Iterative short-read sequencing within cell samples

Abstract

The present disclosure provides a method for in situ multiplex and multi-omics detection and identification using coded padlock probes. The method includes the simultaneous use of RNA-specific and polypeptide-specific padlock probes to detect both RNA and polypeptides in a cell sample. Both types of probes include a barcode that uniquely identifies the RNA or polypeptide that the padlock probe detects. Both types of probes further include a batch-specific sequencing primer binding site to allow sequencing of a desired subset of concatemeric template molecules. The use of batch-specific sequencing primers reduces overcrowded signals and images to generate a strong and resolvable optical image. Performing multiple rounds of sequencing on the same cell sample with different batch-specific sequencing primers allows multiplex and multi-omics sequencing to reveal many target RNAs and their encoded polypeptides.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 332,690, filed April 20, 2022, and U.S. Provisional Patent Application No. 63 / 334,023, filed April 22, 2022, each of which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 52933-752_601_SL.xml, 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.

[0003] The present disclosure provides compositions, devices, and methods for performing iterative short-read sequencing within a cellular sample. In some embodiments, iterative short-read sequencing can be used to discover the RNA content of a cellular sample. Summary of the Invention

[0004] 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 containing (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) (i) determining in situ the sequence of 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) sequencing 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. (c) removing a first sequencing product nucleic acid molecule from the first nucleic acid molecule and a second sequencing product nucleic acid molecule from the second nucleic acid molecule, wherein the first nucleic acid molecule and the second nucleic acid molecule are located within the biological sample after removal; and (d) repeating steps (b) and (c) to detect at least two target nucleic acid sequences in situ in the biological sample. In some embodiments, the biological sample is fixed and permeabilized.In some embodiments, the method further comprises (a) generating a first complementary DNA (cDNA) molecule in situ by reverse transcription of a first messenger RNA (mRNA) molecule within the biological sample, and / or (b) generating a second cDNA molecule in situ by reverse transcription of a second mRNA molecule within the biological sample, wherein the first cDNA molecule or first mRNA molecule comprises a first target nucleic acid sequence or the reverse complement of the first target nucleic acid sequence, and the second cDNA molecule or second mRNA molecule comprises a second target nucleic acid sequence or the reverse complement of the second target nucleic acid sequence. In some embodiments, the method further comprises: (a) contacting in situ the first target nucleic acid sequence or its reverse complement with a first oligonucleotide comprising a first terminal portion and a second terminal portion; and / or (b) contacting in situ the second target nucleic acid sequence or its reverse complement with a second oligonucleotide comprising a first terminal portion and a second terminal portion, wherein the first terminal portion and the second terminal portion of the first oligonucleotide are complementary and bind to two adjacent segments of the first target nucleic acid sequence or its reverse complement, such that the first oligonucleotide forms a circular structure having a gap between its first terminal portion and its second terminal portion; and the first terminal portion and the second terminal portion of the second oligonucleotide are complementary and bind to two adjacent segments of the second target nucleic acid sequence or its reverse complement, such that the second oligonucleotide forms a circular structure having a gap between its first terminal portion and its second terminal portion. In some embodiments, (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 in the first oligonucleotide or the second oligonucleotide has a size of 1 nucleotide. In some embodiments, the gap in the first oligonucleotide or the second oligonucleotide has a size of at least 2 nucleotides.In some embodiments, the first oligonucleotide further comprises a first identifying sequence that identifies the first target nucleic acid sequence, and the second oligonucleotide further comprises a second identifying sequence that identifies the second target nucleic acid sequence. In some embodiments, the first oligonucleotide and the second oligonucleotide further comprise a nucleic acid sequence that is complementary to the nucleic acid sequence of a sequencing primer. In some embodiments, the first oligonucleotide and the second oligonucleotide further comprise a nucleic acid sequence that is complementary 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) that generates concatemers, and the concatemers include at least two repeats of one target nucleic acid sequence or a portion thereof, or its reverse complement, of the at least two target nucleic acid sequences. In some embodiments, the first oligonucleotide and the second oligonucleotide further comprise a reverse complement sequence of a compaction oligonucleotide, wherein (a) a first segment of the compaction oligonucleotide is complementary to and binds to a first portion of the concatemer, and (b) a second segment of the compaction oligonucleotide is complementary to and binds to a second portion of the concatemer, resulting in a reduction in size or a change in shape of the concatemer.

[0005] In some embodiments, the method further comprises (a) in situ ligating a first terminal portion and a second terminal portion of the first oligonucleotide to generate a first circular oligonucleotide within the biological sample, and (b) in situ ligating a first terminal portion and a second terminal portion of the second oligonucleotide to generate a second circular oligonucleotide within the biological sample. In some embodiments, (a) ligating the first terminal portion and the second terminal portion of the first oligonucleotide comprises ligating the first terminal portion and the second terminal portion of the first oligonucleotide via a first nucleic acid enzyme, and (b) ligating the first terminal portion and the second terminal portion of the second oligonucleotide comprises ligating the first terminal portion and the second terminal portion of the second oligonucleotide via 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, 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 polymerization enzyme, or a combination thereof. In some embodiments, the method further comprises (a) in situ amplifying the first circular oligonucleotide to generate the first nucleic acid molecule, and / or (b) in situ amplifying the second circular oligonucleotide to generate the second nucleic acid molecule. In some embodiments, the amplifying 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, (a) a first concatemer comprises at least two repeats of a first nucleic acid unit comprising a first target nucleic acid sequence or a portion thereof, or a reverse complement of the first target nucleic acid sequence or a portion thereof, and / or (b) a second concatemer comprises at least two repeats of a second nucleic acid unit comprising a second target nucleic acid sequence or a portion thereof, or a reverse complement of the second target nucleic acid sequence or a portion thereof.In some embodiments, the first concatemer further comprises a first identifying sequence that identifies the first target nucleic acid sequence, or a sequencing primer or reverse complement thereof, and the second concatemer further comprises a second identifying 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 compaction oligonucleotide, wherein (a) a first segment of the first compaction oligonucleotide is complementary to and binds to a first portion of the first concatemer, and (b) a second segment of the first compaction oligonucleotide is complementary to and binds to a 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 compaction oligonucleotide, wherein (a) a first segment of the second compaction oligonucleotide is complementary to and binds to a first portion of the second concatemer, and (b) a second segment of the second compaction oligonucleotide is complementary to and binds to a 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 determining step comprises (a) determining the sequence of a first nucleic acid molecule or a portion thereof, wherein the first nucleic acid molecule or portion thereof consists of 2 to 30 nucleotides, and (b) determining the sequence of a second nucleic acid molecule or a portion thereof, wherein the second nucleic acid molecule or portion thereof consists of 2 to 30 nucleotides. In some embodiments, 2 to 30 nucleotides of a first nucleic acid molecule or portion thereof comprise a first identifying sequence, and 2 to 30 nucleotides of a second nucleic acid molecule or portion thereof comprise a second identifying sequence.In some embodiments, 2 to 30 nucleotides of the first nucleic acid molecule or portion thereof further comprise at least a portion of the sequence of a first cDNA molecule or a first mRNA molecule, and 2 to 30 nucleotides of the second nucleic acid molecule or 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 step comprises: (a) contacting the first concatemer with a polymerizing enzyme, a plurality of nucleotides, and a primer sequence complementary to a portion of the first concatemer under conditions sufficient to form a binding complex comprising the polymerizing enzyme, one nucleotide of the plurality of nucleotides that is complementary to and binds 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 a nucleotide at 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 step comprises: (a) contacting the second concatemer with a polymerizing enzyme, a plurality of nucleotides, and a primer sequence complementary to a portion of the second concatemer under conditions sufficient to form a binding complex comprising the polymerizing enzyme, one nucleotide of the plurality of nucleotides that is complementary to and binds 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 a nucleotide at 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.

[0006] In some embodiments, the determining step further comprises contacting the nucleotide with an agent that removes the blocking group from the nucleotide to 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 keto 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 of the same type of nucleotide, the same type of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide of the group emits light at a wavelength that differs from the wavelength of light emitted by the fluorescent label of another type of nucleotide of the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, the at least two types of nucleotides being selected from the group comprising dATP, dGTP, dCTP, dTTP, and dUTP, and wherein a fluorescent label of one type of nucleotide of the group emits light at a wavelength that is different from the wavelength of light emitted from a fluorescent label of another type of nucleotide of the group.In some embodiments, the determining step includes: (a) contacting two of the first concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, and two of the primer sequences complementary to a portion of the first concatemers under conditions sufficient to form a multivalent binding complex comprising each of the two polymerizing enzymes, a nucleotide conjugate of the plurality of nucleotide conjugates, and each of the two of the first concatemers hybridized to each of the two of the primer sequences, wherein the nucleotide conjugates comprise a label and at least two of the nucleotide moieties, wherein two of the at least two of the nucleotide moieties are complementary to each other and bound to each nucleotide of the two of the first concatemers; (b) detecting the multivalent binding complex via the labels of the nucleotide conjugates; and (c) identifying nucleobases of nucleotides of the two of the first concatemers that are complementary to each other and bound to each of the at least two of the nucleotide moieties of the nucleotide conjugates. In some embodiments, the determining step includes: (a) contacting two of the second concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, and two of the primer sequences complementary to a portion of the second concatemers under conditions sufficient to form a multivalent binding complex comprising each of the two polymerizing enzymes, a nucleotide conjugate of the plurality of nucleotide conjugates, and each of the two of the second concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugates comprise a label and at least two of the nucleotide moieties, wherein two of the at least two of the nucleotide moieties are complementary to each other and bound to each nucleotide of the two of the second concatemers; (b) detecting the multivalent binding complex via the labels of the nucleotide conjugates; and (c) identifying nucleobases of nucleotides of the two of the second concatemers that are complementary to each other and bound to each of the at least two of the nucleotide moieties of the nucleotide conjugates.In some embodiments, the conditions inhibit the incorporation of at least two of the nucleotide moieties of the nucleotide conjugate into two of the first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core bound to a plurality of nucleotide arms, and each of the nucleotide moieties is bound to one of the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, the detecting step comprises imaging the fluorescent label. In some embodiments, the determining step further includes: (a) removing two of the polymerizing enzymes and the nucleotide conjugate from two of the first concatemers; (b) contacting each of the two first concatemers hybridized to each of the two primer sequences with two of the second polymerizing enzymes and a plurality of unlabeled nucleotides under conditions suitable to form a binding complex comprising each of the two second polymerizing enzymes, 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 the two plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two plurality of unlabeled nucleotides is complementary to and bound to a respective nucleotide of the two first concatemers, and one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety.In some embodiments, the determining step further comprises: (a) removing two of the polymerizing enzymes and the nucleotide conjugate from two of the second concatemers; and (b) contacting each of the two second concatemers hybridized to each of the two primer sequences with two of the second polymerizing enzymes and a plurality of unlabeled nucleotides under conditions sufficient to form a binding complex comprising each of the two second polymerizing enzymes, 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 plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two plurality of unlabeled nucleotides is complementary to and binds to a respective nucleotide of the two second concatemers, and one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety.

[0007] In some embodiments, the determining step comprises (a) polymerizing two of the first concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, a first primer sequence that is complementary to a first portion of the first concatemers, and a second primer sequence that is complementary to a second portion of the first concatemers, under conditions sufficient to form a multivalent binding complex comprising each of the two of the polymerizing enzymes, one nucleotide conjugate of the plurality of nucleotide conjugates, the first portion of the first concatemer hybridized to the first primer sequence, and the second portion of the first concatemer hybridized to the second primer sequence; (b) contacting a nucleotide conjugate comprising a label and at least two nucleotide moieties, two of the at least two nucleotide moieties being complementary to and binding to nucleotides of the first and second portions of the first concatemer; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; and (c) identifying nucleobases of nucleotides of the first and second portions of the first concatemer that are complementary to and binding to each of two of the at least two nucleotide moieties of the nucleotide conjugate.In some embodiments, the determining step includes (a) polymerizing two of the second concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, a first primer sequence that is complementary to a first portion of the second concatemers, and a second primer sequence that is complementary to a second portion of the second concatemers under conditions sufficient to form a multivalent binding complex comprising each of the two of the polymerizing enzymes, one nucleotide conjugate of 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. (b) contacting a nucleotide conjugate comprising a label and at least two of the nucleotide moieties, two of the at least two nucleotide moieties being complementary to and binding to nucleotides of the first and second portions of the second concatemer; (b) detecting the multivalent binding complex via the label of the nucleotide conjugate; and (c) identifying nucleobases of nucleotides of the first and second portions of the second concatemer, which are complementary to and binding to each of two of the at least two nucleotide moieties of the nucleotide conjugate.

[0008] In some embodiments, the conditions inhibit the incorporation of at least two of the nucleotide moieties of the nucleotide conjugate into two of the first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core bound to a plurality of nucleotide arms, each of the nucleotide moieties being bound to one of the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, the detecting step comprises imaging the fluorescent label. In some embodiments, the plurality of nucleotides consists of at least two of the same type of nucleotide, the same type of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide of the group emits light at a wavelength different from the wavelength of light emitted from the fluorescent label of another type of nucleotide of the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, the at least two types of nucleotides being selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and a fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by a fluorescent label of another type of nucleotide from the group. In some embodiments, the determining step further comprises contacting the nucleotide with an agent that removes a blocking group from the nucleotide to 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 keto 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 distinct portions of the same mRNA or cDNA molecule, hi 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 step comprises repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times. In some embodiments, the determining step comprises detecting the first sequencing product nucleic acid molecule or the second sequencing product nucleic acid molecule in situ within the biological sample by imaging. In some embodiments, the determining step comprises simultaneously detecting the first sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule in situ within the biological sample by imaging. In some embodiments, the imaging comprises fluorescent imaging. In some embodiments, 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 protozoan, 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 and paraffin-embedded (FFPE) sample. In some embodiments, the biological sample comprises a fresh cell sample, a fresh frozen cell sample, a sectioned cell sample, or an FFPE cell sample. In some embodiments, the at least two target nucleic acid sequences comprise a target DNA sequence. In some embodiments, the at least two target nucleic acid sequences comprise a target RNA sequence. In some embodiments, the 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 coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA, or 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. 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 wolf 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 group of cells, 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.

[0009] 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 comprise a first target nucleic acid sequence and a second target nucleic acid sequence, and the at least two target polypeptides comprise a first target polypeptide encoded by the first target nucleic acid sequence or its reverse complementary sequence, and a second target polypeptide encoded by the second target nucleic acid sequence or its reverse complementary sequence, the method comprising: (a) providing a biological sample, wherein the biological sample contains: (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; and (iii) a third target nucleic acid sequence or (i) sequencing the sequence of 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) sequencing the sequence of 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) determining in situ the sequence of a third nucleic acid molecule or 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)(i) determining in situ the sequence of a second nucleic acid molecule or 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 portion thereof.and (ii) identifying in situ the sequence of a fourth nucleic acid molecule or 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 complete sequence of the first target nucleic acid sequence and the complete sequence of the second target nucleic acid sequence differ by at least one nucleotide and the complete sequence of the first target polypeptide and the complete sequence of the second target polypeptide differ by at least one amino acid, and step (b) is performed under conditions that prevent step (c).

[0010] 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 comprise a first target nucleic acid sequence and a second target nucleic acid sequence, and the at least two target polypeptides comprise a first target polypeptide encoded by the first target nucleic acid sequence or its reverse complementary sequence, and a second target polypeptide encoded by the second target nucleic acid sequence or its reverse complementary sequence, the method comprising: (a) providing a biological sample, wherein the biological sample contains: (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; and (iii) a third target nucleic acid sequence or (i) sequencing the sequence of 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) sequencing the sequence of 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. (ii) determining in situ the sequence of 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.(c) removing the first sequencing product nucleic acid molecule from the first nucleic acid molecule and the third sequencing product nucleic acid molecule from the third nucleic acid molecule, wherein the first nucleic acid molecule and the third nucleic acid molecule are located in the biological sample after removal; (d) repeating (b) and (c); and (e) (i) identifying in situ the sequence of 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 to 30 nucleotides; and (ii) identifying in situ the sequence of 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. (f) removing the second sequencing product nucleic acid molecule from the second nucleic acid molecule and the fourth sequencing product nucleic acid molecule from the fourth nucleic acid molecule, wherein the second nucleic acid molecule and the fourth nucleic acid molecule are located in the biological sample after removal; and (g) repeating (e) and (f), wherein the complete sequence of the first target nucleic acid sequence and the complete sequence of the second target nucleic acid sequence differ by at least one nucleotide and the complete sequence of the first target polypeptide and the complete sequence of the second target polypeptide differ by at least one amino acid, and step (b) is performed under conditions that prevent step (e).

[0011] 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, the biological sample comprising: (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; 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 first target polypeptide binds to the first target RNA sequence or its reverse complement. (b) generating a first target cDNA sequence by reverse transcription of the first target RNA sequence and a second target cDNA sequence by reverse transcription of the second target RNA sequence; and (c) (i) contacting the first target cDNA sequence with a first oligonucleotide comprising a first end portion and a second end portion; (ii) contacting the second target cDNA sequence with a second oligonucleotide comprising a first end portion and a second end portion. (iii) 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; and (iv) 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 first terminal portion and the second terminal portion of the first oligonucleotide are complementary and bind to two adjacent segments of the first target cDNA sequence, such that the first oligonucleotide forms a circular structure having a gap between the first terminal portion and the second terminal portion, the first oligonucleotide comprises a first identification sequence that identifies the first target RNA sequence, a first sequencing primer, and a nucleic acid amplification primer, and the first terminal portion and the second terminal portion of the second oligonucleotide are complementary and bind to two adjacent segments of the second target cDNA sequence, such that the second oligonucleotideThe first oligonucleotide conjugate forms a circular structure having a gap between the first end portion and the second end portion, the second oligonucleotide comprises a second identification sequence that identifies a second target RNA sequence, a second sequencing primer, and a nucleic acid amplification primer, the first sequencing primer and the second sequencing primer having a difference of at least one nucleotide, the first short nucleic acid comprises a first tag sequence and a second tag sequence, and the first oligonucleotide conjugate specifically binds to the first target polypeptide via the first binding moiety to amplify the first target polypeptide. forming a first binding complex, wherein the first tag sequence and the second tag sequence identify a first binding moiety, the second short nucleic acid comprises a third tag sequence and a fourth tag sequence, and the second oligonucleotide conjugate specifically binds to a second target polypeptide via the second binding moiety to form a second binding complex, wherein the third tag sequence and the fourth tag sequence identify the second binding moiety; and (d) (i) contacting the first binding complex with a third oligonucleotide comprising a first terminal portion and a second terminal portion; and (ii) contacting the second binding complex with a third oligonucleotide comprising a first terminal portion and a second terminal portion. contacting the conjugate with a fourth oligonucleotide comprising a first terminal portion and a second terminal portion, wherein the first terminal portion and the second terminal portion of the third oligonucleotide are complementary and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, such that the third oligonucleotide forms a circular structure having a gap between its first terminal portion and its second terminal portion, and the first terminal portion and the second terminal portion of the fourth oligonucleotide are complementary 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 its first terminal portion and its second terminal portion; (e) ligating the first terminal portion and the second terminal portion of the first oligonucleotide to form a first circular oligonucleotide, ligating the first terminal portion and the second terminal portion of the second oligonucleotide to form a second circular oligonucleotide, and ligating the first terminal portion and the second terminal portion of the third oligonucleotide to form a third circular oligonucleotide;and (f) ligating the first terminal portion and the second terminal portion of the fourth oligonucleotide to generate a fourth circular oligonucleotide, wherein the first circular oligonucleotide and the third circular oligonucleotide comprise a first sequencing primer or its reverse complement, and the second circular oligonucleotide and the fourth circular oligonucleotide comprise a second sequencing primer or its reverse complement, and the sequences of the first sequencing primer and the second sequencing primer differ by at least one nucleotide; and (f)(i) amplifying the first circular oligonucleotide by rolling circle amplification to generate first concatemers comprising a plurality of the first circular oligonucleotides. (ii) amplifying the second circular oligonucleotides by rolling circle amplification to generate second concatemers comprising a plurality of second circular oligonucleotides; (iii) amplifying the third circular oligonucleotides by rolling circle amplification to generate third concatemers comprising a plurality of third circular oligonucleotides; and (iv) amplifying the fourth circular oligonucleotides by rolling circle amplification to generate fourth concatemers comprising a plurality of fourth circular oligonucleotides; and (g)(i) sequencing the sequence of the first concatemers or a portion thereof in a sequence of a first concatemer to generate a first sequencing product nucleic acid molecule that is complementary to and binds to the first concatemer. (ii) 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 binds to the third concatemer, wherein the sequence of the first concatemer or a portion thereof consists of 2 to 30 nucleotides and the sequence of the third concatemer or a portion thereof consists of 2 to 30 nucleotides, and step (g) is performed under conditions that prevent step (j); and (h) removing the first sequencing product nucleic acid molecule from the first concatemer and the third sequencing product nucleic acid molecule from the third concatemer, wherein the first concatemer and the third concatemer are located in the biological sample after removal.(i) repeating (g) and (h) at least once; and (j) (i) 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, and (ii) 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. The method includes: (i) determining in situ the sequence of the second concatemer or a portion thereof, wherein the sequence of the fourth concatemer or a portion thereof consists of 2 to 30 nucleotides, and the sequence of the fourth concatemer or a portion thereof consists of 2 to 30 nucleotides; performing step (j) under conditions that prevent the performance of step (g); the complete sequence of the first target RNA sequence and the complete sequence of the second target RNA sequence differ by at least one nucleotide; and the complete sequence of the first target polypeptide and the complete 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 the fourth sequencing product nucleic acid molecule from the fourth concatemer, wherein the second concatemer and the fourth concatemer are located in the biological sample after removal; and (l) repeating steps (j) and (k) at least once.

[0012] In some embodiments, the determining step includes imaging the first sequencing product nucleic acid molecule or the third sequencing product nucleic acid molecule, and the identifying step includes imaging the second sequencing product nucleic acid molecule or the fourth sequencing product nucleic acid molecule and the fourth sequencing product nucleic acid molecule. In some embodiments, the method further includes 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 sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule within the biological sample. In some embodiments, the biological sample includes an organelle, a cell, a whole cell, a whole group of cells, a tissue, an undamaged tissue, a tumor, an undamaged tumor, an organ, an organism, a protozoan, algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal. In some embodiments, the biological sample includes a fresh sample, a processed sample, a fresh frozen sample, a sectioned sample, or a formalin-fixed and 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 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 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 wolf 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 comprises a compaction oligonucleotide, wherein (a) a first segment of the compaction oligonucleotide is complementary to and binds to a first portion of the first, second, third, or fourth concatemer, and (b) a second segment of the compaction oligonucleotide is complementary to and binds to a second portion of the first, second, third, or fourth concatemer, resulting in a reduction in size or a change in shape of the second concatemer. In some embodiments, the first sequencing product nucleic acid molecule comprises a first identifying sequence that identifies the first target RNA sequence, and the second sequencing product nucleic acid molecule comprises a second identifying sequence that identifies the second target RNA sequence. In some embodiments, the first sequencing product nucleic acid molecule comprises a first target RNA sequence and a first identifying sequence that identifies a portion of the first target RNA sequence, and the second sequencing product nucleic acid molecule comprises a second target RNA sequence and a second identifying sequence that identifies a portion of the second target RNA sequence.In some embodiments, the determining step comprises (a) polymerizing the first, second, third, or fourth concatemer with a polymerizing enzyme, a plurality of nucleotides, and a primer sequence complementary to a portion of the first, second, third, or fourth concatemer, and one nucleotide of the plurality of nucleotides that is complementary to and binds to a nucleotide unit of the first, second, third, or fourth concatemer. (b) contacting a nucleotide at the 3' end of the primer sequence under conditions sufficient to form a binding complex comprising a nucleotide and a first concatemer, a second concatemer, a third concatemer, or a 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 moiety; (b) incorporating the nucleotide at 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 step further comprises contacting the nucleotide with an agent that removes the blocking group from the nucleotide to 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 keto 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 comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.In some embodiments, the determining step includes (a) hybridizing two of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers to two of the polymerizing enzymes, a plurality of nucleotide conjugates, and two of the primer sequences that are complementary to a portion of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers, and one of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers hybridized to each of the two of the polymerizing enzymes, one nucleotide conjugate of the plurality of nucleotide conjugates, and each of the two of the primer sequences. (b) contacting the nucleotide conjugates with a label under conditions sufficient to form a multivalent binding complex comprising the nucleotide conjugates, wherein the nucleotide conjugates comprise a label and at least two of the nucleotide moieties, two of the at least two of the nucleotide moieties being complementary to each other and binding to each of the two nucleotides of the first concatemer; (b) detecting the multivalent binding complex via the labels of the nucleotide conjugates; and (c) identifying the nucleobases of the nucleotides of the two nucleotides of the first concatemers that are complementary to each other and binding to each of the at least two of the nucleotide moieties of the nucleotide conjugates.

