method as described in any of claims 1-109.

152. A non-transitory computer-readable storage medium encoded with a computer program comprising instructions executable by one or more processors, wherein the instructions comprise the method of any one of claims 1-109.

153. A kit for in situ detection of at least two target nucleic acid sequences in a biological sample, wherein the at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, the kit comprising: (a) a first oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target nucleic acid sequence or its reverse complementary sequence or a portion thereof, so that the first oligonucleotide forms a first circular oligonucleotide in the biological sample, wherein the first circular oligonucleotide comprises a gap between the first end and the second end, wherein the first oligonucleotide comprises a first identification sequence for identifying the first target nucleic acid sequence, a first sequencing primer or its reverse complementary sequence, a compaction oligonucleotide or its reverse complementary sequence, or a primer for nucleic acid amplification or its reverse complementary sequence, (b) a second oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target nucleic acid sequence or its reverse complement or a portion thereof, so that the second oligonucleotide forms a second circular oligonucleotide within the biological sample, wherein the second circular oligonucleotide comprises a gap between the first end and the second end, wherein the second oligonucleotide comprises a second identification sequence for identifying the second target nucleic acid sequence, a second sequencing primer or its reverse complement, a compaction oligonucleotide or its reverse complement, or a primer for nucleic acid amplification or its reverse complement; (c) a first nuclease configured to join the first end and the second end of the first oligonucleotide and generate a first circular oligonucleotide, or to join the first end and the second end of the second oligonucleotide and generate a second circular oligonucleotide; (d) a second nuclease configured to amplify the first circular oligonucleotide to produce a first concatemer or amplify the second circular oligonucleotide to produce a second concatemer; (e) a third nuclease, a sequencing primer complementary to a portion of the first concatemer or a portion of the second concatemer, a plurality of nucleotides, or a plurality of nucleotide conjugates, wherein a nucleotide conjugate in the plurality of nucleotide conjugates comprises a core and two nucleotide portions attached to the core, (i) wherein the third nucleic acid enzyme, the nucleotides in the plurality of nucleotides and the sequencing primer are configured to form a binding complex, the binding complex comprising the third nucleic acid enzyme, the nucleotides in the plurality of nucleotides that are complementary to and bound to the nucleotide units of the first or the second concatemer, and the first concatemer or the second concatemer hybridized with the sequencing primer; or (ii) wherein at least two of the third nucleic acid enzymes, the nucleotide conjugate, the sequencing primer, and the first or the second concatemer are configured to form a multivalent binding complex, the multivalent binding complex comprising the third nucleic acid enzyme, the nucleotide conjugate, and the first or the second concatemer hybridized to the sequencing primer, wherein the two nucleotide moieties are complementary to and bind to two nucleotide units of the first or the second concatemer, and each of the two first concatemers or the second concatemers hybridizes to each of the two sequencing primers; (f) a fourth nucleic acid enzyme configured as follows: (i) adding a nucleotide from the plurality of nucleotides to the end of the sequencing primer hybridized to the first concatemer and generating a first sequencing product nucleic acid molecule, wherein the nucleotide is unlabeled and comprises a blocking group; or (ii) adding a nucleotide from the plurality of nucleotides to the end of the sequencing primer hybridized to the second concatemer and generating a second sequencing product nucleic acid molecule, wherein the nucleotide is unlabeled and comprises a blocking group; and (g) a dissociation reagent configured to remove the first sequencing product nucleic acid molecule from the first concatemer or to remove the second sequencing product nucleic acid molecule from the second concatemer.

154. A kit as described in claim 153, which is further configured for in situ detection of at least two target RNA sequences and at least two target polypeptides including a first target polypeptide and a second target polypeptide in a biological sample, the kit comprising: the first target polypeptide encoded by the first target nucleic acid molecule or its reverse complementary sequence, wherein the second target polypeptide is encoded by the second target nucleic acid molecule or its reverse complementary sequence, wherein the full sequence of the first target RNA sequence differs from the full sequence of the second target RNA sequence by at least one nucleotide, and wherein the full sequence of the first target polypeptide differs from the full sequence of the second target polypeptide by at least one amino acid.

155. The kit of claim 153 or 154, wherein the first, second, third or fourth nucleic acid enzyme comprises a nucleic acid ligase, a nucleic acid ligase, a nucleic acid polymerase, a nucleic acid polymerase or a combination thereof.

156. The kit of any one of claims 153-155, wherein the nucleotides in the plurality of nucleotides comprise a fluorescent label.

157. The kit of any one of claims 153-156, wherein the nucleotide in the plurality of nucleotides comprises a removable blocking group at the 3' carbon position of the sugar moiety.

158. A kit as described in any of claims 153-157, wherein the plurality of nucleotides consists of at least two nucleotides of the same type comprising a fluorescent label, wherein the nucleotides of the same type are selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

159. A kit as described in any one of claims 153-158, wherein the plurality of nucleotides comprises at least two types of nucleotides, wherein a type of the at least two types of nucleotides comprises a fluorescent label, wherein the at least two types of nucleotides are selected from the group comprising dATP, dGTP, dCTP, dTTP and dUTP, wherein the wavelength of light emitted by the fluorescent label of one type of nucleotide in the group is different from the wavelength of light emitted by the fluorescent label of another type of nucleotide in the group.