[0013] In some embodiments, the determining step further comprises (a) removing two of the polymerizing enzymes and nucleotide conjugates from two of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers; and (b) separating each of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers hybridized to each of the two of the primer sequences from two of the second polymerizing enzymes and a plurality of unlabeled nucleotides, and separating each of the second polymerizing enzymes and the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers hybridized to each of the two of the primer sequences from two of the second polymerizing enzymes and a plurality of unlabeled nucleotides. and incorporating each of two of the plurality of unlabeled nucleotides into each of two of the primer sequences, wherein each of two of the plurality of unlabeled nucleotides is complementary to and binds to a respective nucleotide of two of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, and wherein one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety.

[0014] In some embodiments, the determining step includes (a) polymerizing two of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, a first primer sequence complementary to a first portion of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers, and a second primer sequence complementary to a first portion of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers. a second primer sequence complementary to a second portion of the concatemer; each of two of the polymerizing enzymes; one nucleotide conjugate of the plurality of nucleotide conjugates; a first portion of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer hybridized to the first primer sequence; and a second primer sequence complementary to a second portion of the concatemer. (b) contacting the nucleotide conjugates under conditions sufficient to form a multivalent binding complex comprising a first portion of the first concatemer, a second portion of the second concatemer, a third concatemer, or a fourth concatemer, wherein the nucleotide conjugates comprise a label and at least two of the nucleotide moieties, and two of the at least two of the nucleotide moieties are complementary to and bind to nucleotides of the first and second portions of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, respectively; (b) detecting the multivalent binding complex via the labels of the nucleotide conjugates; and (c) identifying nucleobases of nucleotides of the first and second portions of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, which are complementary to and bind to each of two of the at least two of the nucleotide moieties of the nucleotide conjugate. In some embodiments, the conditions inhibit incorporation of at least two of the nucleotide moieties of the nucleotide conjugate into two of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer.In some embodiments, the nucleotide conjugate comprises a core bound to a plurality of nucleotide arms, each of the nucleotide moieties being bound 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, the plurality of nucleotides comprises at least two of the same type of nucleotide, the same type of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by the fluorescent label of another type of nucleotide from the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotide, the at least two types of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by the fluorescent label of another type of nucleotide from the group. In some embodiments, the determining step further comprises contacting the nucleotide with an agent that removes the blocking group from the nucleotide to 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 keto 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 of the same type of nucleotide, the same type of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide of the group emits light at a wavelength that differs from the wavelength of light emitted by the fluorescent label of another type of nucleotide of the group.In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, the at least two types of nucleotides being selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted from the fluorescent label of another type of nucleotide from 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. 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 wolf 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 group of cells, 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.

[0015] In another aspect, there is provided herein 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 comprise 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; (b) a first oligonucleotide comprising a first terminal portion and a second terminal portion; and (c) a second oligonucleotide comprising a first terminal portion and a second terminal portion, wherein the first nucleic acid molecule comprises the first target nucleic acid sequence or a reverse complementary sequence thereof, or a portion thereof, and the second nucleic acid molecule comprises the second target nucleic acid sequence or a reverse complementary sequence thereof, or a portion thereof, and the first terminal portion and the second terminal portion of the first oligonucleotide are complementary, and the first target nucleic acid sequence is a second oligonucleotide. The first oligonucleotide binds to two adjacent segments of a second target nucleic acid sequence or its reverse complement, or a portion thereof, such that the first oligonucleotide forms a first circular oligonucleotide in the biological sample, the first circular oligonucleotide comprising a gap between its first and second terminal portions, and the first and second terminal portions of the second oligonucleotide are complementary, and the second oligonucleotide binds 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 in the biological sample, the second circular oligonucleotide comprising a gap between its first and second terminal portions. In some embodiments, the first circular oligonucleotide further comprises a first nucleic acid enzyme configured to ligate the first and second terminal portions of the first oligonucleotide, and the second circular oligonucleotide further comprises a second nucleic acid enzyme configured to ligate the first and second terminal portions 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 ligation enzyme, a nucleic acid polymerase, a nucleic acid polymerization enzyme, 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 nucleic acid unit comprising a first target nucleic acid sequence or a portion thereof, or its reverse complement or a portion thereof, and / or (b) the second concatemer comprises at least two repeats of a second nucleic acid unit comprising a second target nucleic acid sequence or a portion thereof, or its reverse complement or a portion thereof.

[0016] In some embodiments, the first concatemer further comprises a first identifying sequence that identifies the first target nucleic acid sequence, or a sequencing primer or reverse complement thereof, and the second concatemer further comprises a second identifying 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 compaction oligonucleotide, wherein (a) a first segment of the first compaction oligonucleotide is complementary to and binds to a first portion of the first concatemer, and (b) a second segment of the first compaction oligonucleotide is complementary to and binds to a 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 compaction oligonucleotide, wherein (a) a first segment of the second compaction oligonucleotide is complementary to and binds to a first portion of the second concatemer, and (b) a second segment of the second compaction oligonucleotide is complementary to and binds to a 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 polymerizing enzyme, a plurality of nucleotides, and a primer sequence complementary to at least a portion of the first or second concatemer under conditions sufficient to form a binding complex. In some embodiments, the system further comprises an agent configured to remove a blocking group from one nucleotide of the plurality of nucleotides and generate a 3'OH group on the sugar moiety of the nucleotide. 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 keto 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 comprises a fluorescent label. In some embodiments, the plurality of nucleotides consists of at least two of the same type of nucleotides, the same type of nucleotides being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by the fluorescent label of another type of nucleotide from the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, the at least two types of nucleotides being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by the fluorescent label of another type of nucleotide from the group. In some embodiments, the system further comprises a plurality of nucleotide conjugates, wherein one nucleotide conjugate of the plurality of nucleotide conjugates is configured to form a multivalent binding complex comprising two or more of the polymerizing enzymes, the 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 complementary to each other and bind to each nucleotide of the at least two target nucleic acid sequences. In some embodiments, the system further comprises 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 in the biological sample via the first binding moiety to form a first binding complex, and the first tag sequence and the second tag sequence identify the first binding moiety 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 moiety that specifically binds to a second target polypeptide, the second short nucleic acid comprising a third tag sequence and a fourth tag sequence, the second oligonucleotide conjugate specifically binding to the second target polypeptide in the biological sample via the second binding moiety to form a second binding complex, and the third tag sequence and the fourth tag sequence identify the second binding moiety in a nucleic acid sequence reaction. In some embodiments, the first binding moiety or the second binding moiety is an antibody or an antigen-binding fragment thereof. In some embodiments, the system further includes a third oligonucleotide comprising a first terminal portion and a second terminal portion, the first terminal portion and the second terminal portion of the third oligonucleotide being complementary and binding to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, so that the third oligonucleotide forms a circular structure with a gap between the first terminal portion and the second terminal portion. In some embodiments, the system further comprises a fourth oligonucleotide comprising a first end portion and a second end portion, wherein the first end portion and the second end portion of the fourth oligonucleotide are complementary and bind to a third tag sequence and a fourth tag sequence, so that the fourth oligonucleotide forms a circular structure with a gap between the first end portion and the second end portion.In some embodiments, the system further comprises a third circular oligonucleotide derived from the ligation of the first end portion and the second end portion of the third oligonucleotide, and a fourth circular oligonucleotide derived from the ligation of the first end portion and the second end portion of the fourth oligonucleotide, wherein the first circular oligonucleotide and the third circular oligonucleotide comprise a first sequencing primer or its reverse complementary sequence, the second circular oligonucleotide and the fourth circular oligonucleotide comprise a second sequencing primer or its reverse complementary sequence, and the sequences of the first sequencing primer and the second sequencing primer have at least one nucleotide difference, and the ligation is performed by a first nucleic acid enzyme or a second nucleic acid enzyme.In some embodiments, the system further comprises a third amplicon of a third circular oligonucleotide and a fourth amplicon of a fourth circular oligonucleotide. In some embodiments, the third amplicon comprises a third concatemer, and / or the fourth amplicon comprises a fourth concatemer. In some embodiments, (a) the third concatemer comprises at least two repeats of a third nucleic acid unit comprising the 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 nucleic acid unit comprising the 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 tag sequence or a second tag sequence or a reverse complement thereof that identifies the first binding moiety, and the fourth concatemer further comprises a third tag sequence or a fourth tag sequence or a reverse complement thereof that identifies the second binding moiety. In some embodiments, the third concatemer further comprises a third compaction oligonucleotide, wherein (a) a first segment of the third compaction oligonucleotide is complementary to and binds to a first portion of the third concatemer, and (b) a second segment of the third compaction oligonucleotide is complementary to and binds to a second portion of the third concatemer, resulting in a reduction in size or a change in shape of the third concatemer.

[0017] In some embodiments, the fourth concatemer further comprises a fourth compaction oligonucleotide, wherein (a) a first segment of the fourth compaction oligonucleotide is complementary to and binds to a first portion of the fourth concatemer, and (b) a second segment of the fourth compaction oligonucleotide is complementary to and binds to a second portion of the fourth concatemer, resulting in a reduction in size or a change in shape of the fourth concatemer. In some embodiments, the system further comprises a second polymerizing enzyme, a second plurality of nucleotides, and a second primer sequence complementary to at least a portion of the third or fourth concatemer, under conditions sufficient to form a binding complex comprising the third or fourth concatemer hybridized to the second primer sequence, the second polymerizing enzyme, and a second nucleotide of one of the second plurality of nucleotides that is complementary to and bound to a nucleotide of the third or fourth concatemer. In some embodiments, the system further comprises a second agent configured to remove a second blocking group from a second nucleotide of one of the second plurality of nucleotides and generate a 3'OH group on the sugar moiety of the second nucleotide. In some embodiments, 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 keto 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 label. In some embodiments, the second plurality of nucleotides consists of at least two of the same type of nucleotide, the same type of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the second fluorescent label of one type of nucleotide of the group emits light at a wavelength different from the wavelength of light emitted from the second fluorescent label of another type of nucleotide of the group.In some embodiments, the second plurality of nucleotides comprises at least two types of nucleotides, the at least two types of nucleotides being selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and the second fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted from the second fluorescent label of another type of nucleotide from the group. In some embodiments, the system further comprises a second plurality of nucleotide conjugates, wherein a second nucleotide conjugate of the second plurality of nucleotide conjugates is configured to form a multivalent binding complex comprising two or more of the second polymerizing enzyme, a second nucleotide conjugate of the second plurality of nucleotide conjugates, and at least two first short nucleic acids or short nucleic acids or portions thereof, the second nucleotide conjugate comprising a label and at least two nucleotide moieties that are complementary to each other and bound to each nucleotide of at least two of the first short nucleic acids or short nucleic acids or portions thereof. In some embodiments, the system further comprises a solid surface comprising a biological sample immobilized on the solid surface. In some embodiments, the biological sample is permeabilized. In some embodiments, the solid surface further comprises a hydrophilic polymer coating layer attached thereto. In some embodiments, the hydrophilic polymer coating layer 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 attached to the solid surface to detect at least two target nucleic acid sequences and / or at least two target polypeptides in situ in the biological sample. In some embodiments, the first target polypeptide is encoded by a first target nucleic acid molecule or its reverse complement, and the second target polypeptide is encoded by a second target nucleic acid molecule or its reverse complement.

[0018] Provided herein, in another aspect, is a computer-implemented system comprising a computing device having at least one processor, an operating system configured to execute executable instructions, a memory, and a computer program including instructions executable by the computing device, the instructions comprising a method disclosed herein.

[0019] 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, the instructions comprising the methods disclosed herein.

[0020]

[0010] Provided herein, in another aspect, is a kit for in situ detection of at least two target nucleic acid sequences in a biological sample, the at least two target nucleic acid sequences comprising a first target nucleic acid sequence and a second target nucleic acid sequence, the kit comprising: (a) a first oligonucleotide comprising a first terminal portion and a second terminal portion; (b) a second oligonucleotide comprising a first terminal portion and a second terminal portion; and (c) ligating the first terminal portion and the second terminal portion of the first oligonucleotide to generate a first circular oligonucleotide or ligating the first terminal portion and the second terminal portion of the second oligonucleotide; (d) a second nucleic acid enzyme 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 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; and (f) a fourth nucleic acid enzyme configured to amplify (i) the first concatemer or a second concatemer. a fourth nucleic acid enzyme configured to (i) add one nucleotide of the plurality of nucleotides to an end of a sequencing primer hybridized to the first concatemer to generate a first sequencing product nucleic acid molecule, wherein the nucleotide is unlabeled and comprises a blocking group, or (ii) add one nucleotide of the plurality of nucleotides to an end of a sequencing primer hybridized to a second concatemer to generate 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 the second sequencing product nucleic acid molecule from the second concatemer, wherein the first terminal portion and the second terminal portion of the first oligonucleotide are complementary and bind to two adjacent segments of a first target nucleic acid sequence or a reverse complement thereof, or a portion thereof, such that the first oligonucleotide forms a first circular oligonucleotide in the biological sample, and the first circular oligonucleotidethe first oligonucleotide comprises a gap between its first terminal portion and its second terminal portion, and the first oligonucleotide comprises a first identification sequence that identifies a 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; the first terminal portion and the second terminal portion of the second oligonucleotide are complementary and bind to two adjacent segments of the second target nucleic acid sequence or its reverse complementary sequence, or a portion thereof, such that the second oligonucleotide forms a second circular oligonucleotide in the biological sample, and the second circular oligonucleotide comprises a gap between its first terminal portion and its second terminal portion, and the second oligonucleotide comprises a second identification sequence that identifies a second target nucleic acid sequence, a second 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; and two of the nucleotide moieties bound to the core, wherein (i) a third nucleic acid enzyme, one nucleotide of the plurality of nucleotides, and a sequencing primer are configured to form a binding complex comprising the third nucleic acid enzyme, one nucleotide of the plurality of nucleotides that is complementary to and bound to a nucleotide unit of the first concatemer or the second concatemer, and the first concatemer or the second concatemer hybridized to the sequencing primer, or (ii) at least one of the third nucleic acid enzyme At least two of the nucleotide conjugate, the sequencing primer, and the first concatemer or the second concatemer are configured to form a multivalent binding complex comprising a third nucleic acid enzyme, the nucleotide conjugate, and the first concatemer or the second concatemer hybridized to the sequencing primer, wherein the two nucleotide moieties are complementary and bind to two nucleotide units of the first concatemer or the second concatemer, and each of the two of the first concatemers or the second concatemers isThe sequencing primers hybridize to each of the two sequencing primers. In some embodiments, the kit is further configured to detect at least two target RNA sequences and at least two target polypeptides in situ in a biological sample, including a first target polypeptide and a second target polypeptide, wherein the kit includes a first target polypeptide encoded by a first target nucleic acid molecule or its reverse complementary sequence, and the second target polypeptide encoded by a second target nucleic acid molecule or its reverse complementary sequence, wherein the complete sequence of the first target RNA sequence and the complete sequence of the second target RNA sequence differ by at least one nucleotide, and the complete sequence of the first target polypeptide and the complete sequence of the second target polypeptide differ by at least one amino acid. In some embodiments, the first nucleic acid enzyme, the second nucleic acid enzyme, the third nucleic acid enzyme, or the fourth nucleic acid enzyme comprises a nucleic acid ligase, a nucleic acid ligation enzyme, a nucleic acid polymerase, a nucleic acid polymerization enzyme, or a combination thereof. In some embodiments, one nucleotide of the plurality of nucleotides comprises a fluorescent label. In some embodiments, one nucleotide of the plurality of nucleotides comprises a removable blocking group at the 3' carbon position of the sugar moiety. In some embodiments, the plurality of nucleotides consists of at least two of the same type of nucleotides comprising a fluorescent label, the same type of nucleotides being selected from the group comprising dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide of the group emits light at a wavelength different from the wavelength of light emitted from the fluorescent label of another type of nucleotide of the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, one type of the at least two types of nucleotides comprising a fluorescent label, the at least two types of nucleotides being selected from the group comprising dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide of the group emits light at a wavelength different from the wavelength of light emitted from the fluorescent label of another type of nucleotide of the group. In some embodiments, the nucleotide conjugate isThe nucleotide conjugate comprises a detectable label. In some embodiments, the nucleotide conjugate comprises a fluorescent label. 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 wolf 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 group of cells, 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 group of cells, a tissue, an intact tissue, a tumor, an intact tumor, an organ, an organism, a protozoan, 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 and 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 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 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 is an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group,It includes an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto 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 moiety that specifically binds to a first target polypeptide; or (b) a second oligonucleotide conjugate comprising a second short nucleic acid and a second binding moiety that specifically binds to a second target polypeptide, wherein the first short nucleic acid comprises a first tag sequence and a second tag sequence, and the first oligonucleotide conjugate specifically binds to the first target polypeptide via the first binding moiety to form a first binding complex, and the first tag sequence and the second tag sequence identify the first binding moiety; or wherein the second short nucleic acid comprises a third tag sequence and a fourth tag sequence, and the second oligonucleotide conjugate specifically binds to the second target polypeptide via the second binding moiety to form a second binding complex, and the third tag sequence and the fourth tag sequence identify the second binding moiety. In some embodiments, the kit further comprises: (a) a third oligonucleotide comprising a first terminal portion and a second terminal portion; or (b) a fourth oligonucleotide comprising a first terminal portion and a second terminal portion, wherein the first terminal portion and the second terminal portion of the third oligonucleotide are complementary and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, such that the third oligonucleotide forms a circular structure having a gap between the first terminal portion and the second terminal portion; or the first terminal portion and the second terminal portion of the fourth oligonucleotide are complementary 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 terminal portion and the second terminal portion. In some embodiments, the third oligonucleotide comprises a third identification sequence that identifies the first binding portion, and the fourth oligonucleotide comprises a fourth identification sequence that identifies the second binding portion. In some embodiments, the first oligonucleotide and the third oligonucleotide areThe second oligonucleotide and the fourth oligonucleotide comprise the same first sequencing primer or its reverse complement, and the second oligonucleotide and the fourth oligonucleotide comprise the same second sequencing primer or its reverse complement. In some embodiments, the kit further comprises an agent that reacts with a reactive group at the 3' carbon of the sugar moiety in the nucleotide portion of the nucleotide conjugate. In some embodiments, the kit comprises: , and further comprises a reagent for use in a nucleotide incorporation 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 the biological sample, a reagent configured to fix the biological sample, an unblocked nucleotide, a blocked nucleotide, a reagent for use in a nucleotide incorporation reaction, a solution containing a cation, one or more unlabeled nucleotides, one or more buffers for reverse transcription, one or more buffers for nucleic acid incorporation, 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 situ in a biological sample. 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 situ in a biological sample. In some embodiments, the instructions comprise performing sequencing by synthesis. In some embodiments, the instructions comprise performing sequencing by incorporation in a binding reaction in which in situ detection does not occur simultaneously with the nucleotide incorporation step. In some embodiments, the kit further comprises instructions for using the kit using the methods disclosed herein.

[0021] Incorporation by Reference 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. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or supersede any such conflicting material. [Brief explanation of the drawings]

[0022] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings. [Figure 1] Figure 1 shows a schematic diagram of a non-limiting, exemplary workflow for generating concatemers within a cell sample. Target RNA contained in the cell sample is hybridized to a reverse transcription primer (RT primer) and reverse-transcribed to synthesize first-strand cDNA. The first-strand cDNA is hybridized to a target-specific padlock probe to generate a circularized padlock probe with a nick (solid, downward-pointing triangle) between the first and second ends of the hybridized padlock probe. The padlock probe carries an adapter for a universal compaction oligonucleotide binding site, an adapter for a universal sequencing primer binding site, and a target barcode sequence corresponding to a given target cDNA. The nick in the circularized padlock probe is ligated to form a covalently closed circular padlock probe that carries the cDNA sequence corresponding to the target RNA. The sequence of a non-limiting example padlock probe that selectively binds to GAPDH cDNA is shown at the bottom of Figure 1. The covalently closed circular padlock probe can be subjected to rolling circle amplification within the cell sample to generate concatemer molecules. Concatameric molecules can be sequenced iteratively within a cell sample. [Figure 2]

[0023] Figure 2 is a schematic diagram showing a non-limiting exemplary workflow for sequencing concatemers generated inside a cell, such as those shown in Figure 1. The concatemers depicted in Figure 2 comprise tandem repeat units, each comprising (i) a universal sequencing primer binding site (Seq), (ii) a universal compaction oligonucleotide binding site (CO), (iii) an insert sequence corresponding to a given target cDNA, and (iv) a target barcode sequence (BC) corresponding to the given target cDNA. In some embodiments, a universal sequencing primer (solid arrow) is hybridized to the universal sequencing primer binding site, and 30 or fewer sequencing cycles are performed to generate a plurality of first sequencing read products (dashed arrows), each of which contains only the target barcode sequence. A plurality of first sequencing read products are removed from the concatemers, and sequencing is repeated, with 30 or fewer sequencing cycles performed to generate another plurality of first sequencing read products (dashed arrows), where the first sequencing read products contain only the target barcode sequence. A plurality of first sequencing read products are removed from the concatemers, and sequencing is repeated again, with 30 or fewer sequencing cycles performed to generate another plurality of first sequencing read products (dashed arrows), where the first sequencing read products contain only the target barcode sequence. In some embodiments, the repeated sequencing can be performed up to 50 times. The sequences of all of the first sequencing read products can be determined and aligned with a first reference sequence (e.g., a reference barcode sequence) to confirm the presence of the first target RNA molecule within the cell sample. [Figure 3]

[0023] Figure 3 is a schematic diagram showing a non-limiting exemplary workflow for sequencing concatemers generated inside a cell, such as those shown in Figure 1. The concatemers depicted in Figure 3 comprise tandem repeat units, each comprising (i) a universal sequencing primer binding site (Seq), (ii) a universal compaction oligonucleotide binding site (CO), (iii) an insert sequence corresponding to a given target cDNA, and (iv) a target barcode sequence (BC) corresponding to the given target cDNA. In some embodiments, a universal sequencing primer (solid arrow) is hybridized to the universal sequencing primer binding site, and 30 or fewer sequencing cycles are performed to generate a plurality of first sequencing read products (dashed arrows), each of which comprises the target barcode sequence and a portion of the insert sequence. A plurality of first sequencing read products are removed from the concatemers, and sequencing is repeated, with 30 or fewer sequencing cycles performed to generate another plurality of first sequencing read products (dashed arrows), where the first sequencing read products contain the target barcode sequence and a portion of the insert sequence. A plurality of first sequencing read products are removed from the concatemers, and sequencing is repeated again, with 30 or fewer sequencing cycles performed to generate another plurality of first sequencing read products (dashed arrows), where the first sequencing read products contain the target barcode sequence and a portion of the insert sequence. In some embodiments, the repeated sequencing can be performed up to 50 times. The sequences of all of the first sequencing read products 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 within the cell sample. [Figure 4]4 is a schematic diagram showing a non-limiting exemplary workflow for sequencing concatemers generated inside cells, such as those shown in FIG. 1. The concatemers depicted in FIG. 4 comprise tandem repeat units, each comprising (i) a universal sequencing primer binding site (Seq), (ii) a universal compaction oligonucleotide binding site (CO), and (iii) an insert sequence corresponding to a given target cDNA. In some embodiments, a universal sequencing primer (solid arrow) is hybridized to the universal sequencing primer binding site, and 30 or fewer sequencing cycles are performed to generate a plurality of first sequencing read products (dashed arrows), each of which contains a portion of the insert sequence. The plurality of first sequencing read products are removed from the concatemers, and sequencing is repeated, followed by 30 or fewer sequencing cycles to generate another plurality of first sequencing read products (dashed arrows), each of which contains a portion of the insert sequence. A plurality of first sequencing read products are removed from the concatemer, and sequencing is repeated again, performing 30 or fewer sequencing cycles to generate another plurality of first sequencing read products (dashed arrows), where the first sequencing read products contain a portion of the insert sequence. In some embodiments, the repeated sequencing can be performed up to 50 times. The sequences of all of the first sequencing read products can be determined and aligned with a first reference sequence (e.g., the insert sequence corresponding to the target RNA) to confirm the presence of the first target RNA molecule in the cell sample. [Figure 5]5 is a schematic diagram showing a non-limiting exemplary workflow for sequencing concatemers generated inside cells, such as those shown in FIG. 1. The concatemers depicted in FIG. 5 comprise tandem repeat units, each of which comprises (i) a universal sequencing primer binding site (Seq) and (ii) an insert sequence corresponding to a given target cDNA. In some embodiments, a universal sequencing primer (solid arrow) is hybridized to the universal sequencing primer binding site, and 30 or fewer sequencing cycles are performed to generate a plurality of first sequencing read products (dashed arrows), each of which contains a portion of the insert sequence. The plurality of first sequencing read products are removed from the concatemers, and sequencing is repeated, followed by 30 or fewer sequencing cycles to generate another plurality of first sequencing read products (dashed arrows), each of which contains a portion of the insert sequence. A plurality of first sequencing read products are removed from the concatemer, and sequencing is repeated again, performing 30 or fewer sequencing cycles to generate another plurality of first sequencing read products (dashed arrows), where the first sequencing read products contain a portion of the insert sequence. In some embodiments, the repeated sequencing can be performed up to 50 times. The sequences of all of the first sequencing read products can be determined and aligned with a first reference sequence (e.g., the insert sequence corresponding to the target RNA) to confirm the presence of the first target RNA molecule in the cell sample. [Figure 6]Figure 6 shows images of fluorescent sequencing signals emitted from HEK293 cells cultured on a poly-lysine-coated flow cell. The cultured cells were fixed, permeabilized, subjected to reverse transcription, and then subjected to rolling circle amplification on the flow cell, all under conditions suitable for retaining cellular nucleic acids inside the cells. The coated flow cell lacked surface capture primers. The nucleic acids retained in the cells were sequenced using a two-step sequencing method using detectably labeled multivalent molecules and unlabeled nucleotide analogs. Thirty cycles of two-step sequencing reactions were performed using multivalent molecules labeled with one of four fluorophores and unlabeled nucleotide analogs. Figure 6 shows results from a second in situ sequencing experiment (see Example 1). Figure 6 shows the fluorescent signals from cycles 20–25, where the sequence reads CCTCCT. A total of 4,620 fluorescent spots with significant signals were detected in all cycles. More than 90% of the fluorescent spots detected the sample index sequence. [Figure 7] Schematic diagrams of various non-limiting exemplary configurations of multivalent molecules. Left (Class I): Schematic diagram of a multivalent molecule with a "star" or "helter-skelter" configuration. Center (Class II): Schematic diagram of a multivalent molecule with a dendrimer configuration. Right (Class III): Schematic diagram of multiple multivalent molecules formed by reacting streptavidin with a 4-arm or 8-arm PEG-NHS with biotin and dNTPs. Nucleotide units are designated "N," biotin is designated "13," and streptavidin is designated "SA." [Figure 8] FIG. 1 is a schematic diagram of a non-limiting exemplary multivalent molecule comprising a generic core attached to multiple nucleotide arms. [Figure 9] FIG. 1 is a schematic diagram of a non-limiting exemplary multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms. [Figure 10] 1 shows a schematic diagram of a non-limiting exemplary multivalent molecule comprising a core attached to multiple nucleotide arms, the nucleotide arms comprising biotin, a spacer, a linker, and a nucleotide unit. [Figure 11]FIG. 1 is a schematic diagram of a non-limiting exemplary nucleotide arm comprising a core-binding moiety, a spacer, a linker, and a nucleotide unit. [Figure 12] Shown are the chemical structures of non-limiting exemplary spacers (top) and various non-limiting exemplary linkers (bottom), including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker. [Figure 13] 1 shows the chemical structures of various non-limiting exemplary linkers, including linkers 1-9. [Figure 14] 1 shows the chemical structures of various non-limiting exemplary linkers linked / attached to nucleotide units. [Figure 15] 1 shows the chemical structures of various non-limiting exemplary linkers linked / attached to nucleotide units. [Figure 16] 1 shows the chemical structures of various non-limiting exemplary linkers linked / attached to nucleotide units. [Figure 17] 1 shows the chemical structures of non-limiting examples of biotinylated nucleotide arms, in which the nucleotide units are connected to the linker via a propargylamine bond at the 5-position of the pyrimidine base or the 7-position of the purine base. [Figure 18] FIG. 1 is a schematic diagram of a guanine tetrad (e.g., G-tetrad). [Figure 19] FIG. 1 is a schematic diagram of a non-limiting exemplary intramolecular G-quadruplex structure. [Figure 20]1 is a schematic diagram illustrating a workflow for generating circularization padlock probes, including generating first and second cDNAs from first and second target RNA molecules, respectively, and hybridizing the first and second padlock probes to the first and second cDNA molecules, respectively, to generate first and second circularization padlock probes, each of which contains (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 complementary sequence), (iii) a universal binding site for amplification primers (Universal RCA) (or its complementary sequence), and (iv) a universal binding site for compaction oligonucleotides (or its complementary sequence). The second padlock probe contains (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 complementary sequence), (iii) a universal binding site for the amplification primer (Universal RCA) (or its complementary sequence), and (iv) a universal binding site for the compaction oligonucleotide (or its complementary sequence). [Figure 21]Schematic diagram showing a rolling circle and sequencing workflow, which includes generating first and second concatemers by rolling circle amplification using first and second covalently closed circular molecules (respectively). The first and second concatemers are subjected to a first sequencing workflow using a first batch-specific sequencing primer, a sequencing polymerase, and multiple nucleotide reagents. The first concatemers undergo iterative sequencing, but the second concatemers do not. The first and second concatemers are subjected to a second sequencing workflow using a second batch-specific sequencing primer, a sequencing polymerase, and multiple nucleotide reagents. The second concatemers undergo iterative sequencing, but the first concatemers do not. [Figure 22] 1 is a 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 bound to a first oligonucleotide, which carries a first tag sequence 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 bound to a second oligonucleotide, which carries a third tag sequence and a fourth tag sequence. The first tag sequence, the second tag sequence, the third tag sequence, and the fourth tag sequence are different from each other. [Figure 23]1 is a schematic diagram showing first and second antibody-oligonucleotide conjugates bound to respective target polypeptides and respective padlock probes, respectively. The first padlock probe comprises (i) a sequence capable of hybridizing to a 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 complementary sequence), (iv) a universal binding site for amplification primers (Universal RCA) (or its complementary sequence), (v) a universal binding site for compaction oligonucleotides (or its complementary sequence), and (vi) a sequence capable of hybridizing to a second tag sequence. The second padlock probe comprises (i) a sequence capable of hybridizing to the third tag sequence, (ii) a fourth target barcode sequence (Target BC-4) that uniquely identifies a second antibody that selectively binds to the second target polypeptide, (iii) a second batch-specific sequencing primer binding site (Batch Seq-2) (or its complementary sequence), (iv) a universal binding site for the amplification primer (Universal RCA) (or its complementary sequence), (v) a universal binding site for the compaction oligonucleotide (or its complementary sequence), and (vi) a sequence capable of hybridizing to the fourth tag sequence. [Figure 24]This is a schematic diagram showing the rolling circle amplification and sequencing workflow for a first target RNA and a first target polypeptide. The first target cDNA molecule (top left schematic) hybridizes to a target-specific padlock probe for the first cDNA, which contains (i) first and second binding arms that hybridize to the first target cDNA, (ii) a first barcode sequence (BC-1) that uniquely identifies the first target cDNA, (iii) a first batch-specific sequencing primer binding site (Batch Seq-1), (iv) a universal binding site for the amplification primer, and (v) a universal binding site for the compaction oligonucleotide. The hybridized first padlock probe contains a nick or gap, which is enzymatically sealed to generate a first covalently closed circular molecule containing 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 is sequenced using a first batch-specific sequencing primer. A first antibody-oligonucleotide conjugate (top right schematic diagram) binds to the first target polypeptide. The oligonucleotide of the first antibody-oligonucleotide conjugate binds to the target-specific padlock probe of the first target polypeptide oligonucleotide conjugate, which contains (i) a first binding arm and a second binding arm that hybridize to the first tag sequence and the second tag sequence, (ii) a third barcode sequence (BC-3) that uniquely identifies the first antibody-oligonucleotide conjugate that selectively binds to the first target polypeptide, (iii) a first batch-specific sequencing primer binding site (Batch Seq-1), (iv) a universal binding site for the amplification primer, and (v) a universal binding site for the compaction oligonucleotide. The hybridized first padlock probe contains a nick or gap that is enzymatically closed to generate a third covalently closed circular molecule bearing the first tag sequence and the second tag sequence.The covalently closed circular molecule undergoes rolling circle amplification to generate a third oligonucleotide-tagged concatemer molecule (the bottom concatemer molecule), which is then sequenced using the first batch-specific sequencing primer. [Figure 25]This is a schematic diagram showing the rolling circle amplification and sequencing workflow for a second target RNA and a second target polypeptide. The second target cDNA molecule (top left schematic) hybridizes to a target-specific padlock probe for the second cDNA, which contains (i) first and second binding arms that hybridize to the second target cDNA, (ii) a second barcode sequence (BC-2) that uniquely identifies the second target cDNA, (iii) a second batch-specific sequencing primer binding site (Batch Seq-2), (iv) a universal binding site for the amplification primer, and (v) a universal binding site for the compaction oligonucleotide. The hybridized second padlock probe contains a nick or gap, which is enzymatically sealed to generate a second covalently closed circular molecule containing 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 is sequenced using a second batch-specific sequencing primer. A second antibody-oligonucleotide conjugate (top right schematic diagram) binds to the second target polypeptide. The oligonucleotide of the second antibody-oligonucleotide conjugate binds to the target-specific padlock probe of the second target polypeptide oligonucleotide conjugate, which contains (i) a first binding arm and a second binding arm that hybridize to the third tag sequence and the fourth tag sequence, (ii) a fourth barcode sequence (BC-4) that uniquely identifies the second antibody-oligonucleotide conjugate that selectively binds to the second target polypeptide, (iii) a second batch-specific sequencing primer binding site (Batch Seq-2), (iv) a universal binding site for the amplification primer, and (v) a universal binding site for the compaction oligonucleotide. The hybridized fourth padlock probe contains a nick or gap that is enzymatically closed to generate a fourth covalently closed circular molecule bearing the third tag sequence and the fourth tag sequence.The covalently closed circular molecule undergoes rolling circle amplification to generate a fourth oligonucleotide-tagged concatemer molecule (the bottom concatemer molecule), which is then sequenced using a second batch-specific sequencing primer. [Figure 26] 1 illustrates a computer system that is programmed or otherwise configured to perform the methods provided herein. [Figure 27] 1 depicts a non-limiting exemplary method of conjugating an antibody to multiple oligonucleotides via multiple linkers. Detection efficiency can be increased by adding linker moieties directly or via the multivalent structures described herein. This can reduce non-detection due to rolling circle amplification. [Figure 28] The present disclosure provides a non-limiting exemplary method.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 target RNA.The first oligonucleotide tag and the second oligonucleotide tag can be used to circularize the padlock probe disclosed herein.This method can increase binding specificity. [Figure 29] The present disclosure provides a non-limiting exemplary method.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 target RNA.The first oligonucleotide tag and the second oligonucleotide tag can be used to circularize the padlock probe disclosed herein.This method can increase binding specificity. DETAILED DESCRIPTION OF THE INVENTION

[0023] definition The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0024] Unless otherwise defined, technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art. Generally, the terms related to molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization techniques described herein are well known and commonly used in the art. The techniques and procedures described herein are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000), which is incorporated by reference in its entirety. Also see Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), which is incorporated by reference in its entirety. The nomenclature utilized in connection with the laboratory procedures and techniques described herein is that well known and commonly used in the art.

[0025] Unless otherwise required by context herein, singular terms shall include the plural and plural terms shall include the singular. The singular forms "a," "an," and "the," as well as use of the singular form of any word, include plural referents unless expressly and unambiguously limited to one referent.

[0026] It is understood that the use of alternative language (eg, "or") is to be construed as meaning either one or both of the alternatives or any combination thereof.

[0027] The term "and / or" used herein should be interpreted as meaning the specific disclosure of each of the specified features or components, regardless of the presence or absence of others. For example, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (A alone), or "B" (B alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: "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" (A alone), "B" (B alone), or "C" (C alone).

[0028] As used in this specification and the appended claims, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, when used herein, are intended to be open-ended, such that one or more items in a list do not exclude other items that may be substituted for or added to the listed items. Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0029] As used herein, the terms "about" and "approximately" refer to a value or composition that falls within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations, according to practice in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% (i.e., ±10%) or more, depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a specific value or composition is provided in this disclosure, unless otherwise specified, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that particular value or composition. Furthermore, when ranges and / or subranges of values ​​are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0030] The term "polymerase" and variations thereof, as used herein, includes enzymes that contain a nucleotide (or nucleoside)-binding domain, and the polymerase can form a complex with a template nucleic acid and a complementary nucleotide. A 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. A polymerase can be any enzyme that can catalyze the polymerization of nucleotides (including their analogs) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase contains one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, a polymerase contains other enzymatic activities, such as, for example, 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, as well as any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion, or other engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes that contain a nucleotide-binding domain. In some embodiments, polymerases can be isolated from cells or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases can be expressed in prokaryotic, eukaryotic, viral, or phage organisms. In some embodiments, polymerases can be post-translationally modified proteins or fragments thereof. Polymerases can be derived from prokaryotic, eukaryotic, viral, or phage organisms. Polymerases include DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0031] As used herein, the term "strand displacement" refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acid and synthesize a new strand in a template-directed manner. Strand-displacing polymerases displace the complementary strand from the template strand and catalyze new strand synthesis. Strand-displacing polymerases include mesophilic and thermophilic polymerases. Strand-displacing polymerases include wild-type enzymes as well as exonuclease-minus mutants, mutant versions, chimeric enzymes, and variants containing cleavage enzymes. Examples of strand-displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment (exo-) of Bsu DNA polymerase, large fragment (exo-) of Bca DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or a mutant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

[0032] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," as well as other related terms, are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular 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), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids (PNAs) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides comprise natural or non-natural bases and / or sugars. Nucleic acids comprise naturally occurring internucleoside linkages, such as phosphodiester linkages. Nucleic acids can lack phosphate groups. Nucleic acids comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise one type of polynucleotide or a mixture of two or more different species of polynucleotides.

[0033] "Operably linked" and "operably linked" or related terms, as used herein, refer to the juxtaposition of components. Juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically ligated together, where the bond linking the two components comprises a phosphodiester bond. A first nucleic acid component and a second nucleic acid component can bind to each other, where the first nucleic acid component can confer a function to the second nucleic acid component. For example, the bond between a primer binding sequence and a desired sequence forms a nucleic acid library molecule having a portion capable of binding to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or nucleic acid sequence of interest) can be ligated to a vector, where the bond allows for expression or function of the transgene sequence contained in the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. 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, host cell regulatory sequences control the level, timing, and / or location of expression of the transgene.

[0034] "Bound," "linked," "attached," "attached," and variations thereof include any type of fusion, bond, adhesion, or association between any combination of compounds or molecules that is stable enough to withstand use in a particular procedure. Procedures can include, but are not limited to, nucleotide binding, nucleotide incorporation, deblocking (e.g., removal of chain-terminating moieties), washing, removal, flow, detection, imaging, and / or identification, or any combination thereof. Such binding can include, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity binding, binding or association involving van der Waals forces, mechanical binding, etc., or any combination thereof. In some embodiments, such binding occurs intramolecularly, for example, by joining the ends of a single- or double-stranded linear nucleic acid molecule to form a circular molecule. In some embodiments, such binding can occur between different molecular combinations or between a molecule and a non-molecular entity, including, but not limited to, binding between a nucleic acid molecule and a solid surface, binding between a protein and a detectable reporter moiety, binding between a nucleotide and a detectable reporter moiety, etc. Some examples of conjugation can be found, for example, in Hermanson, G., "Bioconjugate Techniques", Second Edition (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 entireties.

[0035] As used herein, the term "primer" and related terms refer to an oligonucleotide capable of hybridizing to a DNA and / or RNA polynucleotide template to form a duplex molecule. Primers contain natural nucleotides and / or nucleotide analogs. Primers may be recombinant nucleic acid molecules. Primers can be of any length but typically range from 4 to 50 nucleotides. A typical primer contains a 5' end and a 3' end. The 3' end of a primer can contain a 3' OH moiety that functions 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 moiety or can contain a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one or more nucleotides along the length of the primer can be labeled with a detectable reporter moiety. Primers can be in solution (e.g., soluble primers) or immobilized on a support (e.g., capture primers).

[0036] The terms "template nucleic acid," "template polynucleotide," "target nucleic acid," "target polynucleotide," "template strand," and other variations refer to a nucleic acid strand that serves as the base nucleic acid molecule for any of the iterative sequencing methods described herein. A template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. A template nucleic acid can be obtained from natural sources, recombinant forms, or chemically synthesized to contain any type of nucleic acid analog. A template nucleic acid can be linear, circular, or in other forms. A template nucleic acid can include an insert having an insert sequence. A template nucleic acid can further include at least one adapter sequence. An insert can be isolated in any form, including from a chromosome, genome, organelle (e.g., mitochondria, chloroplast, or ribosome), recombinant molecule, cloned, amplified, cDNA, RNA, e.g., precursor mRNA or mRNA, oligonucleotide, total 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 prokaryotic, eukaryotic (e.g., human, plant, and animal), fungal, viral cells, tissues, normal or diseased cells or tissues, biological fluids including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid samples, sweat, semen, environmental samples, and 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, ovaries, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testes, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary gland, thymus, skin, heart, larynx, or other organs. Template nucleic acids can be subjected to nucleic acid analysis, including sequencing and compositional analysis.

[0037] The term "adapter" and related terms refer to an oligonucleotide capable of operably binding to a target polynucleotide, where the adapter confers functionality to the co-ligated adapter-target molecule. Adapters can comprise DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adapters can contain at least one ribonucleoside residue. Adapters can be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. Adapters can be configured in linear, stem-loop, hairpin, or Y-shaped configurations. Adapters can be any length, from 4 to 100 nucleotides or more. Adapters can have blunt ends, overhanging ends, or a combination of both. Overhanging ends include 5' overhangs and 3' overhangs. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, can have a 5' phosphate group or lack a 5' phosphate group. The adapter may include a 5' tail that does not hybridize to the target polynucleotide (e.g., a tailed adapter), or the adapter may be non-tailed. The adapter may include a sequence complementary to at least a portion of 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 primer. The adapter may include at least one inosine residue. The adapter may include at least one phosphorothioate, phosphorothiolate, and / or phosphoramidate linkage. The adapter may include a barcode sequence that can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex assays. The adapter may include a unique identifying sequence (e.g., a unique molecular index, UMI, or unique molecular tag) that can be used to uniquely identify the nucleic acid molecule to which the adapter is attached. In some embodiments, the unique identifying sequence can be used to increase error correction and accuracy, reduce the rate of false-positive variant calls, and / or increase the sensitivity of variant detection.The adaptor can comprise at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from the group consisting of Type I, Type II, Type III, Type IV, Hs, or Type IIB.

[0038] In some embodiments, any of the 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 to 50 nucleotides in length, or about 5 to 40 nucleotides in length, or about 5 to 25 nucleotides in length.

[0039] The term "universal sequence" and related terms refer to a sequence in a nucleic acid molecule that is common to two or more polynucleotide molecules. For example, an adapter having a universal sequence can be operably linked to multiple polynucleotides such that a population of co-linked molecules retains the same universal adapter sequence. Examples of universal adapter sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or immobilized capture primers).

[0040] When used in reference to nucleic acid molecules, the terms "hybridize" or "hybridizing" or "hybridization," or other related terms, refer to hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule with a double-stranded region. Hybridization can involve Watson-Crick or Hoogsteen binding to form a double-stranded double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or two different regions of a single nucleic acid, can be fully complementary or partially complementary. Complementary nucleic acid strands need not hybridize to each other over their entire length. Complementary base pairing can be standard AT or CG base pairing or other forms of base pairing interactions. Double-stranded nucleic acids can contain mismatched base pair nucleotides.

[0041] 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 to the terminal 3'OH end of a nucleic acid chain, resulting in the 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 a nucleic acid molecule can be used, including primer extension catalyzed by DNA polymerase or RNA polymerase.

[0042] The term "nucleotide" and related terms refer to a molecule comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Classical nucleotide or non-classical nucleotide is consistent with this use of the term. Phosphate in some embodiments includes monophosphate, diphosphate, or triphosphate, or the corresponding phosphate analogs. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be unlabeled or labeled with a detectable reporter moiety.

[0043] Nucleotides (and nucleosides) typically contain a heterocyclic base that contains a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring normally found in nucleic acids, including naturally occurring, substituted, modified, or engineered variants, or analogs thereof. The base of a nucleotide (or nucleoside) can form Watson-Crick and / or Hoogsteen hydrogen bonds with an appropriate complementary base. Non-limiting examples of bases include, but are not limited to, purines and pyrimidines, such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N-Δ-isopentenyladenine (6iA), N-Δ-isopentenyl-2-methylthioadenine (2ms6iA), N-methyladenine, guanine (G), isoguanine, N-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine, and O-methylguanine, 7-deazapurines, such as 7-deazaadenine (7-deaza-A) and 7-deaza- 7-deazaguanine (7-deaza-G), pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, 4-methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil, D), indoles such as nitroindole and 4-methylindole, pyrroles such as nitropyrrole, nebularine, inosine, hydroxymethylcytosine, 5-methylcytosine, base (Y), as well as methylated, glycosylated, and acylated moieties. Further 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.

[0044] Nucleotides (and nucleosides) typically comprise a sugar moiety, e.g., a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48, which is incorporated herein by reference in its entirety), an acyclic moiety (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 another sugar moiety (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. Pat. No. 5,558,991, all of which are incorporated herein by reference in their entireties. 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.

[0045] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, typically linked to the 5' carbon of the sugar moiety via an ester or phosphoramidate bond. In some embodiments, the nucleotide is an analog having a phosphorus chain linked together with intervening O, S, NH, methylene, or ethylene atoms. In some embodiments, the phosphorus atoms in the chain comprise a substituted side chain group comprising O, S, or BH3. In some embodiments, the chain comprises a phosphate group substituted with an analog comprising a phosphoramidate group, a phosphorothioate group, a phosphorodithioate group, and an O-methylphosphoramidite group.