160. The kit of any one of claims 153-159, wherein the nucleotide conjugate comprises a detectable label.

161. The kit of any one of claims 153-160, wherein the nucleotide conjugate comprises a fluorescent label.

162. The kit of any one of claims 153-161, wherein the biological sample comprises a human sample, a simian sample, an ape sample, a dog sample, a cat sample, a cow sample, a horse sample, a mouse sample, a pig sample, a goat sample, a lupin sample, a frog sample, a fish sample, a plant sample, an insect sample, a bacterial sample, an algae sample, a viral sample, a protozoan sample, or a fungal sample.

163. The kit of any one of claims 153-162, wherein the biological sample comprises an organelle, a cell, a whole cell, a whole group of cells, a tissue, an intact tissue, a tumor, a whole tumor, an organ, or an organism.

164. The kit of any one of claims 153-163, wherein the biological sample comprises an organelle, a cell, a whole cell, a whole cell group, a tissue, a complete tissue, a tumor, a complete tumor, an organ, an organism, a protozoa, an algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal.

165. The kit of any one of claims 153-164, wherein the biological sample comprises a fresh sample, a processed sample, a fresh frozen sample, a sectioned sample, or a formalin-fixed paraffin-embedded (FFPE) sample.

166. A kit as described in any one of claims 153-165, wherein the first target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA, and / or wherein the first target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA.

167. A kit as described in any of claims 153-161, wherein the second target nucleic acid sequence comprises DNA, cDNA, RNA, coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA, and / or wherein the second target RNA sequence comprises coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA or immature microRNA.

168. A kit as described in any one of claims 157-167, wherein the blocking group comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group or a silyl group.

169. The kit of any one of claims 153-168, further comprising: (a) a first oligonucleotide conjugate comprising a first short nucleic acid and a first binding moiety that specifically binds to the first target polypeptide, wherein the first short nucleic acid comprises a first tag sequence and a second tag sequence, wherein the first oligonucleotide conjugate specifically binds to the first target polypeptide through the first binding moiety to form a first binding complex, wherein the first and second tag sequences identify the first binding moiety; or (b) a second oligonucleotide conjugate, comprising a second short nucleic acid and a second binding portion that specifically binds to the second target polypeptide, wherein the second short nucleic acid includes a third tag sequence and a fourth tag sequence, wherein the second oligonucleotide conjugate specifically binds to the second target polypeptide through the second binding portion to form a second binding complex, wherein the third and fourth tag sequences identify the second binding portion.

170. The kit of claim 169, further comprising: (a) a third oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the third oligonucleotide are complementary to and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, so that the third oligonucleotide forms a ring structure having a gap between the first end and the second end; or (b) a fourth oligonucleotide comprising a first end and a second end, wherein the first end and the second end of the fourth oligonucleotide are complementary to and bind to the third tag sequence and the fourth tag sequence, so that the fourth oligonucleotide forms a ring structure with a gap between the first end and the second end.

171. The kit of claim 170, wherein the third oligonucleotide comprises a third identification sequence that identifies the first binding moiety, and wherein the fourth oligonucleotide comprises a fourth identification sequence that identifies the second binding moiety.

172. A kit as described in claim 171, wherein the first oligonucleotide and the third oligonucleotide comprise the same first sequencing primer or its reverse complement sequence, and wherein the second oligonucleotide and the fourth oligonucleotide comprise the same second sequencing primer or its reverse complement sequence.

173. The kit of claim 171, further comprising a reagent that reacts with the reactive group at the 3' carbon of the sugar moiety in the nucleotide portion of the nucleotide conjugate.

174. The kit of claim 173, further comprising reagents for the nucleotide binding reaction.

175. The kit of claim 174, wherein the reagent comprises a cation.

176. The kit of any one of claims 153-175, further comprising a reverse transcriptase, a primer for reverse transcription, a sequencing primer, a reagent configured to permeabilize the biological sample, a reagent configured to immobilize the biological sample, unblocked nucleotides, blocked nucleotides, reagents for the nucleotide incorporation reaction, a solution comprising a cation, one or more unlabeled nucleotides, one or more buffers for reverse transcription, one or more buffers for nucleic acid binding, one or more buffers for nucleic acid amplification, or one or more buffers for nucleic acid dissociation.

177. The kit of any one of claims 153-176, further comprising instructions for using the kit to detect the at least two target nucleic acid sequences in the biological sample in situ.

178. The kit of any one of claims 153-176, further comprising instructions for using the kit to detect the at least two target nucleic acid sequences and the at least two target polypeptides in the biological sample in situ.

179. The kit of claim 177 or 178, wherein the instructions comprise sequencing by synthesis.

180. The kit of claim 177 or 178, wherein the instructions comprise performing sequencing by binding reaction wherein in situ detection is performed at a different time than the nucleotide incorporation step.

181. The kit of any one of claims 153-180, further comprising instructions for using the kit using the method provided in any one of claims 1-109.

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