[0046] The terms "reporter moiety" and "reporter moieties" or related terms refer to a compound that generates or causes the generation of a detectable signal. Reporter moieties are often called "labels." Any suitable reporter moiety may be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, haptens, affinity tags, atoms, or enzymes. A reporter moiety generates a detectable signal that results from a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzymatic activity, or a proximity event). A proximity event is the proximity of two reporter moieties to each other, or their association with each other, or their interaction with each other. In some embodiments, reporter moieties may be selected so that each reporter moiety absorbs excitation radiation and / or fluoresces at a wavelength that is distinguishable from other reporter moieties, thereby allowing the presence of different reporter moieties to be monitored in the same or different reactions. Two or more different reporter moieties may be selected that have spectrally distinct emission profiles or that have minimally overlapping spectral emission profiles. The reporter moiety may be attached (e.g., operably attached) to a nucleotide, a nucleoside, a nucleic acid, an enzyme (e.g., a polymerase or reverse transcriptase), or a support (e.g., a surface).

[0047] The reporter moiety (or label) can comprise a fluorescent label or fluorophore. Non-limiting exemplary oral moieties that can serve as fluorescent labels or fluorophores include, but are not limited to, fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluoresceinthiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, rhodamine, fluorescein thiosemicarbazide ... Rhodamine and rhodamine derivatives, such as TRITC, TMR, Lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, Lissamine rhodamine B sulfonyl chloride, Lissamine rhodamine B sulfonyl hydrazine, 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, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow Yellow) and derivatives, such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanines and derivatives, such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives,For example, 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 dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbenes, DEG dyes, NR dyes, near-infrared dyes, and others known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999); or Hermanson, Bioconjugate Techniques, 2nd Edition (both of which are incorporated herein by reference in their entireties), or derivatives thereof, or any combination thereof. Cyanine dyes may exist in sulfonated or non-sulfonated forms and may consist of two indolenine, benzo-indolium, pyridinium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (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(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-indolin-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-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)- Cy7 (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-5-sulfonate), and Cy8 (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 number identifies the number of carbon atoms between the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are non-limiting examples of exceptions to this rule.

[0048] In some embodiments, the reporter moieties may be FRET pairs so that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may include excitation exchange (Förster) transfer or electron exchange (Dexter) transfer.

[0049] When used in reference to nucleic acids, the terms "amplify," "amplifying," "amplification," and other related terms include making multiple copies of an original polynucleotide template molecule, where the copies contain sequences complementary to the template sequence or where the copies contain sequences that are identical to the template sequence. In some embodiments, the copies contain sequences that are substantially identical to the template sequence or to sequences that are complementary to the template sequence.

[0050] The term "support" as used herein refers to a substrate designed for the deposition of biomolecules or biological samples for assay and / or analysis. Examples of biomolecules deposited on a support include nucleic acids (e.g., DNA, RNA), polypeptides, sugars, 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.

[0051] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, comprising the inner surface of a capillary or capillary.

[0052] In some embodiments, the surface of the support can be substantially smooth, hi some embodiments, the support can be regularly or irregularly textured, including protrusions, etchings, pores, a three-dimensional scaffold, or any combination thereof.

[0053] In some embodiments, the support comprises beads having any shape, including spherical, hemispherical, cylindrical, barrel-shaped, toroidal, disk-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or wire-shaped.

[0054] The support can be fabricated from any material, including, but not limited to, glass, fused silica, silicon, polymer (e.g., polystyrene (PS), macroporous polystyrene (1VIPPS), 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 compositions of both glass and plastic substrates are contemplated.

[0055] The support can have multiple (e.g., two or more) nucleic acid templates immobilized thereon. The multiple immobilized nucleic acid templates may have the same sequence or different sequences. In some embodiments, individual nucleic acid template molecules in the multiple nucleic acid templates are immobilized at different sites on the support. In some embodiments, two or more individual nucleic acid molecules in the multiple nucleic acid templates are immobilized at sites on the support.

[0056] The term "array" refers to a support comprising a plurality of sites arranged at predetermined locations on the support to form an array of sites. The sites may be dispersed 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, the plurality of predetermined sites are arranged on the support in an organized manner. In some embodiments, the plurality of predetermined sites are arranged in any organized pattern, including a rectilinear pattern, a hexagonal pattern, a lattice pattern, a pattern with mirror symmetry, a pattern with rotational symmetry, etc. The pitch between different pairs of sites may be the same or different. In some embodiments, the support comprises at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, or more, where the sites are located at predetermined locations on the support. In some embodiments, a plurality of predetermined sites (e.g., 10 to 10 sites or more) on the support are immobilized with nucleic acid templates to form a nucleic acid template array. In some embodiments, the nucleic acid templates are immobilized at a plurality of predetermined sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to surface capture primers. In some embodiments, the nucleic acid templates are immobilized at a plurality of predetermined sites, e.g., 10 to 10 sites or more. In some embodiments, the immobilized nucleic acid template is clonally amplified to generate clusters of immobilized nucleic acids at multiple predetermined sites, hi some embodiments, the individual immobilized nucleic acid clusters comprise linear clusters or comprise single- or double-stranded concatemers.

[0057] In some embodiments, a support comprising a plurality of sites located at random locations on the support is referred to herein as a support having randomly located sites thereon. The locations of the randomly located sites on the support are not predetermined. The plurality of randomly located sites are arranged on the support in a disordered and / or unpredictable manner. In some embodiments, the support comprises at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, at least 10 sites, or more, where the sites are randomly located on the support. In some embodiments, the plurality of randomly located sites on the support (e.g., 10 to 10 sites or more) are immobilized with a nucleic acid template to form a nucleic acid template-immobilized support. In some embodiments, the nucleic acid template is immobilized at multiple randomly located sites by hybridization to 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 immobilized at multiple randomly located sites, for example, at 10 to 10 sites or more. In some embodiments, the immobilized nucleic acid template is clonally amplified to generate immobilized nucleic acid clusters at multiple randomly located sites. In some embodiments, the individual immobilized nucleic acid clusters comprise linear clusters or single- or double-stranded concatemers.

[0058] In some embodiments, multiple immobilized surface capture primers on a support are in fluid communication with each other, allowing solutions of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, etc.) to be flowed over the support such that the multiple immobilized surface capture primers on the support can react with reagents essentially simultaneously in a massively parallel manner. In some embodiments, the fluid communication of multiple immobilized surface capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) essentially simultaneously on the multiple immobilized surface capture primers.

[0059] In some embodiments, multiple immobilized nucleic acid clusters on a support are in fluid communication with each other, allowing solutions of reagents (e.g., enzymes, nucleotides, divalent cations, etc.) to be flowed over the support so that multiple immobilized nucleic acid clusters on the support can react with reagents essentially simultaneously in a massively parallel manner. In some embodiments, the fluid communication of multiple immobilized nucleic acid clusters can be used to perform nucleotide binding assays and / or nucleotide polymerization reactions (e.g., primer extension or sequencing) essentially simultaneously on multiple immobilized nucleic acid clusters, and optionally to perform massively parallel sequencing detection and imaging.

[0060] When used in reference to immobilized enzymes, the term "immobilized" and related terms refer to an enzyme (e.g., a polymerase) that is bound to a support through covalent or non-covalent interactions, or that is bound to a coating on a support, or that is embedded within a matrix formed by a coating on a support.

[0061] When used in reference to immobilized nucleic acids, the term "immobilized" and related terms refer to nucleic acid molecules that are bound to a support through covalent or non-covalent interactions, or to a coating on a support, or embedded within a matrix formed by a coating on a support, and 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).

[0062] In some embodiments, one or more nucleic acid templates are immobilized on a support, e.g., immobilized at sites on a support. In some embodiments, one or more nucleic acid templates are clonally amplified. In some embodiments, one or more nucleic acid templates are clonally amplified off-support (e.g., in solution) and then deposited on a support and immobilized thereon. In some embodiments, a clonal amplification reaction of one or more nucleic acid templates is performed on a support and immobilized thereon. In some embodiments, one or more nucleic acid templates are clonally amplified (e.g., in solution or on a support) using a nucleic acid amplification reaction including any one or any combination of polymerase chain reaction (PCR), multiplex displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-strand binding (SSB) protein-dependent amplification.

[0063] The term "duration" and related terms refer to the length of time that a binding complex formed between a target nucleic acid, a polymerase, and a conjugated or unconjugated nucleotide remains stable without the binding components dissociating from the binding complex. This duration indicates the stability of the binding complex and the strength of the binding interaction. Duration can be measured by observing the occurrence and / or duration of the binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeling reagent containing one or more nucleotides is present in the binding complex, and therefore, a signal from the label can be detected during the duration of the binding complex. One non-limiting example of a label is a fluorescent label.

[0064] Introduction 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. The methods, compositions, systems, and kits described herein can be used to obtain information about the location of nucleic acid sequences, such as RNA or DNA, and proteins encoded by the 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 target proteins or polypeptides can be identified by padlock probes as described herein and further amplified and / or sequenced. In this way, information about the location and / or sequence of target proteins, RNA sequences, and DNA sequences can be collected.

[0065] In some embodiments, the present disclosure provides methods, compositions, systems, and kits for use in processing and / or analyzing nucleic acid sequences. The methods, systems, kits, and compositions described herein can be used in in situ repeat short-read sequencing. Sequencing can be performed on one or more cells, tissues, or tumors. The nucleic acid sequence can be, for example, a DNA sequence or an RNA sequence. The methods, systems, kits, and compositions described herein can be useful in utilizing supersequence sequencing technology. Such methods, compositions, kits, and systems can further be found in US Patent Publication US20210139884.

[0066] method The present disclosure provides methods for analyzing nucleic acid sequences. The methods 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 a single nucleic acid sequence. Identifying may include performing any of the methods disclosed herein. In some embodiments, the methods disclosed herein do not require the use of barcoded nucleic acids and instead utilize spatially encoded reagents. For example, spatially encoded primers on a solid surface may be identified by their relative position on the solid surface. The methods described herein may use in situ iterative short-read sequencing within a cellular sample, including a single cell, multiple cells, tissues, or tumors. In some embodiments, the method includes repeatedly performing a short number of sequencing cycles of the same region of a template molecule. By performing the iterative short sequencing cycles, the RNA content of a cellular sample can be discovered. The methods described herein may be used in performing transcriptomics workflows that utilize massively parallel sequencing technology. Compared with long-read sequencing workflow, the iterative short sequencing cycle described herein uses less sequencing reagent, which reduces cost and saves time.The method for carrying out iterative short sequencing cycle has many uses, including but not limited to, detecting specific RNA of interest, mutant RNA sequence, splice variant and their abundance level.

[0067] One non-limiting example of the objective of the methods described herein is to detect and image the spatial localization of RNA within a cell sample using massively parallel sequencing. The workflow can include the following general steps: The cell sample can be placed on a solid support that can be placed on a fluorescence microscope configured to detect fluorescent signals. The cells can be treated with a chemical fixation reagent to retain the target RNA inside the cells. The cell sample can be further permeabilized to enable manipulation of the target RNA inside the cells. The target RNA can be converted into first-strand cDNA that can selectively hybridize to target-specific padlock probes to generate circularized padlock probes corresponding to specific cDNAs. The padlock probes can carry at least universal adapters for sequencing primer binding sites. The padlock probes can further carry target barcode sequences, each corresponding to a given target cDNA. The circularized padlock probes can be subjected to ligation and / or fill-in reactions to form covalently closed circular padlock probes, which can be amplified within the cell sample by rolling circle amplification to generate single-stranded concatemers. Rolling circle amplification can be performed in the presence of one or more compaction oligonucleotides. The concatemer can retain the tandem repeat unit of the target cDNA, the universal sequencing primer binding site, and the target barcode sequence. The concatemer can be sequenced within a cell sample, and a short number of sequencing cycles are performed for each round, performing multiple rounds of short-read sequencing. The entire length of the target barcode and cDNA region does not need to be sequenced. Instead, at least a portion of the target barcode region can be sequenced repeatedly. In some embodiments, the cDNA region does not need to be sequenced. In some embodiments, the target barcode and a portion of the cDNA region are sequenced repeatedly. The entire length of the cDNA region may not need to be sequenced. It may not be necessary to assemble sequencing reads or obtain the full-length sequence of the target cDNA.The redundant sequencing information obtained from short sequencing reads can eliminate the need to sequence the complementary strands of concatemers, and therefore pairwise sequencing may not be necessary.

[0068] The methods described herein can offer several advantages over other in situ transcriptomics workflows. For example, cell samples can be placed on a solid support, such as a planar support comprising glass or plastic, which can be processed into any shape and size. Assembling a hybridization chamber on the support may not be necessary. Preparation of chemically cleaned glass beads for cell attachment may also not be necessary. The support can be passivated with a coating that promotes cell attachment to the support. The coating may not need to be formulated to contain a tethered capture primer. The support with the cell sample attached thereon can be fluidly connected to an automated fluid dispensing system and easily adapted to fit into an existing flow cell holder / cradle configured on a fluorescence microscope. Any combination of steps for performing in situ iterative short-read sequencing, including cell seeding, cell fixation, cell permeabilization, reverse transcription, padlock probe hybridization, padlock probe ligation, rolling circle amplification, and sequencing, can be performed in an automated mode using the fluid dispensing system.

[0069] Another advantage of the methods described herein is the formation of concatemers within a cell sample. Single-stranded concatemers may fold into compact DNA nanoballs, each carrying many tandem copies of a polynucleotide unit along its length, the polynucleotide unit containing the cDNA sequence of interest and at least a universal sequencing primer binding site. Each polynucleotide unit can bind to a sequencing primer, a sequencing polymerase, and a detectably labeled nucleotide reagent (e.g., a detectably labeled multivalent molecule) to form a detectable sequencing complex (e.g., a detectable ternary complex). Each nanoball carries many detectable sequencing complexes. The compact nature of the nanoballs can therefore increase the local concentration of the detectably labeled nucleotide reagent used during the sequencing workflow, thereby increasing the signal intensity emitted from the nanoballs. This gives rise to discrete detectable signals that can be imaged as fluorescent spots within the cell sample. Each spot corresponds to a concatemer, and each concatemer corresponds to a target RNA molecule in the cell sample. Multiple spots can be detected and imaged simultaneously in a cell sample.

[0070] Additionally, short portions of the cDNA region in the concatemer can be resequenced at least once from the same starting position (e.g., iterative sequencing) to generate overlapping sequencing reads that can be aligned to a reference sequence. For example, the same portion of the concatemer molecule can be sequenced at least twice, three times, four times, five times, or even up to 50 times. The starting sequencing site can be anywhere in the concatemer and can be directed by a sequencing primer designed to anneal to a selected position within the concatemer. Iterative short sequencing reads can increase the redundancy of sequencing information for individual bases in the cDNA region. Iterative sequencing of one strand of the concatemer template molecule can provide sufficient base coverage to reveal the presence of target RNA in a cell sample, eliminating the need for pairwise sequencing of complementary strands.

[0071] Concatemer template molecules may contain multiple sequencing primer binding sites along the same concatemer molecule, which can be used to generate multiple usable sequencing reads to increase sequencing depth. In summary, repeatedly sequencing one strand of a concatemer template can increase sequencing base coverage and sequencing depth compared to sequencing a single copy of a template molecule.

[0072] The method described herein can be carried out in uniplex or multiplex mode.Two or more different target RNAs can be simultaneously detected and imaged in a cell sample by using different reverse transcription primers, different target-specific padlock probes, and universal sequencing primers.For example, the presence of housekeeping RNA and at least one target RNA in a cell sample can be simultaneously detected and imaged using any of the iterative short-read sequencing methods described herein.

[0073] In the methods described herein, RNA may not need to be extracted from the cell sample, and sequencing information may not need to be tracked or remapped to an image of the cell sample. Rather, RNA may be retained within the cell sample to enable direct imaging of the spatial location of target RNA within the cell. Furthermore, RNA within the cell sample may not need to be fragmented, and enrichment of target RNA may not be necessary. The use of target-specific and / or random-sequence reverse transcription primers allows for the detection of both polyA RNA and non-polyA RNA in either uniplex or multiplex mode.

[0074] The methods described herein offer several advantages over other in situ transcriptomics workflows, including a simpler workflow, fewer reagents, lower cost, shorter time, gentler conditions on the cell sample, and no need for specialized equipment.

[0075] Methods for performing in situ iterative short-read sequencing The present disclosure provides a method for performing in situ sequencing in biological samples.One or more nucleic acids can be analyzed in biological samples, for example, cells.Nucleic acid can include, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).Sequencing can be performed repeatedly, so that multiple sequencing cycles of nucleic acid are performed.

[0076] 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 can include a first target nucleic acid sequence and a second target nucleic acid sequence. The biological sample can be a cell sample. The method can include performing a sequencing reaction within the cell sample, and the cDNA amplicon can be a concatemeric molecule. The biological sample can include a first nucleic acid molecule, a second nucleic acid molecule, or a combination thereof. The first nucleic acid molecule can include a first target nucleic acid sequence or a portion thereof, or a reverse complementary sequence or a portion thereof. The second nucleic acid molecule can include a second target nucleic acid sequence or a portion thereof, or a reverse complementary sequence or a portion thereof. The method can include in situ determining the sequence of 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. The method may include determining the sequence of a second nucleic acid molecule or a portion thereof in a biological sample 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 complete sequence of the first target nucleic acid sequence and the complete 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. The first nucleic acid molecule and the second nucleic acid molecule may be located within the biological sample after removal. The method may further include repeating the step of determining the sequence of the first nucleic acid and the sequence of the second nucleic acid in situ. In some embodiments, the repeating step includes repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times. The method may further include repeating the steps of 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.In some embodiments, the repeating step comprises repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times.

[0077] The present disclosure provides a method for in situ detection of at least two different target nucleic acid molecules in a biological sample. The present disclosure provides a method for in situ detection of at least two different target RNA molecules in a cellular sample, the method comprising providing a cellular sample deposited on a solid support, the cellular sample having at least a first plurality of DNA amplicons corresponding to a first target RNA molecule and a second plurality of DNA amplicons corresponding to a second target RNA molecule. In some embodiments, the cellular sample can contain a first target RNA molecule. In some embodiments, the cellular sample can contain at least a first plurality of DNA amplicons that can correspond to the first target RNA molecule. In some embodiments, the cellular sample can contain a second target RNA molecule. In some embodiments, the cellular sample can contain at least a second plurality of DNA amplicons that can correspond to the second target RNA. In some embodiments, the cellular sample can be deposited on a solid support.

[0078] In some embodiments, the cell sample has 2-25 different target RNA molecules, or 25-50 different target RNA molecules, or 50-75 different target RNA molecules, or 75-100 different target RNA molecules. In some embodiments, the cell sample has 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 has more than 10,000 different target RNA molecules. In some embodiments, the cell sample comprises whole cells, a plurality of whole cells, an intact tissue, or an intact tumor. In some embodiments, the cell sample comprises a fresh cell sample, a fresh frozen cell sample, a sectioned cell sample, or an 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 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 cultured in simple or complex cell culture media.

[0079] 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.

[0080] In some embodiments, the first plurality of DNA amplicons comprises a first target DNA sequence corresponding to a first target RNA molecule. In some embodiments, the first plurality of DNA amplicons comprises a first target DNA sequence corresponding to the 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 comprises a first target DNA sequence corresponding to the first target RNA molecule and at least one universal adapter sequence, such as a universal sequencing primer binding site (or its complementary sequence). In some embodiments, the first plurality of DNA amplicons comprises a first target DNA sequence corresponding to the first target RNA molecule and a universal primer binding site (or its complementary sequence) of a rolling circle amplification primer. In some embodiments, the first plurality of DNA amplicons comprises a first target DNA sequence corresponding to the first target RNA molecule and a universal compaction oligonucleotide binding site (or its complementary sequence).

[0081] In some embodiments, the second plurality of DNA amplicons comprises a second target DNA sequence corresponding to a second target RNA molecule. In some embodiments, the second plurality of DNA amplicons comprises 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 comprises 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 complementary sequence). In some embodiments, the second plurality of DNA amplicons comprises a second target DNA sequence corresponding to a second target RNA molecule and a universal primer binding site (or its complementary sequence) of a rolling circle amplification primer. In some embodiments, the second plurality of DNA amplicons comprises a second target DNA sequence corresponding to a second target RNA molecule and a universal compaction oligonucleotide binding site (or its complementary sequence).

[0082] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cellular sample further includes (b) sequencing a first plurality of DNA amplicons within the cellular sample. The sequencing step may include performing 2 to 30 or fewer sequencing cycles to generate a plurality of first sequencing read products, and sequencing a second plurality of DNA amplicons within the cellular sample, which may include performing 2 to 30 or fewer sequencing cycles to generate a plurality of second sequencing read products. In some embodiments, (b) may include sequencing the first plurality of DNA amplicons within the cellular sample, which may include performing 2 to 30 or fewer sequencing cycles. In some embodiments, 2 to 30 sequencing cycles can generate a plurality of first sequencing read products. In some embodiments, (b) may include sequencing the second plurality of DNA amplicons within the cellular sample, which may include performing 2 to 30 or fewer sequencing cycles. In some embodiments, 2 to 30 sequencing cycles can generate multiple second sequencing read products. 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 cellular 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 cellular sample. 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 cellular sample, and 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 cellular sample.

[0083] In some embodiments, the first target reference sequence comprises the first target barcode sequence. In some embodiments, the first target reference sequence comprises the first target barcode sequence and at least a portion of the first target RNA sequence. In some embodiments, the first target reference sequence comprises at least a portion of the first target RNA sequence.

[0084] 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.

[0085] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cell sample can 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) can include removing a plurality of first sequencing read products from the first DNA amplicon. In some embodiments, (c) can include retaining the first DNA amplicon within the cell sample. In some embodiments, (c) can include removing a plurality of second sequencing read products from the second DNA amplicon. In some embodiments, (c) can include retaining the second DNA amplicon within the cell sample.

[0086] In some embodiments, the method for in situ detecting at least two different target RNA molecules in a cellular sample further includes (c) removing a plurality of first sequencing read products from the first DNA amplicon and retaining the first DNA amplicon within the cellular sample, and removing a plurality of second sequencing read products from the first DNA amplicon and retaining the second DNA amplicon within the cellular sample.

[0087] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cell sample can further include (d) iteratively sequencing the first plurality of DNA amplicons and the second plurality of DNA amplicons by repeating (b) and (c) at least once. In some embodiments, the repeating step includes repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times. In some embodiments, (d) can include iteratively sequencing the first plurality of DNA amplicons by repeating (b) and (c) at least once. In some embodiments, (d) can include iteratively sequencing the second plurality of DNA amplicons by repeating (b) and (c) at least once. In some embodiments, (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, (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.

[0088] In some embodiments, the method for in situ detection of at least two different target RNA molecules in a cell sample can further include (d) iteratively sequencing the first plurality of DNA amplicons and the second plurality of DNA amplicons by repeating steps (b) and (c) at least once. In some embodiments, steps (b) and (c) can be repeated at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times. In some embodiments, steps (b) and (c) can be repeated up to 10, up to 20, up to 30, up to 40, or up to 50 times.

[0089] In some embodiments, the sequences of the first sequencing read product and the second sequencing read product can be aligned after each round of generating first sequencing read products and second sequencing read products that are 30 bases or less in length (e.g., after step (b)), or after generating a set of replicate sequencing read products, wherein the first sequencing read product and the second sequencing read product are 30 bases or less in length (e.g., after step (d)).

[0090] The present disclosure provides methods for in situ detection of at least two different target RNA molecules in a cell sample, the method comprising: (a) providing a cell sample having a plurality of RNAs, including at least a first target RNA molecule and a second target RNA molecule, wherein the cell sample has been fixed and permeabilized. In some embodiments, the cell sample has 2 to 25 different target RNA molecules, or 25 to 50 different target RNA molecules, or 50 to 75 different target RNA molecules, or 75 to 100 different target RNA molecules. In some embodiments, the cell sample has 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 has more than 10,000 different target RNA molecules. In some embodiments, the cell sample comprises whole cells, a plurality of whole cells, an intact tissue, or an intact tumor. In some embodiments, the cell sample comprises a fresh cell sample, a fresh frozen cell sample, a sectioned cell sample, or an 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 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 plurality of RNAs comprises at least a first target RNA molecule. In some embodiments, the plurality of RNAs comprises at least a second target RNA molecule. In some embodiments, the cell sample is cultured before performing step (b) described below.

[0091] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample further includes (b) generating, within the cell sample, a plurality of cDNA molecules comprising at least a first target cDNA molecule corresponding to a first target RNA molecule, wherein the plurality of cDNA molecules comprises a second target cDNA molecule corresponding to a second target RNA molecule. One cDNA molecule of the plurality of cDNA molecules may be generated by reverse transcription of a messenger RNA (mRNA) molecule within the biological sample, wherein the cDNA molecule or mRNA molecule comprises a target nucleic acid sequence or a reverse complementary sequence of the target nucleic acid sequence. The plurality of cDNA molecules may be generated by reverse transcription of one or more messenger RNA (mRNA) molecules within the biological sample, wherein the plurality of cDNA molecules or one or more mRNA molecules comprise a target nucleic acid sequence or a reverse complementary sequence of the 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 a biological sample, wherein the plurality of cDNA molecules or the one or more mRNA molecules comprises a target nucleic acid sequence or a reverse complement of the 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 step of step (b) includes contacting a plurality of RNAs within the cellular 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 cellular sample (e.g., FIG. 1). In some embodiments, the plurality of reverse transcription primers comprises a first sub-population of target-specific reverse transcription primers that selectively hybridize to a first target RNA and a second sub-population of target-specific reverse transcription primers that selectively hybridize to a second target RNA. In some embodiments, the plurality of reverse transcription primers comprises a first sub-population of random-sequence reverse transcription primers that hybridize to the first target RNA and a second sub-population of random-sequence reverse transcription primers that hybridize to the second target RNA.

[0092] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample can include (b) generating, within the cell sample, a plurality of cDNA molecules, each of which can include at least a first target cDNA molecule that can correspond to a first target RNA molecule. The plurality of cDNA molecules can include a second target cDNA molecule that can correspond to a second target RNA molecule. In some embodiments, the plurality of cDNA molecules can include at least a first target cDNA molecule. In some embodiments, the first target cDNA molecule can correspond to the first target RNA molecule. In some embodiments, the plurality of cDNA molecules can include at least a second target cDNA molecule. In some embodiments, the second target cDNA molecule can correspond to the second target RNA molecule. In some embodiments, the method can include generating at least 2 to 10,000 different target cDNA molecules that can correspond to 2 to 10,000 different target RNA molecules. In some embodiments, the generating step of step (b) can include contacting the plurality of RNA molecules within the cell sample with (i) a plurality of reverse transcription primers, (ii) a plurality of reverse transcriptases, and (iii) a plurality of nucleotides. In some embodiments, the generating step of 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., FIG. 1 ). In some embodiments, the generating step of step (b) can comprise contacting a plurality of RNAs within the cell sample with a plurality of reverse transcription primers. In some embodiments, the generating step of step (b) can comprise contacting a plurality of RNAs within the cell sample with a plurality of reverse transcriptases. In some embodiments, the generating step of step (b) can comprise contacting a plurality of RNAs within the cell sample with a plurality of nucleotides. In some embodiments, the plurality of reverse transcription primers can comprise a first subpopulation of target-specific reverse transcription primers capable of selectively hybridizing to a first target RNA.In some embodiments, the plurality of reverse transcription primers can comprise a second subpopulation of target-specific reverse transcription primers capable of selectively hybridizing to a second target RNA. In some embodiments, the plurality of reverse transcription primers can comprise a first subpopulation of target-specific reverse transcription primers. In some embodiments, the first subpopulation of target-specific reverse transcription primers can selectively hybridize to the first target RNA. In some embodiments, the plurality of reverse transcription primers can comprise a second subpopulation of target-specific reverse transcription primers. In some embodiments, the second subpopulation of target-specific reverse transcription primers can selectively hybridize to the second target RNA. In some embodiments, the plurality of reverse transcription primers can comprise a first subpopulation of random-sequence reverse transcription primers capable of hybridizing to the first target RNA and a second subpopulation of random-sequence reverse transcription primers capable of hybridizing to the second target RNA. In some embodiments, the plurality of reverse transcription primers can comprise a first subpopulation of random-sequence reverse transcription primers. In some embodiments, the first subpopulation of random-sequence reverse transcription primers can hybridize to the first target RNA. In some embodiments, the plurality of reverse transcription primers can comprise a second subpopulation of random-sequence reverse transcription primers, which can hybridize to a second target RNA.

[0093] In some embodiments, the method for detecting at least two different target RNA molecules in a cellular sample can include (c) contacting a plurality of cDNA molecules in the cellular 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 can comprise at least a first plurality of target-specific padlock probes. In some embodiments, the plurality of target-specific padlock probes can comprise at least a second plurality of target-specific padlock probes. In some embodiments, the method can include contacting a plurality of cDNA molecules in the cellular sample with at least 2 to 10,000 different target-specific padlock probes.

[0094] In some embodiments, each padlock probe in the plurality of first target-specific padlock probes comprises a first end and a second end (e.g., a first padlock binding arm 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 step (c) comprises hybridizing the first and second ends of the first target-specific padlock probe to proximal positions on the first target cDNA molecule to form a circularized first target-specific padlock probe having a nick or gap between the hybridized first and second ends (e.g., FIG. 1). In some embodiments, the first target-specific padlock probe comprises a first target barcode sequence corresponding to the first target cDNA sequence. In some embodiments, the first target-specific padlock probe comprises a first target barcode sequence located adjacent to one of the regions of the first target-specific padlock probe that selectively hybridize 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 complementary sequence). In some embodiments, the first target-specific padlock probe comprises a universal primer binding site (or its complementary sequence) for a rolling circle amplification primer. In some embodiments, the first target-specific padlock probe comprises a universal compaction oligonucleotide binding site (or its complementary sequence).

[0095] 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 terminal portion and a second terminal portion. The first terminal portion and the second terminal portion of the first oligonucleotide may be complementary. The first terminal portion and the second terminal portion of the first oligonucleotide are such that the first oligonucleotide binds to two adjacent segments of the first target nucleic acid sequence or its reverse complement, thereby forming a circular structure having a gap between the first terminal portion and the second terminal portion. 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 terminal portion and a second terminal portion. In some embodiments, the first terminal portion and the second terminal portion of the second oligonucleotide are complementary. In some embodiments, the first and second terminal portions bind to two adjacent segments of a second target nucleic acid sequence or its reverse complement, such that the second oligonucleotide forms a circular structure with a gap between the first and second terminal portions. 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 or second oligonucleotide is one nucleotide in size. In some embodiments, the gap in the first oligonucleotide is one nucleotide in size. In some embodiments, the gap in the first oligonucleotide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more nucleotides in size. In some embodiments, the gap in 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 in the first oligonucleotide or the second oligonucleotide is at least two nucleotides in size. In some embodiments, the gap in the first oligonucleotide and the second oligonucleotide is at least two nucleotides in size. In some embodiments, the first oligonucleotide further comprises a first identification sequence that identifies the first target nucleic acid sequence. In some embodiments, the second oligonucleotide further comprises a second identification sequence that identifies the second target nucleic acid sequence. In some embodiments, the first oligonucleotide further comprises a nucleic acid sequence that is complementary to the nucleic acid sequence of a sequencing primer. In some embodiments, the second oligonucleotide further comprises a nucleic acid sequence that is complementary to the nucleic acid sequence of a primer for nucleic acid amplification. In some embodiments, the second oligonucleotide further comprises a nucleic acid sequence that is complementary 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 generates concatemers. In some embodiments, the concatemer comprises at least two repeats of one target nucleic acid sequence or a portion thereof, or its reverse complement, of the at least two target nucleic acid sequences. In some embodiments, the first oligonucleotide further comprises a reverse complement sequence of a compaction oligonucleotide. In some embodiments, the second oligonucleotide further comprises a reverse complement sequence of a compaction oligonucleotide. In some embodiments, the first segment of the compaction oligonucleotide is complementary to and binds to the first portion of the concatemer. In some embodiments, the second segment of the compaction oligonucleotide is complementary to and binds to the second portion of the concatemer.In some embodiments, binding of the concatemer to the first and / or second portions results in a decrease in size or a change in shape of the concatemer. In some embodiments, the method further comprises in situ ligating a first terminal portion and a second terminal portion of the first oligonucleotide to generate a first circular oligonucleotide within the biological sample. In some embodiments, the method further comprises in situ ligating a first terminal portion and a second terminal portion of the second oligonucleotide to generate a second circular oligonucleotide within the biological sample. In some embodiments, ligating the first terminal portion and the second terminal portion of the first oligonucleotide comprises ligating the first terminal portion and the second terminal portion of the first oligonucleotide via a first nucleic acid enzyme. In some embodiments, ligating the first terminal portion and the second terminal portion of the second oligonucleotide comprises ligating the first terminal portion and the second terminal portion of the second oligonucleotide via a second nucleic acid enzyme. In some embodiments, the first nucleic acid enzyme and the second nucleic acid enzyme are the same type of enzyme. In some embodiments, the step of ligating the first and second terminal portions of the second oligonucleotide comprises ligating the first and second terminal portions of the second oligonucleotide via a second nucleic acid enzyme, wherein the first and second nucleic acid enzymes are different types of enzymes. In some embodiments, the first nucleic acid enzyme comprises a nucleic acid ligase, a nucleic acid ligation enzyme, a nucleic acid polymerase, a nucleic acid polymerization enzyme, or a combination thereof. In some embodiments, the second nucleic acid enzyme comprises a nucleic acid ligase, a nucleic acid ligation enzyme, a nucleic acid polymerase, a nucleic acid polymerization enzyme, or a combination thereof. In some embodiments, the method further comprises in situ amplifying the first circular oligonucleotide to generate the first nucleic acid molecule. In some embodiments, the method further comprises in situ amplifying the second circular oligonucleotide to generate the second nucleic acid molecule.In some embodiments, the amplifying step 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, the first concatemer comprises at least two repeats of a first nucleic acid unit comprising a first target nucleic acid sequence or a portion thereof, or a reverse complement of the first target nucleic acid sequence or a portion thereof. In some embodiments, the second concatemer comprises at least two repeats of a second nucleic acid unit comprising a second target nucleic acid sequence or a portion thereof, or a reverse complement of the second target nucleic acid sequence or a portion thereof. In some embodiments, the first concatemer further comprises a first identifying sequence that identifies the first target nucleic acid sequence, or a sequencing primer or reverse complement thereof, and the second concatemer further comprises a second identifying 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 compaction oligonucleotide. In some embodiments, the first segment of the first compaction oligonucleotide is complementary to and binds to a first portion of the first concatemer. In some embodiments, the second segment of the first compaction oligonucleotide is complementary to and binds to a 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 compaction oligonucleotide. In some embodiments, the first segment of the second compaction oligonucleotide is complementary to and binds to a first portion of the second concatemer. In some embodiments, the second segment of the second compaction oligonucleotide is complementary to and binds to a second portion of the second concatemer, resulting in a reduction in size or a change in shape of the second concatemer.

[0096] In some embodiments, each padlock probe in the plurality of second target-specific padlock probes comprises a first end 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 step (c) comprises hybridizing the first end and the second end of the second target-specific padlock probe to proximal positions on the second target cDNA molecule to form a circularized second target-specific padlock probe having a nick or gap 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 one of the regions of the second target-specific padlock probe that selectively hybridize to the second target cDNA molecule. In some embodiments, the second target-specific padlock probe comprises at least one universal adapter sequence, such as at least one universal sequencing primer binding site (or its complementary sequence). In some embodiments, the second target-specific padlock probe comprises a universal primer binding site (or its complementary sequence) for a rolling circle amplification primer. In some embodiments, the second target-specific padlock probe comprises a universal compaction oligonucleotide binding site (or its complementary sequence).

[0097] In some embodiments, the target-specific padlock probe comprises a universal sequencing primer binding site and a target barcode sequence adjacent to each other, such that the target barcode region of the concatemer is sequenced first. The target barcode sequence can be any length, for example, 3-15 bases, or 15-25 bases, or 25-40 bases, or longer.

[0098] In some embodiments, the method for detecting at least two different target RNA molecules in a cell sample can include (d) performing an enzymatic reaction to close a nick or gap in at least the first and second circularized target-specific padlock probes, 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) can include performing an enzymatic reaction to close a nick or gap in the at least first circularized target-specific padlock probe, thereby generating at least a first covalently closed circular padlock probe. In some embodiments, step (d) can include performing an enzymatic reaction to close a nick or gap in the at least second circularized target-specific padlock probe, thereby generating at least a second covalently closed circular padlock probe. In some embodiments, closing the nick can include performing an enzymatic ligation reaction. In some embodiments, closing the gaps can 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 gaps can include performing a polymerase-catalyzed fill-in reaction using the first target cDNA molecule as a template. In some embodiments, closing the gaps can include performing a polymerase-catalyzed fill-in reaction using the second target cDNA molecule as a template. In some embodiments, the method can include performing an enzymatic reaction to close nicks or gaps in at least 2-10,000 circularized target-specific padlock probes, thereby generating at least 2-10,000 covalently closed circular padlock probes within the cell sample.

[0099] In some embodiments, a method for detecting at least two different target RNA molecules in a cellular sample can include (e) performing a rolling circle amplification reaction within the cellular sample using first and second covalently closed circular padlock probes as template molecules, thereby generating a plurality of concatemer molecules including at least a first concatemer molecule corresponding to the first target RNA molecule, and the plurality of concatemer molecules can include at least a second concatemer molecule corresponding to the second target RNA molecule. In some embodiments, step (e) can include performing a rolling circle amplification reaction within the cellular sample using the first covalently closed circular padlock probe as a template molecule. In some embodiments, rolling circle amplification of the first covalently closed circular padlock probe can generate a plurality of concatemer molecules. In some embodiments, the plurality of concatemer molecules can include the first concatemer molecule. In some embodiments, the first concatemer molecule can correspond to the first target RNA molecule. In some embodiments, step (e) can include performing a rolling circle amplification reaction within the cell sample using the second covalently closed circular padlock probe as a template molecule. In some embodiments, rolling circle amplification of the second covalently closed circular padlock probe can generate a plurality of concatemer molecules. In some embodiments, the plurality of concatemer molecules can include a second concatemer molecule. In some embodiments, the second concatemer molecule can correspond to a second target RNA molecule. In some embodiments, the first concatemer molecule can include a tandem repeat unit, where the unit can include a sequence of the first target cDNA and a universal sequencing primer binding site (or a complementary sequence thereof). In some embodiments, the second concatemer molecule can include a tandem repeat unit, where the unit can include a sequence of the second target cDNA and a universal sequencing primer binding site (or a complementary sequence thereof).In some embodiments, the method can include performing a rolling circle amplification reaction within a cell sample using at least 2-10,000 covalently closed circular padlock probes as template molecules, thereby generating at least 2-10,000 concatemer molecules corresponding to at least 2-10,000 target RNA molecules. In some embodiments, the rolling circle amplification can be performed in the presence of a plurality of compaction oligonucleotides. In some embodiments, each compaction oligonucleotide can comprise 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 to a portion of the concatemer molecule, and the second region can hybridize to another portion of the concatemer molecule. In some embodiments, hybridization of the first and second regions of the compaction oligonucleotide can compact the concatemer molecule. In some embodiments, the compaction oligonucleotide can compact the size of the concatemer molecule. In some embodiments, compaction oligonucleotides can compress the shape of concatemeric molecules. In some embodiments, compaction oligonucleotides can compress the size and shape of concatemeric molecules. In some embodiments, compaction oligonucleotides can compress concatemeric molecules to form compressed nanoballs.

[0100] In some embodiments, a method for detecting at least two different target RNA molecules in a cellular sample can include (f) sequencing a plurality of concatemeric molecules within the cellular sample, which can include sequencing the first concatemeric molecules by performing 2 to 30 or fewer sequencing cycles to generate a plurality of first sequencing read products, and sequencing the second concatemeric molecules by performing 2 to 30 or fewer sequencing cycles to generate a plurality of second sequencing read products. In some embodiments, step (f) can include sequencing the plurality of concatemeric molecules within the cellular sample. In some embodiments, sequencing the plurality of concatemeric molecules within the cellular sample can include sequencing the first concatemeric molecules by performing 2 to 30 or fewer sequencing cycles to generate a plurality of first sequencing read products. In some embodiments, step (f) can include sequencing the plurality of concatemeric molecules within the cellular sample. In some embodiments, sequencing the plurality of concatemer molecules within the cell sample can include sequencing the second concatemer molecules by performing 2 to 30 or fewer sequencing cycles to generate a plurality of second sequencing read products. In some embodiments, the sequencing step of step (f) can include sequencing first concatemer molecules of 2 to 30 bases or fewer to generate a plurality of first sequencing read products, which can include sequencing second concatemer molecules of 2 to 30 bases or fewer to generate a plurality of second sequencing read products. In some embodiments, the sequencing step of step (f) can include sequencing first concatemer molecules of 2 to 30 bases or fewer to generate a plurality of first sequencing read products.In some embodiments, the sequencing step of step (f) can include sequencing second concatemer molecules of 2 to 30 bases or less to generate a plurality of second sequencing read products. In some embodiments, the method can include sequencing at least 2 to 10,000 concatemer molecules within a cell sample, which can include performing 2 to 30 sequencing cycles on the 2 to 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., FIG. 2). In some embodiments, at least a portion or the entire length of the first target barcode of the first concatemer molecule is sequenced (e.g., FIG. 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., FIG. 3). In some embodiments, at least a portion of the first cDNA region of the first concatemer molecule is sequenced (e.g., Figure 4 or 5). In some embodiments, only the second target barcode region of the second concatemer molecule is sequenced (e.g., Figure 2). In some embodiments, at least a portion or the entire length of the second target barcode of 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 5).

[0101] In some embodiments, the sequencing step of step (f) is 1.0 mm 2 Sequencing at least a portion of the first and second nucleic acid concatemers using an optical imaging system that includes a larger field of view (FOV).

[0102] In some embodiments, in the sequencing step of step (f), the plurality of first and second sequencing read products may be detectable by imaging, and the sequencing step may comprise decoding the plurality of first and second sequencing read products from images obtained during 2 to 30 or fewer sequencing cycles. In some embodiments, in the sequencing step of step (f), the plurality of first sequencing read products may be detectable by imaging. In some embodiments, in the sequencing step of step (f), the plurality of first sequencing read products may be detectable by imaging, and the sequencing step may comprise decoding the plurality of first sequencing read products from images obtained during 2 to 30 or fewer sequencing cycles. In some embodiments, in the sequencing step of step (f), the plurality of second sequencing read products may be detectable by imaging. In some embodiments, in the sequencing step of step (f), the plurality of second sequencing read products may be detectable by imaging, and the sequencing step may include decoding the plurality of second sequencing read products from images obtained during 2 to 30 or fewer sequencing cycles.

[0103] In some embodiments, in the sequencing step of step (f), the plurality of first and second sequencing read products may be detectable by imaging, and the sequencing step may comprise decoding the plurality of first and second sequencing read products from images obtained during 2 to 30 or fewer sequencing cycles. In some embodiments, in the sequencing step of step (f), the plurality of first sequencing read products may be detectable by imaging. In some embodiments, in the sequencing step of step (f), the plurality of first sequencing read products may be detectable by imaging, and the sequencing step may comprise decoding the plurality of first sequencing read products from images obtained during 2 to 30 or fewer sequencing cycles. In some embodiments, in the sequencing step of step (f), the plurality of second sequencing read products may be detectable by imaging. In some embodiments, in the sequencing step of step (f), the plurality of second sequencing read products may be detectable by imaging, and the sequencing step may include decoding the plurality of second sequencing read products from images obtained during 2 to 30 or fewer sequencing cycles.

[0104] 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.

[0105] In some embodiments, the sequencing step (f) can include (1) contacting a plurality of concatemer molecules within the cellular 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 concatemers. In some embodiments, the sequencing step (f) can include (1) contacting a plurality of concatemer molecules within the cellular sample with a plurality of universal sequencing primers. In some embodiments, the sequencing step (f) can include (1) contacting a plurality of concatemer molecules within the cellular sample with a plurality of sequencing polymerases. In some embodiments, the sequencing step (f) can include (1) contacting a plurality of concatemer molecules within the cellular sample with a plurality of nucleotide reagents. In some embodiments, the sequencing step can further include (2) performing 2 to 30 or fewer sequencing cycles to generate at least a first plurality of sequencing read products. In some embodiments, the sequencing step can further include (2) performing 2 to 30 or fewer sequencing cycles to generate at least a first plurality of sequencing read products and a second plurality of sequencing read products. In some embodiments, the sequencing step can further include (3) removing the first plurality of sequencing read products from the concatemers and retaining the plurality of concatemers within the cellular sample. In some embodiments, the sequencing step can further include (3) removing the first plurality of sequencing read products from the first concatemer molecules. In some embodiments, the sequencing step (3) can further include retaining the first concatemer molecules within the cellular sample.In some embodiments, the sequencing step can further include (3) removing the first plurality of sequencing read products from the concatemers and retaining the first concatemer molecules within the cellular sample, and removing the second plurality of sequencing read products from the second concatemer molecules and retaining the second concatemer molecules within the cellular sample. In some embodiments, the sequencing step can further include (3) removing the second plurality of sequencing read products from the second concatemer molecules. In some embodiments, the sequencing step (3) can further include retaining the second concatemer molecules within the cellular sample. In some embodiments, the sequencing step can further include (4) repeating steps (1) to (3) at least once. In some embodiments, (4) can include repeating steps (1) to (3) at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times. In some embodiments, (4) can include repeating (1)-(3) up to 10 times, up to 20 times, up to 30 times, up to 40 times, or up to 50 times.

[0106] In some embodiments, iterative sequencing can be performed using a binding sequencing procedure, labeled and / or unlabeled chain-terminating nucleotides, or multivalent molecules. A description of these three sequencing methods is provided below.

[0107] In some embodiments, multiple universal sequencing primers can be hybridized to concatemeric template molecules using a hybridization reagent comprising an SSC buffer (e.g., 2x saline-sodium citrate) buffer with formamide (e.g., 10-20% formamide). Hybridization conditions include a temperature of about 20-30°C for about 10-60 minutes.

[0108] In some embodiments, the multiple sequencing read products can be removed from the concatemers, and the multiple concatemers can be maintained inside the cell sample at a temperature that promotes nucleic acid denaturation, such as 30-90°C, using a dehybridization reagent containing an SSC buffer (e.g., saline-sodium citrate) buffer, with or without formamide.

[0109] In some embodiments, the plurality of nucleotide reagents in step (f) comprises a plurality of detectably labeled or unlabeled nucleotides. In some embodiments, each nucleotide is conjugated to a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the plurality of detectably labeled nucleotide analogs comprises a plurality of chain-terminating nucleotides, wherein the chain-terminating moiety is conjugated to the 3' nucleotide sugar position to form a 3' blocked nucleotide analog. In some embodiments, the chain-terminating moiety can be removed to convert the 3' blocked nucleotide analog to an extendable nucleotide having a 3' OH group on the sugar. In some embodiments, the labeled nucleotide analogs are conjugated to different fluorophores corresponding to the nucleobases adenine, cytosine, guanine, thymine, or uracil, and the different fluorophores emit fluorescent signals during the sequencing of step (f). In some embodiments, a sequencing cycle includes (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 (e.g., deblocking) the chain-terminating portion 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 cellular 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 within a cellular sample to generate multiple first sequencing reads.In some embodiments, 2 to 30 or fewer sequencing cycles can be performed on a plurality of concatemers within the cellular sample to generate a plurality of second sequencing read products. In some embodiments, the sequence of the first sequencing read product can be determined and aligned with a first reference sequence to confirm the presence of a first target RNA molecule within the cellular sample. In some embodiments, the sequence of the second sequencing read product can be determined and aligned with a second reference sequence to confirm the presence of a first target polypeptide within the cellular sample. In some embodiments, the sequences of the first and second sequencing read products can be aligned after each round of generating the first and second sequencing read products. In some embodiments, the first and second sequencing read products can be 30 bases or less in length. In some embodiments, the sequences of the first and second sequencing read products can be aligned after generating a set of replicate sequencing read products. In some embodiments, the first and second sequencing read products can be 30 bases or less in length. In some embodiments, the sequence of the first sequencing read product can be aligned after each round of generating the first sequencing read product. In some embodiments, the first sequencing read product can be 30 bases or less in length. In some embodiments, the sequence of the first sequencing read product can be aligned after generating a set of replicate sequencing read products. In some embodiments, the first sequencing read product can be 30 bases or less in length. In some embodiments, the sequence of the second sequencing read product can be aligned after each round of generating the second sequencing read product. In some embodiments, the second sequencing read product can be 30 bases or less in length. In some embodiments, the sequence of the second sequencing read product can be aligned after generating a set of replicate sequencing read products. In some embodiments, the second sequencing read product can be 30 bases or less in length.In some embodiments, the sequencing reaction can be performed on a sequencing device with a detector that captures fluorescent signals from the sequencing reaction inside the cell sample. The sequencing device can be configured to relay the fluorescent signal data captured by the detector to a computer system programmed to display an image of different fluorescent spots co-located in the cell sample, each corresponding to a different target RNA molecule or a different target polypeptide. In some embodiments, when sequencing is performed using different fluorescently labeled nucleotide reagents corresponding to different nucleic acid bases (e.g., A, G, C, T / U), the image can show fluorescent spots of different colors co-located in the same cell sample in different sequencing cycles.

[0110] In some embodiments, asynchronous phasing and / or prephasing events can occur during synchronous sequencing reactions on clonally amplified template amplicons, and the sequencing reaction comprises a polymerase-catalyzed sequencing reaction using detectably labeled chain terminator nucleotides. In some embodiments, the sequencing reaction for one template molecule in clonally amplified template molecules precedes (e.g., prephasing) or follows (e.g., phasing) the sequencing of other template molecules in clonally amplified template molecules. During sequencing, typically, fluorescent signals corresponding to the incorporation of labeled chain terminator nucleotides are detected. Thus, phasing and prephasing events can be detected and monitored using the incorporation of labeled chain terminator nucleotides.

[0111] In some embodiments, the plurality of nucleotide reagents in step (f) comprises a plurality of multivalent molecules each comprising a core attached to a plurality of nucleotide arms, the nucleotide arms being attached to the nucleotide units. In some embodiments, each multivalent molecule is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the core of the multivalent molecule is labeled with a fluorophore, and the fluorophore attached to a given core of the multivalent molecule corresponds to the nucleotide base of the nucleotide arm (e.g., adenine, guanine, cytosine, thymine, or uracil). In some embodiments, at least one of the nucleotide arms of the multivalent molecule comprises a linker and / or a nucleotide base attached to a fluorophore, and the fluorophore attached to a given nucleotide base corresponds to the nucleotide base of the nucleotide arm (e.g., adenine, guanine, cytosine, thymine, or uracil).In some embodiments, a sequencing cycle includes (1) contacting the concatemer / sequencing primer duplex with a first sequencing polymerase to form a complexed polymerase; (2) contacting the complexed polymerase with a detectably labeled multivalent molecule under conditions suitable for binding complementary nucleotide units of the multivalent molecule to the complexed polymerase, thereby forming a multivalent binding complex, wherein the conditions are suitable for inhibiting incorporation of complementary nucleotide units onto the terminus of the sequencing primer; and (3) contacting a detectably labeled multivalent molecule with a conjugated polymerase to form a multivalent binding complex. (4) removing the first sequencing polymerase and the bound detectably labeled multivalent molecule, 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 the unlabeled chain-terminating nucleotide into the terminal portion of the sequencing primer; and (6) removing (e.g., deblocking) the chain-terminating portion, retaining the concatemer / sequencing primer duplex. In some embodiments, the sequencing cycle can include contacting the concatemer / sequencing primer duplex with a first sequencing polymerase to form a complexed polymerase. In some embodiments, a sequencing cycle can include contacting a conjugated polymerase with a detectably labeled multivalent molecule under conditions suitable for binding complementary nucleotide units of the multivalent molecule to the conjugated polymerase, thereby forming a multivalent binding complex, wherein the conditions are suitable for inhibiting incorporation of the complementary nucleotide units onto the terminus of the sequencing primer. In some embodiments, a sequencing cycle can include detecting and imaging the fluorescent signal and color emitted by the bound detectably labeled multivalent molecule.In some embodiments, a sequencing cycle can include removing a first sequencing polymerase and the bound detectably labeled multivalent molecule, and retaining the concatemer / sequencing primer duplex. In some embodiments, a sequencing cycle can include contacting the retained concatemer / sequencing primer duplex with a second sequencing polymerase and unlabeled chain-terminating nucleotides under conditions suitable for polymerase-catalyzed incorporation of the unlabeled chain-terminating nucleotides into the terminal portion of the sequencing primer. In some embodiments, a sequencing cycle can include removing (e.g., deblocking) the chain-terminating portion and retaining the concatemer / sequencing primer duplex. In some embodiments, an individual cycle time can be achieved in less than 30 minutes. In some embodiments, the field of view (FOV) is 1 mm. 2 It can exceed the range of large areas (>10mm 2The cycle time for scanning the chromatogram can be less than 5 minutes. In some embodiments, 2 to 30 or fewer sequencing cycles can be performed on a plurality of concatemers within the cellular sample to generate a plurality of first sequencing read products and a plurality of second sequencing read products. In some embodiments, 2 to 30 or fewer sequencing cycles can be performed on a plurality of concatemers within the cellular sample to generate a plurality of first sequencing read products. In some embodiments, 2 to 30 or fewer sequencing cycles can be performed on a plurality of concatemers within the cellular sample to generate a plurality of second sequencing read products. In some embodiments, the first sequencing read products can be sequenced and aligned to a first reference sequence to confirm the presence of a first target RNA molecule within the cellular sample. In some embodiments, the second sequencing read products can be sequenced and aligned to a second reference sequence to confirm the presence of a second target RNA molecule within the cellular sample. 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 30 bases or less in length, or after generating a set of replicate sequencing read products, and the first and second sequencing read products can be 30 bases or less in length. 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. In some embodiments, the first and second sequencing read products can be 30 bases or less in length. In some embodiments, the sequences of the first and second sequencing read products can be aligned after generating a set of replicate sequencing read products. In some embodiments, the first and second sequencing read products can be 30 bases or less in length.In some embodiments, the sequence of the first sequencing read product can be aligned after each round of generating the first sequencing read product. In some embodiments, the first sequencing read product can be 30 bases or less in length. In some embodiments, the sequence of the first sequencing read product can be aligned after generating a set of replicate sequencing read products. In some embodiments, the first sequencing read product can be 30 bases or less in length. In some embodiments, the sequence of the second sequencing read product can be aligned after each round of generating the second sequencing read product. In some embodiments, the second sequencing read product can be 30 bases or less in length. In some embodiments, the sequence of the second sequencing read product can be aligned after generating a set of replicate sequencing read products. In some embodiments, the second sequencing read product can be 30 bases or less in length. In some embodiments, the sequencing reaction can be performed on a sequencing device having a detector capable of capturing fluorescent signals from the sequencing reaction within the cell sample. The sequencing device can be configured to relay the fluorescent signal data captured by the detector to a computer system that can be programmed to display images of different fluorescent spots that can be co-located in a cell sample, where each 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 a first RNA molecule and a second RNA molecule).

[0112] In some embodiments, when sequencing using detectably labeled multivalent molecules, the conditions for step (2), in which multivalent binding complexes are formed, and step (3), in which the bound detectably labeled multivalent molecules are imaged and detected, are milder than those for sequencing workflows using detectably labeled chain-terminating nucleotides. For example, steps (2) and (3) can be performed at moderate temperatures, such as about 35-45°C or about 39-42°C. Steps (2) and (3) can be performed at moderate temperatures, such as about 35-45°C or about 39-42°C. These moderate temperatures can help 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 images inside the cell sample. In some embodiments, the DNA nanoballs do not resolve during multiple sequencing cycles. In some embodiments, the DNA nanoball spot images do not expand during multiple sequencing cycles. In some embodiments, the DNA nanoball spot images remain discrete during multiple sequencing cycles. The spot images can be represented as Gaussian spots, and the size can be measured as FWHM. A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanoball spot can be about 10 μm or less.

[0113] In some embodiments, asynchronous phasing and / or prephasing events can occur during synchronized polymerase-catalyzed sequencing reactions using detectably labeled multivalent molecules. During sequencing, a fluorescent signal corresponding to the binding of complementary nucleotide units of the multivalent molecule to the complexed polymerase can be detected, thereby forming a multivalent binding complex. Thus, phasing and prephasing events can be detected and monitored using the binding of labeled multivalent molecules. In some embodiments, when performing up to 30 sequencing cycles using detectably labeled multivalent molecules, the phasing and / or prephasing rate 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, the phasing and / or prephasing rate for performing up to 30 sequencing cycles using labeled chain terminator nucleotides can be about 5%.

[0114] In some embodiments, the sequencing step (f) can include determining the sequence of a first nucleic acid molecule or a portion thereof, wherein the first nucleic acid molecule or portion thereof consists of 2 to 30 nucleotides. In some embodiments, the sequencing step (f) can include determining the sequence of a second nucleic acid molecule or a portion thereof, wherein the second nucleic acid molecule or portion thereof consists of 2 to 30 nucleotides. In some embodiments, 2 to 30 nucleotides of the first nucleic acid molecule or portion thereof comprises a first identifying sequence, and 2 to 30 nucleotides of the second nucleic acid molecule or portion thereof comprises a second identifying sequence. In some embodiments, 2 to 30 nucleotides of the first nucleic acid molecule or portion thereof further comprises at least a portion of the sequence of a first cDNA molecule or a first mRNA molecule, and 2 to 30 nucleotides of the second nucleic acid molecule or portion thereof further comprises at least a portion of the sequence of a second cDNA molecule or a second mRNA molecule.

[0115] In some embodiments, the determining step comprises contacting the first concatemer with a polymerizing enzyme, a plurality of nucleotides, and a primer sequence complementary to a portion of the first concatemer under conditions sufficient to form a binding complex comprising the polymerizing enzyme, one nucleotide of the plurality of nucleotides that is complementary to and binds 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. In some embodiments, the method further comprises incorporating a nucleotide at 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.

[0116] In some embodiments, the determining step comprises contacting the second concatemer with a polymerizing enzyme, a plurality of nucleotides, and a primer sequence complementary to a portion of the second concatemer under conditions sufficient to form a binding complex comprising the polymerizing enzyme, one nucleotide of the plurality of nucleotides that is complementary to and binds 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. In some embodiments, the method further comprises incorporating a nucleotide at 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, the determining step further comprises contacting the nucleotide with an agent that removes the blocking group from the nucleotide to 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 keto 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, the plurality of nucleotides consists of at least two of the same type of nucleotides, the same type of nucleotides being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by the fluorescent label of another type of nucleotide from the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, the at least two types of nucleotides being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP.In some embodiments, the fluorescent label of one type of nucleotide of the group emits light at a wavelength that is different from the wavelength of light emitted from the fluorescent label of another type of nucleotide of the group.

[0117] In some embodiments, the determining step comprises contacting two of the first concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, and two of the primer sequences complementary to a portion of the first concatemers under conditions sufficient to form a multivalent binding complex comprising each of the two polymerizing enzymes, a nucleotide conjugate of the plurality of nucleotide conjugates, and each of the two of the first concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugates comprise a label and at least two nucleotide moieties, two of the at least two nucleotide moieties being complementary to each other and bound to each nucleotide of the two of the first concatemers. In some embodiments, the determining step comprises detecting the multivalent binding complex by the label of the nucleotide conjugate and identifying the nucleobases of the nucleotides of the two of the first concatemers that are complementary to each other and bound to each of the at least two nucleotide moieties of the nucleotide conjugates. In some embodiments, the conditions inhibit the incorporation of at least two of the nucleotide moieties of the nucleotide conjugate into two of the first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core bound to a plurality of nucleotide arms, and each of the nucleotide moieties is bound 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, the determining step further includes removing two of the polymerizing enzymes and the nucleotide conjugates from two of the first concatemers; contacting each of the two first concatemers hybridized to each of the two primer sequences with two of the second polymerizing enzymes and a plurality of unlabeled nucleotides under conditions suitable to form a binding complex comprising each of the two second polymerizing enzymes, 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 plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the plurality of unlabeled nucleotides is complementary to and bound to a respective nucleotide of the two first concatemers, and one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety.

[0118] In some embodiments, the determining step comprises contacting two of the second concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, and two of the primer sequences complementary to a portion of the second concatemers under conditions sufficient to form a multivalent binding complex comprising each of the two polymerizing enzymes, a nucleotide conjugate of the plurality of nucleotide conjugates, and each of the two of the second concatemers hybridized to each of the two primer sequences, wherein the nucleotide conjugates comprise a label and at least two of the nucleotide moieties, wherein two of the at least two of the nucleotide moieties are complementary to each other and bound to each nucleotide of the two of the second concatemers; detecting the multivalent binding complex via the labels of the nucleotide conjugates; and identifying the nucleobases of the nucleotides of the two of the second concatemers that are complementary to each other and bound to each of the at least two of the nucleotide moieties of the nucleotide conjugates. In some embodiments, the conditions inhibit the incorporation of at least two of the nucleotide moieties of the nucleotide conjugate into two of the first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core bound to a plurality of nucleotide arms, and each of the nucleotide moieties is bound to one of the plurality of nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, the detecting step comprises imaging the fluorescent label.In some embodiments, the determining step further includes removing two of the polymerizing enzymes and the nucleotide conjugates from two of the first concatemers; contacting each of the two first concatemers hybridized to each of the two primer sequences with two of the second polymerizing enzymes and a plurality of unlabeled nucleotides under conditions suitable to form a binding complex comprising each of the two second polymerizing enzymes, 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 plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the plurality of unlabeled nucleotides is complementary to and bound to a respective nucleotide of the two first concatemers, and one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety.

[0119] In some embodiments, the determining step further includes removing two of the polymerizing enzymes and the nucleotide conjugate from two of the second concatemers; contacting each of the two second concatemers hybridized to each of the two primer sequences with two of the second polymerizing enzymes and a plurality of unlabeled nucleotides under conditions sufficient to form a binding complex comprising each of the two second polymerizing enzymes, 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 plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the two plurality of unlabeled nucleotides is complementary to and binds to a respective nucleotide of the two second concatemers, and one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety. In some embodiments, the conditions inhibit the incorporation of at least two of the nucleotide moieties of the nucleotide conjugate into two of the first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core bound to a plurality of nucleotide arms, and each of the nucleotide moieties is bound 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, the determining step further includes removing two of the polymerizing enzymes and the nucleotide conjugates from two of the first concatemers; contacting each of the two first concatemers hybridized to each of the two primer sequences with two of the second polymerizing enzymes and a plurality of unlabeled nucleotides under conditions suitable to form a binding complex comprising each of the two second polymerizing enzymes, 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 plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the plurality of unlabeled nucleotides is complementary to and bound to a respective nucleotide of the two first concatemers, and one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety.

[0120] In some embodiments, the determining step comprises polymerizing two of the first concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, a first primer sequence that is complementary to a first portion of the first concatemers, and a second primer sequence that is complementary to a second portion of the first concatemers under conditions sufficient to form a multivalent binding complex comprising each of the two of the polymerizing enzymes, one nucleotide conjugate of the plurality of nucleotide conjugates, the first portion of the first concatemer hybridized to the first primer sequence, 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 moieties, two of the at least two nucleotide moieties being complementary to and bound to nucleotides in the first and second portions of the first concatemer; detecting the multivalent binding complex via the label of the nucleotide conjugate; and identifying nucleobases of nucleotides in the first and second portions of the first concatemer that are complementary to and bound to two of the at least two nucleotide moieties of the nucleotide conjugate, respectively. In some embodiments, the conditions inhibit incorporation of at least two nucleotide moieties of the nucleotide conjugate into the first or two of the second concatemers. In some embodiments, the nucleotide conjugate comprises a core bound to multiple nucleotide arms, each of the nucleotide moieties being bound to one nucleotide arm of the multiple nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, the detecting step comprises imaging the fluorescent label.In some embodiments, the determining step further includes removing two of the polymerizing enzymes and the nucleotide conjugates from two of the first concatemers; contacting each of the two first concatemers hybridized to each of the two primer sequences with two of the second polymerizing enzymes and a plurality of unlabeled nucleotides under conditions suitable to form a binding complex comprising each of the two second polymerizing enzymes, 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 plurality of unlabeled nucleotides into each of the two primer sequences, wherein each of the plurality of unlabeled nucleotides is complementary to and bound to a respective nucleotide of the two first concatemers, and one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety.

[0121] In some embodiments, the determining step comprises hybridizing two of the second concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, a first primer sequence that is complementary to a first portion of the second concatemers, and a second primer sequence that is complementary to a second portion of the second concatemers under conditions sufficient to form a multivalent binding complex comprising each of the two of the polymerizing enzymes, a nucleotide conjugate of 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. The method includes contacting a nucleotide conjugate comprising a label and at least two nucleotide moieties, wherein two of the at least two nucleotide moieties are complementary to and bound to nucleotides in the first and second portions of a second concatemer; detecting the multivalent binding complex via the label of the nucleotide conjugate; and identifying nucleobases of nucleotides in the first and second portions of the second concatemer that are complementary to and bound to two of the at least two nucleotide moieties of the nucleotide conjugate, respectively. In some embodiments, the conditions inhibit incorporation of at least two nucleotide moieties of the nucleotide conjugate into two of the first or second concatemers. In some embodiments, the nucleotide conjugate comprises a core bound to multiple nucleotide arms, and each nucleotide moiety is bound to one nucleotide arm of the multiple nucleotide arms. In some embodiments, the detectable label comprises a fluorescent label. In some embodiments, the detecting step comprises imaging the fluorescent label. In some embodiments, the plurality of nucleotides consists of at least two of the same type of nucleotides, the same type of nucleotides being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and wherein the fluorescent label of one type of nucleotide of the group emits light at a wavelength that is different from the wavelength of light emitted from the fluorescent label of another type of nucleotide of the group.In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, the at least two types of nucleotides being selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and a fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by a fluorescent label of another type of nucleotide from the group. In some embodiments, the determining step further comprises contacting the nucleotide with an agent that removes a blocking group from the nucleotide to 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 keto group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0122] 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, the determining step comprises detecting the first sequencing product nucleic acid molecule or the second sequencing product nucleic acid molecule in situ within the biological sample by imaging. In some embodiments, the determining step comprises simultaneously detecting the first sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule in situ within the biological sample by imaging. In some embodiments, the imaging comprises fluorescent imaging. In some embodiments, 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 protozoan, 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 and paraffin-embedded (FFPE) sample. In some embodiments, the biological sample comprises a fresh cell sample, a fresh frozen cell sample, a sectioned cell sample, or an FFPE cell sample. In some embodiments, the at least two target nucleic acid sequences comprise target DNA sequences. In some embodiments, the at least two target nucleic acid sequences comprise target RNA sequences.In some embodiments, the 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 coding RNA, non-coding RNA, mRNA, tRNA, rRNA, miRNA, gRNA, snRNA, siRNA, antisense RNA, mature microRNA, or 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.

[0123] In some embodiments, the method for detecting at least two different target RNA molecules in a cellular sample can further include (g) removing a plurality of first sequencing read products from the first concatemer molecules and retaining the first concatemer molecules in the cellular sample, and removing a plurality of second sequencing read products from the second concatemer molecules and retaining the second concatemer molecules in the cellular sample. In some embodiments, (g) can include removing a plurality of first sequence read products from the first concatemer molecules. In some embodiments, step (g) can further include retaining the first concatemer molecules in the cellular sample. In some embodiments, step (g) can include removing a plurality of second sequence read products from the second concatemer molecules. In some embodiments, step (g) can further include retaining the second concatemer molecules in the cellular sample.

[0124] In some embodiments, a method for detecting at least two different target RNA molecules in a cellular sample includes (h) repeatedly sequencing a plurality of concatemers by repeating steps (f) and (g) at least once, wherein the sequence of a plurality of first sequencing read products confirms the presence of the first target RNA molecule in the cellular sample, and the sequence of a plurality of second sequencing read products confirms the presence of a second target RNA molecule in the cellular sample. In some embodiments, the repeating step includes repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times. In some embodiments, step (h) can include repeatedly sequencing a plurality of concatemers by repeating steps (f) and (g) at least once. In some embodiments, the repeating step comprises repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times. In some embodiments, the sequence of the plurality of first sequencing read products can confirm the presence of a first target RNA molecule in a cellular sample. In some embodiments, the sequence of the plurality of second sequencing read products can confirm the presence of a second target RNA molecule in a cellular sample. In some embodiments, the sequence of the plurality of first sequencing read products can confirm the presence of a first target RNA molecule in a cellular sample, and the sequence of the plurality of second sequencing read products can confirm the presence of a second target RNA molecule in the sample.

[0125] 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, and 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 (a) providing a biological sample. In some embodiments, the biological sample includes a first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or a reverse complement of the first target nucleic acid sequence or a portion thereof. In some embodiments, the biological sample includes a second nucleic acid molecule comprising the second target nucleic acid sequence or a portion thereof, or a reverse complement of the second target nucleic acid sequence or a portion thereof. In some embodiments, the biological sample includes 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, and the presence of the third target nucleic acid sequence or its reverse complement identifies the presence of a first target polypeptide in the biological sample. In some embodiments, the biological sample includes a fourth target nucleic acid sequence or a portion thereof, or 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, and the presence of the fourth target nucleic acid sequence or its reverse complement identifies the presence of a second target polypeptide in the biological sample. In some embodiments, the method further includes (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 a third nucleic acid molecule or portion thereof in situ 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.In some embodiments, the method further comprises (b) identifying in situ the sequence of 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. In some embodiments, step (c) further comprises identifying in situ the sequence of 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 a portion thereof. In some embodiments, the complete sequence of the first target nucleic acid sequence and the complete sequence of the second target nucleic acid sequence differ by at least one nucleotide. In some embodiments, the complete sequence of the first target polypeptide and the complete 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.

[0126] The present disclosure provides a method for in situ detection of at least two target nucleic acid sequences 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 the first target nucleic acid sequence or its reverse complement. In some embodiments, the at least two target polypeptides include a second target polypeptide encoded by the second target nucleic acid sequence or its reverse complement. In some embodiments, the method includes (a) providing a biological sample containing (i) a first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or a reverse complement of the first target nucleic acid sequence or a portion thereof. In some embodiments, the biological sample includes (ii) a second nucleic acid molecule comprising the second target nucleic acid sequence or a portion thereof, or a reverse complement of the second target nucleic acid sequence or a portion thereof. In some embodiments, the biological sample includes (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. In some embodiments, 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 further includes (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, and the presence of the fourth target nucleic acid sequence or its reverse complement identifies the presence of a second target polypeptide in the biological sample. In some embodiments, the method further includes (b)(i) determining the sequence of the first nucleic acid molecule or a portion thereof in situ 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 to 30 nucleotides.In some embodiments, step (b) further comprises determining the sequence of a third nucleic acid molecule or a portion thereof in situ 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 portion thereof consists of 2 to 30 nucleotides. In some embodiments, the method further comprises (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, the first nucleic acid molecule and the third nucleic acid molecule are located in the biological sample after removal. In some embodiments, the method further comprises (d) repeating steps (b) and (c). In some embodiments, the method further comprises (e) (i) identifying in situ the sequence of a second nucleic acid molecule or 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 portion thereof, wherein the second nucleic acid molecule or portion thereof consists of 2 to 30 nucleotides. In some embodiments, step (e) further comprises (ii) identifying in situ the sequence of a fourth nucleic acid molecule or 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. In some embodiments, the fourth nucleic acid molecule or portion thereof consists of 2 to 30 nucleotides. In some embodiments, the method further comprises (f) removing the second sequencing product nucleic acid molecule from the second nucleic acid molecule and the fourth sequencing product nucleic acid molecule from the fourth nucleic acid molecule, wherein the second nucleic acid molecule and the fourth nucleic acid molecule are located in the biological sample after removal. In some embodiments, the method further comprises (g) repeating steps (e) and (f). In some embodiments, the complete sequence of the first target nucleic acid sequence and the complete sequence of the second target nucleic acid sequence differ by at least one nucleotide.In some embodiments, the complete sequence of the first target polypeptide and the complete sequence of the second target polypeptide differ by at least one amino acid. In some embodiments, performing step (b) is under conditions that prevent performing step (e).

[0127] The present disclosure provides methods for in situ detection of at least two target RNA sequences and at least two polypeptides in a biological sample. In some embodiments, the method includes (a) providing a biological sample immobilized on a surface, permeabilized, and immobilized. In some embodiments, the biological sample includes (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. In some embodiments, the biological sample includes (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. In some embodiments, the method further includes (b) generating a first target cDNA sequence by reverse transcription of the first target RNA sequence and a second target cDNA sequence by reverse transcription of the second target RNA sequence. In some embodiments, the method further comprises (c) contacting the first target cDNA sequence with a first oligonucleotide comprising a first terminal portion and a second terminal portion, wherein the first terminal portion and the second terminal portion of the first oligonucleotide are complementary and bind to two adjacent segments of the first target cDNA sequence, such that the first oligonucleotide forms a circular structure with a gap between its first terminal portion and its second terminal portion, and the first oligonucleotide comprises a first identification sequence that identifies the first target RNA sequence, a first sequencing primer, and a nucleic acid amplification primer. In some embodiments, step (c) further comprises contacting the second target cDNA sequence with a second oligonucleotide comprising a first terminal portion and a second terminal portion, wherein the first terminal portion and the second terminal portion of the second oligonucleotide are complementary and bind to two adjacent segments of the second target cDNA sequence, such that the second oligonucleotide forms a circular structure with a gap between its first terminal portion and its second terminal portion.In some embodiments, the second oligonucleotide comprises a second identification sequence that identifies 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 at least one nucleotide difference. 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 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, and the first oligonucleotide conjugate specifically binds to the first target polypeptide via the first binding moiety to form a first binding complex, and the first tag sequence and the second tag sequence identify the first binding moiety. 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 moiety that specifically binds to the second target polypeptide, wherein the second short nucleic acid comprises a third tag sequence and a fourth tag sequence, and the second oligonucleotide conjugate specifically binds to the second target polypeptide via the second binding moiety to form a second binding complex, wherein the third tag sequence and the fourth tag sequence identify the second binding moiety. In some embodiments, the method further comprises step (d) contacting the first binding complex with a third oligonucleotide comprising a first terminal portion and a second terminal portion, wherein the first terminal portion and the second terminal portion of the third oligonucleotide are complementary and bind to the first tag sequence and the second tag sequence of the first oligonucleotide conjugate, such that the third oligonucleotide forms a circular structure with a gap between the first terminal portion and the second terminal portion.In some embodiments, step (d) comprises contacting the second binding complex with a fourth oligonucleotide comprising a first terminal portion and a second terminal portion, wherein the first terminal portion and the second terminal portion of the fourth oligonucleotide are complementary 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 terminal portion and the second terminal portion. In some embodiments, the method further comprises (e) ligating the first and second terminal portions of the first oligonucleotide to form a first circular oligonucleotide, the first and second terminal portions of the second oligonucleotide to form a second circular oligonucleotide, the first and second terminal portions of the third oligonucleotide to form a third circular oligonucleotide, and the first and second terminal portions of the fourth oligonucleotide to form a fourth circular oligonucleotide, wherein the first and third circular oligonucleotides comprise a first sequencing primer or its reverse complement, and the second and fourth circular oligonucleotides comprise a second sequencing primer or its reverse complement, and the sequences of the first and second sequencing primers differ by at least one nucleotide. In some embodiments, the method further comprises (f) amplifying the first circular oligonucleotide by rolling circle amplification to generate 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 generate a second concatemer comprising a plurality of the second circular oligonucleotides. In some embodiments, step (f) further comprises amplifying the third circular oligonucleotide by rolling circle amplification to generate a third concatemer comprising a plurality of the third circular oligonucleotides.In some embodiments, step (f) further comprises amplifying the fourth circular oligonucleotide by rolling circle amplification to generate a fourth concatemer comprising a plurality of fourth circular oligonucleotides. In some embodiments, the method further comprises (g) 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 binds to the first concatemer, wherein the sequence of the first concatemer, or portion thereof, consists of 2 to 30 nucleotides. In some embodiments, step (g) further comprises determining the sequence of a third concatemer, or a portion thereof, in situ 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 portion thereof, consists of 2 to 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 (h) removing the first sequencing product nucleic acid molecule from the first concatemer and the third sequencing product nucleic acid molecule from the third concatemer, wherein the first concatemer and the third concatemer are located in the biological sample after removal. In some embodiments, the method further comprises (i) repeating (g) and (h) at least once. In some embodiments, the method further comprises (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 to 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 portion thereof, consists of 2 to 30 nucleotides. In some embodiments, step (j) is performed under conditions that prevent step (g) from being performed.In some embodiments, the complete sequence of the first target RNA sequence and the complete sequence of the second target RNA sequence differ by at least one nucleotide. In some embodiments, the complete sequence of the first target polypeptide and the complete sequence of the second target polypeptide differ by at least one amino acid. In some embodiments, the method further comprises (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, the second concatemer and the fourth concatemer are placed in a biological sample after removal. In some embodiments, the method further comprises (i) repeating (j) and (k) at least once.

[0128] In some embodiments, the determining step includes imaging the first sequencing product nucleic acid molecule or the third sequencing product nucleic acid molecule, and the identifying step includes imaging the second sequencing product nucleic acid molecule or the fourth sequencing product nucleic acid molecule and the fourth sequencing product nucleic acid molecule. In some embodiments, the method further includes 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 sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule within the biological sample. In some embodiments, the biological sample includes an organelle, a cell, a whole cell, a whole group of cells, a tissue, an undamaged tissue, a tumor, an undamaged tumor, an organ, an organism, a protozoan, algae, a bacterium, a virus, a plant, a fungus, an insect, or an animal. In some embodiments, the biological sample includes a fresh sample, a processed sample, a fresh frozen sample, a sectioned sample, or a formalin-fixed and 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 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 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 wolf 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 concatemer, the second concatemer, the third concatemer, or the fourth concatemer comprises a compaction oligonucleotide, and a first segment of the compaction oligonucleotide is complementary to and binds to a first portion of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer. In some embodiments, the second segment of the compaction oligonucleotide is complementary to the second portion of the first, second, third, or fourth concatemer, and binds to the second portion, causing the size or shape of the second concatemer to decrease. In some embodiments, the first sequencing product nucleic acid molecule comprises a first identifying sequence that identifies the first target RNA sequence, and the second sequencing product nucleic acid molecule comprises a second identifying sequence that identifies the second target RNA sequence. In some embodiments, the first sequencing product nucleic acid molecule comprises a first identifying sequence that identifies the first target RNA sequence and a portion of the first target RNA sequence, and the second sequencing product nucleic acid molecule comprises a second identifying sequence that identifies the second target RNA sequence and a portion of the second target RNA sequence.In some embodiments, the determining step further comprises contacting the first, second, third, or fourth concatemer with a polymerizing enzyme, 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 comprising the polymerizing enzyme, one nucleotide of the plurality of nucleotides that is complementary to and binds to a nucleotide unit of the first, second, third, or fourth concatemer, and the first, second, third, or fourth concatemer hybridized 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 moiety. In some embodiments, the determining step further comprises incorporating a nucleotide at the 3' end of the primer sequence. In some embodiments, the determining step further comprises identifying the nucleobase of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide. In some embodiments, the determining step further comprises contacting the nucleotide with an agent that removes a blocking group from the nucleotide to 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 keto 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 comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0129] In some embodiments, the determining step comprises contacting two of the first, second, third, or fourth concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, and two of the primer sequences complementary to a portion of the first, second, third, or fourth concatemers under conditions sufficient to form a multivalent binding complex comprising each of the two polymerizing enzymes, one nucleotide conjugate from the plurality of nucleotide conjugates, and each of the 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 of the nucleotide moieties. In some embodiments, two of the at least two of the nucleotide moieties are complementary to each other and bind to each of the nucleotides in the first concatemer. In some embodiments, the determining step comprises detecting the multivalent binding complex by labeling the nucleotide conjugate. In some embodiments, the determining step further comprises identifying the nucleobases of the two nucleotides of the first concatemer that are complementary to each other and that are bound to at least two of the nucleotide moieties of the nucleotide conjugate.

[0130] In some embodiments, the determining step further comprises removing two of the polymerizing enzymes and the nucleotide conjugate from two of the first, second, third, or fourth concatemers. In some embodiments, the determining step further comprises contacting each of two of the first, second, third, or fourth concatemers hybridized to each of two of the primer sequences with two of the second polymerizing enzymes and a plurality of unlabeled nucleotides under conditions suitable for forming a binding complex comprising each of the two of the second polymerizing enzymes, each of two of the first, second, third, or fourth concatemers hybridized to each of two of the primer sequences, and two of the plurality of unlabeled nucleotides, and incorporating each of two of the plurality of unlabeled nucleotides into each of two of the primer sequences. In some embodiments, each of two of the plurality of unlabeled nucleotides is complementary and binds to each of two nucleotides of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer. In some embodiments, one unlabeled nucleotide of the plurality of unlabeled nucleotides comprises a removable blocking group on the 3' carbon of the sugar moiety.

[0131] In some embodiments, the determining step comprises polymerizing two of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers with two of the polymerizing enzymes, a plurality of nucleotide conjugates, a first primer sequence complementary to a first portion of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers, and a second primer sequence complementary to a second portion of the first concatemers, the second concatemers, the third concatemers, or the fourth concatemers. The method includes contacting the two conjugated enzymes under conditions sufficient to form a multivalent binding complex comprising each of two conjugated enzymes, one nucleotide conjugate of 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. In some embodiments, the nucleotide conjugate comprises a label and at least two of the nucleotide moieties. In some embodiments, two of the at least two of the nucleotide moieties are complementary to each other and bind to nucleotides in the first and second portions of the first, second, third, or fourth concatemer. In some embodiments, the determining step further comprises detecting the multivalent binding complex by the label of the nucleotide conjugate. In some embodiments, the determining step further comprises identifying nucleobases of nucleotides in the first and second portions of the first, second, third, or fourth concatemer that are complementary to each other and that bind to at least two of the nucleotide portions of the nucleotide conjugate. In some embodiments, the conditions inhibit the incorporation of at least two of the nucleotide portions of the nucleotide conjugate into two of the first, second, third, or fourth concatemers.In some embodiments, the nucleotide conjugate comprises a core bound to a plurality of nucleotide arms, each of the nucleotide moieties being bound 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, the plurality of nucleotides comprises at least two of the same type of nucleotide, the same type of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by the fluorescent label of another type of nucleotide from the group. In some embodiments, the plurality of nucleotides comprises at least two types of nucleotide, the at least two types of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted by the fluorescent label of another type of nucleotide from the group. In some embodiments, the determining step further comprises contacting the nucleotide with an agent that removes the blocking group from the nucleotide to 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 keto 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 of the same type of nucleotide, the same type of nucleotide being selected from the group including dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide of the group emits light at a wavelength that differs from the wavelength of light emitted by the fluorescent label of another type of nucleotide of the group.In some embodiments, the plurality of nucleotides comprises at least two types of nucleotides, the at least two types of nucleotides being selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and the fluorescent label of one type of nucleotide from the group emits light at a wavelength different from the wavelength of light emitted from the fluorescent label of another type of nucleotide from 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.

[0132] 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 wolf 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 group of cells, 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.

[0133] In situ batch sequencing of RNA and polypeptides The present disclosure provides a method for in situ multiplex and multi-omics detection and identification using coded padlock probes. The padlock probes are designed to selectively detect target RNAs or polypeptides. The padlock probes may contain one or more batch-specific primer binding sites specific to primers. The primers may be spatially localized, and therefore the padlock probes may not require barcode sequences. However, in some embodiments, the padlock probes may contain barcode sequences.

[0134] The RNA-specific padlock probe selectively hybridizes to the cDNA corresponding to the target RNA. The RNA-specific probe carries a barcode that uniquely identifies the cDNA. The RNA-specific padlock probe also carries a batch-specific sequencing primer binding site. The padlock probe may contain one or more batch-specific primer binding sites specific to the primer. The primer may be spatially localized, and therefore the padlock probe may not require a barcode sequence. However, in some embodiments, the padlock probe may contain a barcode sequence.

[0135] The target polypeptide is detected using an antibody-oligonucleotide conjugate and a polypeptide-specific padlock probe that selectively hybridizes to the oligonucleotide conjugated to the antibody.The polypeptide-specific padlock probe carries a barcode that uniquely identifies the antibody that selectively binds to the target polypeptide.The polypeptide-specific padlock probe also carries a batch-specific sequencing primer, which is the same batch-specific sequencing primer carried by the corresponding RNA-specific padlock probe, to enable simultaneous sequencing and detection of the target RNA and the polypeptide encoded by the target RNA.Therefore, the padlock probe allows simultaneous detection and identification of the RNA and the polypeptide encoded by the target RNA.

[0136] Both types of padlock probes are used to generate concatemers with batch-specific sequencing binding sites and multiple copies of barcodes, which can collapse into DNA nanoballs with compact shapes and sizes that result in increased signal intensity and color differentiation during sequencing.

[0137] For in situ sequencing, optical resolution limitations hinder the ability to perform highly multiplexed sequencing. Batch-specific sequencing primer binding sites on the padlock probe allow for sequencing of a desired subset (e.g., batch) of concatemers using selected batch-specific sequencing primers, reducing overcrowded signals and images. The use of batch-specific sequencing primers generates robust and resolvable optical images. Performing multiple rounds of sequencing on the same cell sample using different batch-specific sequencing primers enables multiplex and multi-omics sequencing to reveal many target RNAs and their encoded polypeptides.

[0138] The batch specific sequencing method described herein has many applications.For example, the number of spots that are imaged and associated with sequencing can be counted.Counted spots can be used as a measure of the RNA and polypeptide level in cell samples.

[0139] For example, a pairwise sequencing kit can be used to perform 150 forward sequencing cycles and 150 reverse sequencing cycles (e.g., a total of 300 sequencing cycles per kit) to reveal more than 300,000 molecules. A non-limiting example cell has approximately 30 copies of 5,000 different transcripts for a total of approximately 150,000 transcripts, and the resulting calls correspond to approximately 15,000 polypeptides encoded by the transcripts for a total of 300,000 total RNA and polypeptide molecules. Using the pairwise kit, 300 sequencing cycles (not in pairwise mode) can be performed to reveal 300,000 different RNA and polypeptide molecules in a cell sample. See Table 1. Table 1 lists the estimated total sequencing cycles and total time required to decode the RNA and polypeptides in a cell sample.

[0140] [Table 1]

[0141] 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, the method comprising: (a) providing a cellular sample deposited on a solid support, the cellular sample comprising: (i) a first plurality of DNA amplicons corresponding to a first target RNA molecule; (ii) a second plurality of DNA amplicons corresponding to a second target RNA molecule; (iii) a third plurality of DNA amplicons corresponding to a first polypeptide encoded by the first target RNA molecule; and (iv) a fourth plurality of DNA amplicons corresponding to a second polypeptide encoded by the second target RNA molecule.

[0142] In some embodiments, the method further includes (b) sequencing the first plurality of DNA amplicons and the third plurality of DNA amplicons within the cellular sample under conditions that inhibit sequencing of the second plurality of DNA amplicons and the fourth plurality of DNA amplicons, wherein sequencing the first plurality of DNA amplicons within the cellular sample comprises generating a plurality of first sequencing read products, wherein 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 cellular sample, and sequencing the third plurality of DNA amplicons within the cellular sample comprises generating a plurality of second sequencing read products, wherein 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 cellular sample.

[0143] In some embodiments, the method further includes (c) sequencing the second plurality of DNA amplicons and the fourth plurality of DNA amplicons within the cellular sample under conditions that inhibit sequencing of the first plurality of DNA amplicons and the third plurality of DNA amplicons, wherein sequencing the second plurality of DNA amplicons within the cellular sample comprises generating a plurality of third sequencing read products, wherein sequences of the third sequencing read products are aligned with a third target reference sequence to confirm the presence of the second target RNA in the cellular sample, and sequencing the fourth plurality of DNA amplicons within the cellular sample comprises generating a plurality of fourth sequencing read products, wherein 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 cellular sample.

[0144] 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, the method comprising: (a) providing a cellular sample deposited on a solid support, the cellular sample comprising: (i) a first plurality of DNA amplicons corresponding to a first target RNA molecule; (ii) a second plurality of DNA amplicons corresponding to a second target RNA molecule; (iii) a third plurality of DNA amplicons corresponding to a first polypeptide encoded by the first target RNA molecule; and (iv) a fourth plurality of DNA amplicons corresponding to a second polypeptide encoded by the second target RNA molecule.

[0145] In some embodiments, the method further includes (b) sequencing the first and third plurality of DNA amplicons within the cellular sample under conditions that inhibit sequencing of the second and fourth plurality of DNA amplicons. In some embodiments, step (b) comprises sequencing the first plurality of DNA amplicons within the cellular sample, which comprises performing 2 to 30 or fewer sequencing cycles to generate a plurality of first sequencing read products, the sequences of which are aligned with a first target reference sequence to confirm the presence of the first target RNA in the cellular sample. In some embodiments, step (b) comprises sequencing the first plurality of DNA amplicons within the cellular sample, which comprises performing 1 to 250 sequencing cycles to generate a plurality of first sequencing read products, the sequences of which are aligned with a first target reference sequence to confirm the presence of the first target RNA in the cellular sample. In some embodiments, step (b) comprises sequencing a third plurality of DNA amplicons within the cellular sample, comprising performing 2 to 30 or fewer sequencing cycles to generate a plurality of second sequencing read products, the sequences of which are aligned with the second target reference sequence to confirm the presence of the first target polypeptide in the cellular sample. In some embodiments, step (b) comprises sequencing a third plurality of DNA amplicons within the cellular sample, comprising performing 1 to 250 sequencing cycles to generate a plurality of second sequencing read products, the sequences of which are aligned with the second target reference sequence to confirm the presence of the first target polypeptide in the cellular sample.

[0146] In some embodiments, the method further comprises (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 moiety can be added to the first sequencing read product and the second sequencing read product to inhibit further sequencing reactions. For example, a nucleotide analog can be incorporated if the nucleotide analog inhibits the incorporation of a subsequent nucleotide. Non-limiting exemplary blocking nucleotide analogs include dideoxynucleotides or nucleotides with 2' or 3' chain terminating moieties.

[0147] In some embodiments, the method further comprises (d) iteratively sequencing the first plurality of DNA amplicons and the third plurality of DNA amplicons by repeating steps (b) and (c) at least once. In some embodiments, the repeating comprises repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times.

[0148] In some embodiments, the method further includes (e) sequencing a second plurality of DNA amplicons and a fourth plurality of DNA amplicons within the cellular sample under conditions that inhibit sequencing of the first plurality of DNA amplicons and the third plurality of DNA amplicons. In some embodiments, step (e) comprises sequencing the second plurality of DNA amplicons within the cellular sample, which comprises performing 2 to 30 or fewer sequencing cycles to generate a plurality of third sequencing read products, the sequences of which are aligned with a third target reference sequence to confirm the presence of the second target RNA in the cellular sample. In some embodiments, step (e) comprises sequencing the second plurality of DNA amplicons within the cellular sample, which comprises performing 1 to 250 sequencing cycles to generate a plurality of third sequencing read products, the sequences of which are aligned with a third target reference sequence to confirm the presence of the second target RNA in the cellular sample. In some embodiments, step (e) comprises sequencing a fourth plurality of DNA amplicons within the cellular sample, comprising performing 2 to 30 or fewer 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 cellular sample. In some embodiments, step (e) comprises sequencing a fourth plurality of DNA amplicons within the cellular sample, comprising performing 1 to 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 cellular sample.

[0149] In some embodiments, the method includes (f) removing a plurality of third sequencing read products from the second DNA amplicon and retaining the second DNA amplicon in the cell sample, and removing a plurality of fourth sequencing read products from the fourth DNA amplicon and retaining the fourth DNA amplicon in the cell sample. In some embodiments, a 3' blocking moiety can be added to the third sequencing read product and the fourth sequencing read product to inhibit further sequencing reactions. For example, a nucleotide analog can be incorporated if the nucleotide analog inhibits the incorporation of a subsequent nucleotide. Non-limiting exemplary blocking nucleotide analogs include dideoxynucleotides or nucleotides with 2' or 3' chain terminating moieties.

[0150] In some embodiments, the method includes (g) repeatedly sequencing the second plurality of DNA amplicons and the fourth plurality of DNA amplicons by repeating steps (e) and (f) at least once. In some embodiments, the repeating includes repeating at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times.

[0151] 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,...

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, and the method is (a) (i) A first nucleic acid molecule comprising the first target nucleic acid sequence or a portion thereof, or its reverse complementary sequence or a portion thereof, (ii) A second nucleic acid molecule comprising the second target nucleic acid sequence or a part thereof, or its inverse complementary sequence or a part thereof. The process of providing the aforementioned biological sample, (b) (i) A step of determining in situ the sequence of the first nucleic acid molecule or a part thereof in the biological sample in order to generate a first sequencing product nucleic acid molecule that is complementary to the first nucleic acid molecule or a part thereof and binds to the first nucleic acid molecule or a part thereof, and (ii) A step of determining in situ the sequence of the second nucleic acid molecule or a portion thereof in the biological sample in order to generate a second sequencing product nucleic acid molecule that is complementary to the second nucleic acid molecule or a portion thereof and binds to the second nucleic acid molecule or a portion thereof, wherein the complete sequence of the first target nucleic acid sequence and the complete sequence of the second target nucleic acid sequence have a difference of at least one nucleotide. The decision-making process, (c) A step of removing the first sequencing product nucleic acid molecule from the first nucleic acid molecule and the second sequencing product nucleic acid molecule from the second nucleic acid molecule, wherein the first nucleic acid molecule and the second nucleic acid molecule are located inside the biological sample after removal, (d) A step of repeating (b) and (c) in order to detect the at least two target nucleic acid sequences in the biological sample in situ, Methods that include...

2. The method according to claim 1, wherein the biological sample is fixed and subjected to permeabilization.

3. a. A step of generating a first complementary DNA (cDNA) molecule in situ by reverse transcription of a first messenger RNA (mRNA) molecule inside the biological sample, and / or, b. A step of generating a second cDNA molecule in situ by reverse transcription of a second mRNA molecule inside the biological sample. It further includes, The first cDNA molecule or the first mRNA molecule comprises the first target nucleic acid sequence or the reverse complementary sequence of the first target nucleic acid sequence. The method according to claim 1, 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. a. A step of in situ contacting the first target nucleic acid sequence or its reverse complementary sequence with a first oligonucleotide including a first terminal portion and a second terminal portion, and / or, b. The step of in situ contacting the second target nucleic acid sequence or its reverse complementary sequence with a second oligonucleotide including the first and second terminal portions. It further includes, The first and second terminal portions of the first oligonucleotide are complementary to and bind to two adjacent segments of the first target nucleic acid sequence or its inverse complementary sequence, and as a result, the first oligonucleotide forms a cyclic structure having a nick or gap between the first and second terminal portions. The method according to claim 3, wherein the first and second terminal portions of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target nucleic acid sequence or its inverse complementary sequence, and as a result, the second oligonucleotide forms a cyclic structure having a nick or gap between the first and second terminal portions.

5. The method according to claim 4, wherein the gap in the first oligonucleotide or the second oligonucleotide is the size of one nucleotide or at least two nucleotides.

6. (i) The first oligonucleotide further comprises a first identification sequence for identifying the first target nucleic acid sequence, and the second oligonucleotide further comprises a second identification sequence for identifying the second target nucleic acid sequence, (ii) The first oligonucleotide and the second oligonucleotide further comprise nucleic acid sequences complementary to the nucleic acid sequence of the sequencing primer, and / or (iii) The method according to claim 4, wherein the first oligonucleotide and the second oligonucleotide further comprise nucleic acid sequences complementary to the nucleic acid sequence of the primer for nucleic acid amplification.

7. The method according to claim 6, wherein the primer for nucleic acid amplification is a primer for rolling circle amplification (RCA) that generates a concatemer, the concatemer comprising at least two repeats of one of the at least two target nucleic acid sequences or a portion thereof, or its inverse complementary sequence.

8. The first oligonucleotide and the second oligonucleotide further comprise the reverse complementary sequence of the compaction oligonucleotide, a. The first segment of the compaction oligonucleotide is complementary to the first portion of the concatemer and binds to the first portion of the concatemer, and b. The method according to claim 7, wherein the second segment of the compaction oligonucleotide is complementary to the second portion of the concatemer and binds to the second portion of the concatemer, resulting in a reduction in the size or a change in the shape of the concatemer.

9. (a) A step of in situ linking the first terminal portion and the second terminal portion of the first oligonucleotide in order to generate a first cyclic oligonucleotide inside the biological sample, (b) A step of in situ linking the first terminal portion and the second terminal portion of the second oligonucleotide in order to generate a second cyclic oligonucleotide inside the biological sample. The method according to claim 4, further comprising:

10. (a) a step of amplifying the first cyclic oligonucleotide in situ to produce the first nucleic acid molecule, and / or (b) further comprising the step of amplifying the second cyclic oligonucleotide in situ to produce the second nucleic acid molecule, The method according to claim 9, wherein the amplification step includes rolling circle amplification (RCA), the first nucleic acid molecule comprises a first concatemer, and the second nucleic acid molecule comprises a second concatemer.

11. The step determined in situ in step (b) is: a. Determining the sequence of the first nucleic acid molecule or a part thereof, wherein the first nucleic acid molecule or a part thereof consists of 2 to 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 to 30 nucleotides. Includes, (i) Two to thirty nucleotides of the first nucleic acid molecule or a part thereof contain the first identified sequence, and two to thirty nucleotides of the second nucleic acid molecule or a part thereof contain the second identified sequence, or (ii) The method according to claim 6, wherein 2 to 30 nucleotides of the first nucleic acid molecule or a part thereof further comprises at least a portion of the sequence of the first cDNA molecule or the first mRNA molecule, and 2 to 30 nucleotides of the second nucleic acid molecule or a part thereof further comprises at least a portion of the sequence of the second cDNA molecule or the second mRNA molecule.

12. The in situ determination step of step (b) includes determining the sequence of the first concatemer, (a1) 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 comprising the polymerase, one nucleotide from the plurality of nucleotides that is complementary to the nucleotide unit of the first concatemer and binds to the nucleotide unit of the first concatemer, and the first concatemer hybridized to the primer sequence, wherein the nucleotide includes a fluorescent label and a removable blocking group at the 3' carbon position of the sugar moiety, (b1) Incorporating the nucleotide into the 3' end of the primer sequence, (c1) Identifying the nucleic acid base of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide. Including, and, The in situ determination step (b) includes determining the sequence of the second concatemer, and the determination is (a2) 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 comprising the polymerase, one of the plurality of nucleotides complementary to the nucleotide unit of the second concatemer and bound to the nucleotide unit of the second concatemer, and the second concatemer hybridized to the primer sequence, wherein the nucleotide includes a fluorescent label and a removable blocking group at the 3' carbon position of the sugar moiety, (b2) Incorporating the nucleotide into the 3' end of the primer sequence, (c2) Identifying the nucleic acid base of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide. The method according to claim 10, including the method described in claim 10.

13. The in situ determination step of step (b) includes determining the arrangement of the first concatemer, the determination being: (a1) Contacting two of the first concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to a portion of the first concatemer, under conditions sufficient to form a polyvalent complex containing each of the two polymerases, one nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two primer sequences, wherein the nucleotide conjugates include a label and at least two nucleotide moieties, and at least two of the nucleotide moieties are complementary to and bind to each of the nucleotides of the two first concatemers. (b1) Detecting the multivalent binding complex via the label of the nucleotide conjugate, (c1) Identifying two nucleic acid bases of the first concatemer that are complementary to each of the two of the at least two nucleotide portions of the nucleotide conjugate and that bind to each of the two of the at least two nucleotide portions of the nucleotide conjugate. Including, and, The in situ determination step (b) includes determining the sequence of the second concatemer, and the determination is (a2) Contacting two of the second concatemers with two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to a portion of the second concatemer, under conditions sufficient to form a polyvalent complex containing each of the two polymerases, one 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 conjugates include a label and at least two nucleotide moieties, and at least two of the nucleotide moieties are complementary to and bind to each of the nucleotides of the two second concatemers. (b2) Detecting the polyvalent binding complex via the label of the nucleotide conjugate, (c2) Identifying two nucleic acid bases of the second concatemer that are complementary to each of the two of the at least two nucleotide portions of the nucleotide conjugate and that bind to each of the two of the at least two nucleotide portions of the nucleotide conjugate. The method according to claim 10, including the method described in claim 10.

14. (i) The condition inhibits the incorporation of at least two of the nucleotide portions of the nucleotide conjugate into two of the first concatemer and / or the second concatemer, (ii) The nucleotide conjugate comprises a core bonded to a plurality of nucleotide arms, each of which nucleotide portions is bonded to one of the plurality of nucleotide arms, and / or (iii) The method according to claim 13, wherein the detectable label includes a fluorescent label.

15. Determining the sequence of the first concatemer further means (d1) Removing the two polymerases and the nucleotide conjugate from the two first concatemers, (e1) 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 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. (f1) Incorporating each of the two of the plurality of unlabeled nucleotides into each of the two of the primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to each of the two nucleotides of the first concatemer and is bound to each of the two nucleotides of the first concatemer, and one of the unlabeled nucleotides of the plurality of unlabeled nucleotides contains a removable blocking group at the 3' carbon of the sugar portion. Including, and, Determining the sequence of the second concatemer further involves, (d2) Removing the two polymerases and the nucleotide conjugate from the two second concatemers, (e2) 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 comprising each of the two second polymerases, each of the two second concatemers hybridized to each of the two primer sequences, and the two of the plurality of unlabeled nucleotides. (f2) Incorporating each of the two of the plurality of unlabeled nucleotides into each of the two of the primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to each of the two of the second concatemers and is bound to each of the two of the second concatemers, and one of the unlabeled nucleotides of the plurality of unlabeled nucleotides contains a removable blocking group at the 3' carbon of the sugar portion. The method according to claim 13, including the method described in claim 13.

16. The method according to claim 1, wherein the repeating step is repeated at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or at least 50 times.

17. The method according to claim 1, wherein the biological sample is immobilized on the surface.

18. 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, and the at least two target polypeptides include a first target polypeptide encoded by the first target nucleic acid sequence or its inverse complementary sequence and a second target polypeptide encoded by the second target nucleic acid sequence or its inverse complementary sequence, and the method is (a) A step of providing the biological sample, wherein the biological sample is (i) A first nucleic acid molecule comprising the first target nucleic acid sequence or a part thereof, or the reverse complementary sequence of the first target nucleic acid sequence or a part thereof, (ii) A second nucleic acid molecule comprising the second target nucleic acid sequence or a part thereof, or the reverse complementary sequence of the second target nucleic acid sequence or a part thereof, (iii) A third nucleic acid molecule comprising a third target nucleic acid sequence or a part thereof, or a reverse complementary sequence of the third target nucleic acid sequence or a part thereof, wherein 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, (iv) A fourth nucleic acid molecule comprising a fourth target nucleic acid sequence or a part thereof, or a reverse complementary sequence or a part thereof of the fourth target nucleic acid sequence, wherein the presence of the fourth target nucleic acid sequence or its reverse complementary sequence identifies the presence of the second target polypeptide in the biological sample. Processes including, (b) (i) A step of determining in situ the sequence of the first nucleic acid molecule or a portion thereof in the biological sample in order to generate a first sequencing product nucleic acid molecule that is complementary to the first nucleic acid molecule or a portion thereof and binds to the first nucleic acid molecule or a portion thereof, and (ii) A step of determining in situ the sequence of the third nucleic acid molecule or a part thereof in the biological sample in order to generate a third sequencing product nucleic acid molecule that is complementary to the third nucleic acid molecule or a part thereof and binds to the third nucleic acid molecule or a part thereof, (c) (i) The step of in situ identifying the sequence of the second nucleic acid molecule or a part thereof in the biological sample in order to generate a second sequencing product nucleic acid molecule that is complementary to the second nucleic acid molecule or a part thereof and binds to the second nucleic acid molecule or a part thereof, and (ii) The step of in situ identifying the sequence of the fourth nucleic acid molecule or a part thereof in the biological sample in order to generate a fourth sequencing product nucleic acid molecule that is complementary to the fourth nucleic acid molecule or a part thereof and binds to the fourth nucleic acid molecule or a part thereof. Includes, A method wherein the complete sequences of the first target nucleic acid sequence and the complete sequences of the second target nucleic acid sequence differ by at least one nucleotide, the complete sequences of the first target polypeptide and the complete sequences of the second target polypeptide differ by at least one amino acid, and the execution of step (b) is performed under conditions that prevent the execution of step (c).

19. The first nucleic acid molecule or a part thereof consists of 2 to 30 nucleotides, The third nucleic acid molecule or a part thereof consists of 2 to 30 nucleotides. The aforementioned method, (b') A step of 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 the first nucleic acid molecule and the third nucleic acid molecule are located in the biological sample after removal. (b'') The process includes repeating (b) and (b'), The second nucleic acid molecule or a part thereof consists of 2 to 30 nucleotides. The aforementioned fourth nucleic acid molecule or a part thereof consists of 2 to 30 nucleotides, and, The aforementioned method, (c') A step of removing the second sequencing product nucleic acid molecule from the second nucleic acid molecule and the fourth sequencing product nucleic acid molecule from the fourth nucleic acid molecule, wherein the second nucleic acid molecule and the fourth nucleic acid molecule are located in the biological sample after removal. The method according to claim 18, further comprising the step of repeating (c''), (c) and (c').

20. The at least two target nucleic acid molecules comprise at least two target RNA sequences, The aforementioned method, (a) A step of providing the biological sample that is immobilized on a surface, permeable, and immobilized, wherein the biological sample is (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, (ii) comprising a second target RNA sequence of the at least two target RNA sequences and a second target polypeptide of the at least two polypeptides, The first target polypeptide is encoded by the first target RNA sequence or its reverse complementary sequence. The second target polypeptide is encoded by the second target RNA sequence or its reverse complementary sequence. The process, (b) A step of generating a first target cDNA sequence by reverse transcription of the first target RNA sequence and a second target cDNA sequence by reverse transcription of the second target RNA sequence, (c) (i) A step of contacting the first target cDNA sequence with a first oligonucleotide including a first terminal portion and a second terminal portion. (ii) A step of contacting the second target cDNA sequence with a second oligonucleotide including the first terminal portion and the second terminal portion. (iii) A step of 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, and (iv) A step of 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, The first and second terminal portions of the first oligonucleotide are complementary to two adjacent segments of the first target cDNA sequence and bind to the two adjacent segments of the first target cDNA sequence, thereby forming a cyclic structure with a nick or gap between the first and second terminal portions, and the first oligonucleotide comprises a first identification sequence for identifying the first target RNA sequence, a first sequencing primer sequence, and a nucleic acid amplification primer sequence. The first and second terminal portions of the second oligonucleotide are complementary to two adjacent segments of the second target cDNA sequence and bind to the two adjacent segments of the second target cDNA sequence, thereby forming a cyclic structure with a nick or gap between the first and second terminal portions, and the second oligonucleotide comprises a second identification sequence for identifying the second target RNA sequence, a second sequencing primer sequence, and a nucleic acid amplification primer sequence, wherein the first and second sequencing primers have a difference of at least one nucleotide. The first short nucleic acid comprises a first tag sequence and a second tag sequence, the first alkyl conjugate specifically binds to the first target polypeptide via the first binding site to form a first binding complex, the first tag sequence and the second tag sequence identify the first binding site, and The second short nucleic acid comprises a third tag sequence and a fourth tag sequence, the second oligonucleotide conjugate specifically binds to the second target polypeptide via the second binding site to form a second binding complex, and the third tag sequence and the fourth tag sequence identify the second binding site, the steps of (d) (i) A step of bringing the first binding complex into contact with a third oligonucleotide including a first terminal portion and a second terminal portion, and (ii) A step of bringing the second binding complex into contact with a fourth oligonucleotide including the first and second terminal portions, The first and second terminal portions of the third oligonucleotide are complementary to the first and second tag sequences of the first oligonucleotide conjugate, and bind to the first and second tag sequences of the first oligonucleotide conjugate, thereby forming a cyclic structure with a gap or nick between the first and second terminal portions, and The first and second terminal portions of the fourth oligonucleotide are complementary to the third and fourth tag sequences, and bind to the third and fourth tag sequences, thereby forming a cyclic structure in which the fourth oligonucleotide has a gap or nick between the first and second terminal portions. (e) A step of generating a first cyclic oligonucleotide by linking the first and second terminal portions of the first oligonucleotide, generating a second cyclic oligonucleotide by linking the first and second terminal portions of the second oligonucleotide, generating a third cyclic oligonucleotide by linking the first and second terminal portions of the third oligonucleotide, and generating a fourth cyclic oligonucleotide by linking the first and second terminal portions of the fourth oligonucleotide, wherein the first and third cyclic oligonucleotides include a first sequencing primer sequence or its inverse complementary sequence, the second and fourth cyclic oligonucleotides include a second sequencing primer sequence or its inverse complementary sequence, and the sequences of the first and second sequencing primers differ by at least one nucleotide. (f) (i) A step of amplifying the first cyclic oligonucleotide by rolling circle amplification in order to produce a first concatemer comprising a plurality of sequences of the first cyclic oligonucleotide, (ii) A step of amplifying the second cyclic oligonucleotide by rolling circle amplification in order to produce a second concatemer comprising a plurality of sequences of the second cyclic oligonucleotide, (iii) A step of amplifying the third cyclic oligonucleotide by rolling circle amplification in order to produce a third concatemer comprising a plurality of sequences of the third cyclic oligonucleotide, and (iv) A step of amplifying the fourth cyclic oligonucleotide by rolling circle amplification in order to produce a fourth concatemer comprising a plurality of sequences of the fourth cyclic oligonucleotide, (g) (i) A step of determining the sequence of the first concatemer or a portion thereof in situ in order to generate a first sequencing product nucleic acid molecule that is complementary to the first concatemer and binds to the first concatemer, and (ii) A step of determining the sequence of the third concatemer or a portion thereof in situ in order to generate a third sequencing product nucleic acid molecule that is complementary to the third concatemer and binds to the third concatemer, The sequence of the first concatemer or a part thereof consists of 2 to 30 nucleotides, and The sequence of the third concatemer or a part thereof consists of 2 to 30 nucleotides. The execution of process (g) is subject to conditions that prevent the execution of process (j). The process, (h) A step of removing the first sequencing product nucleic acid molecule from the first concatemer and the third sequencing product nucleic acid molecule from the third concatemer, wherein the first concatemer and the third concatemer are located in the biological sample after removal. (i) (g) and (h) are repeated at least once, (j) (i) the step of determining the sequence of the second concatemer or a portion thereof in situ in order to generate a second sequencing product nucleic acid molecule that is complementary to the second concatemer and binds to the second concatemer, and (ii) A step of determining the sequence of the fourth concatemer or a portion thereof in situ in order to generate a fourth sequencing product nucleic acid molecule that is complementary to the fourth concatemer and binds to the fourth concatemer, The sequence of the second concatemer or a part thereof consists of 2 to 30 nucleotides, and The sequence of the fourth concatemer or a part thereof consists of 2 to 30 nucleotides. The execution of step (j) is performed under conditions that prevent the execution of step (g), wherein the complete sequences of the first target RNA sequence and the second target RNA sequence differ by at least one nucleotide, and the complete sequences of the first target polypeptide and the second target polypeptide differ by at least one amino acid. The process, (k) A step of removing the second sequencing product nucleic acid molecule from the second concatemer and the fourth sequencing product nucleic acid molecule from the fourth concatemer, wherein the second concatemer and the fourth concatemer are located in the biological sample after removal. A step of repeating (l), (j), and (k) at least once, The method according to claim 18, including the method described in claim 18.

21. The method according to claim 18, wherein the determining step includes imaging the first sequencing product nucleic acid molecule or the third sequencing product nucleic acid molecule, and the identifying step includes imaging the second sequencing product nucleic acid molecule or the fourth sequencing product nucleic acid molecule.

22. The method of claim 18, further comprising the step of simultaneously imaging the first sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule in order to analyze the spatial distribution of the first sequencing product nucleic acid molecule and the second sequencing product nucleic acid molecule within the biological sample.

23. The first concatemer, the second concatemer, the third concatemer, or the fourth concatemer comprises a compaction oligonucleotide sequence. (a) The first segment of the compaction oligonucleotide is complementary to and bound to the first portion of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer. (b) The method according to claim 20, wherein the second segment of the compaction oligonucleotide is complementary to the second portion of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, and binds to the second portion to result in a reduction in size or a change in shape of the second concatemer.

24. The first sequencing product nucleic acid molecule comprises a first identification sequence for identifying the first target RNA sequence, and the second sequencing product nucleic acid molecule comprises a second identification sequence for identifying the second target RNA sequence, or The method according to claim 20, wherein the first sequencing product nucleic acid molecule comprises the first target RNA sequence and a first identification sequence for identifying a portion of the first target RNA sequence, and the second sequencing product nucleic acid molecule comprises the second target RNA sequence and a second identification sequence for identifying a portion of the second target RNA sequence.

25. The step of determining the above is: (a) Contacting the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer with a polymerase, a plurality of nucleotides, and a primer sequence complementary to a portion of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, under conditions sufficient to form a binding complex comprising the polymerase, one of the plurality of nucleotides complementary to the nucleotide unit of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer and binding to the nucleotide unit, and the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer hybridized to the primer sequence, wherein the nucleotide includes a fluorescent label and a removable blocking group at the 3' carbon position of the sugar moiety, (b) Incorporating the nucleotide at the 3' end of the primer sequence, (c) Identifying the nucleic acid base of the incorporated nucleotide by imaging the fluorescent label of the incorporated nucleotide. The method according to claim 20, including the method described in claim 20.

26. The step of determining the above is: (a) Two of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer are hybridized to two polymerases, a plurality of nucleotide conjugates, and two primer sequences complementary to a portion of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, with each of the two polymerases, one nucleotide conjugate from the plurality of nucleotide conjugates, and each of the two primer sequences. Contacting under conditions sufficient to form a polyvalent complex comprising each of the concatemers, the second concatemer, the third concatemer, or the fourth concatemer, wherein the nucleotide conjugate comprises a label and at least two nucleotide moieties, the two of which are complementary to and bind to each of the two nucleotides of the first concatemer, (b) detecting the polyvalent binding complex via the label of the nucleotide conjugate, (c) Identifying two nucleic acid bases of the first concatemer that are complementary to each of the two of the at least two nucleotide portions of the nucleotide conjugate and that bind to each of the two of the at least two nucleotide portions of the nucleotide conjugate. The method according to claim 20, including the method described in claim 20.

27. ​​The determination step further includes: (d) Removing two polymerases and the nucleotide conjugate from two of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, (e) Each of the two first concatemers, second concatemers, third concatemers, or fourth concatemers hybridized to each of the two primer sequences forms a binding complex with two second polymerases and a plurality of unlabeled nucleotides, each of the two second polymerases, each of the two first concatemers, second concatemers, third concatemers, or fourth concatemers hybridized to each of the two primer sequences, and two of the plurality of unlabeled nucleotides. The method according to claim 26, comprising contacting under conditions suitable for making the product, and incorporating each of the two of the plurality of unlabeled nucleotides into each of the two of the primer sequences, wherein each of the two of the plurality of unlabeled nucleotides is complementary to and bound to each of the two nucleotides of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, and one of the unlabeled nucleotides of the plurality of unlabeled nucleotides contains a removable blocking group at the 3' carbon of the sugar portion.

28. The step of determining the above is: (a) Two of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer are hybridized to two polymerases, a plurality of nucleotide conjugates, a first primer sequence complementary to the first portion of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, and a second primer sequence complementary to the second portion of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, each of the two polymerases, one nucleotide conjugate from the plurality of nucleotide conjugates, the first concatemer, the second Contacting under conditions sufficient to form a polyvalent conjugate comprising a concatemer, the first portion of the third concatemer, or the fourth concatemer, and the second portion of the first concatemer, second concatemer, third concatemer, or fourth concatemer hybridized to the second primer sequence, wherein the nucleotide conjugate comprises a label and at least two nucleotide moieties, two of which are complementary to and bind to the nucleotides of the first and second portions of the first, second, third, or fourth concatemer, respectively. (b) detecting the polyvalent binding complex via the label of the nucleotide conjugate, (c) Identifying the nucleic acid bases of the first and second portions of the first concatemer, second concatemer, third concatemer, or fourth concatemer that are complementary to and bind to each of at least two of the nucleotide portions of the nucleotide conjugate. The method according to claim 20, including the method described in claim 20.

29. (i) The condition inhibits the incorporation of at least two of the nucleotide portions of the nucleotide conjugate into two of the first concatemer, the second concatemer, the third concatemer, or the fourth concatemer, (ii) The nucleotide conjugate comprises a core bonded to a plurality of nucleotide arms, each of which nucleotide portions is bonded to one of the plurality of nucleotide arms, and / or (iii) The method according to claim 27, wherein the detectable label includes a fluorescent label.

30. The method according to claim 18, wherein the biological sample is immobilized on the surface.

31. A system for in situ detection of at least two target nucleic acid sequences in a biological sample, The at least two target nucleic acid sequences include a first target nucleic acid sequence and a second target nucleic acid sequence, and the system is (a) A biological sample containing a first nucleic acid molecule and a second nucleic acid molecule, (b) A first oligonucleotide comprising a first terminal portion and a second terminal portion, (c) comprising a second oligonucleotide including a first terminal portion and a second terminal portion, The first nucleic acid molecule comprises the first target nucleic acid sequence or its reverse complementary sequence, or a part thereof, and the second nucleic acid molecule comprises the second target nucleic acid sequence or its reverse complementary sequence, or a part thereof. The first and second terminal portions 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 part thereof, and as a result, the first oligonucleotide forms a first cyclic oligonucleotide in the biological sample, and the first cyclic oligonucleotide includes a gap between the first and second terminal portions. A system wherein the first and second terminal portions of the second oligonucleotide are complementary to and bind to two adjacent segments of the second target nucleic acid sequence or its inverse complementary sequence or a part thereof, and as a result, the second oligonucleotide forms a second cyclic oligonucleotide in the biological sample, the second cyclic oligonucleotide having a gap between the first and second terminal portions.

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