Methods and systems for sample processing or analysis

Abstract

The present disclosure provides methods and systems for detecting nucleic acid sequences in biological samples having a three-dimensional matrix. The present disclosure also provides methods and systems for processing samples for nucleic acid sequence detection.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 711,994, filed on July 30, 2018, which is incorporated herein by reference in its entirety. Background Art

[0003] The padlock probe can be a linear cyclizable oligonucleotide having a free 5' end and a 3' end that can be used for connection so as to present a circular conformation. In order for cyclization to occur (e.g., by connection), the padlock probe can have a free 5' phosphate group or a 5' adenylated end. In order to allow the end of the padlock probe to be juxtaposed for connection, the padlock probe can be configured to have its 5' terminal region and 3' terminal region complementary to its target sequence (e.g., RNA in a cell sample to be analyzed or a synthetic complementary deoxyribonucleic acid (cDNA) molecule). These complementary regions can allow the padlock probe to specifically bind to its target sequence by hybridizing with a specific sequence in the target. Summary of the Invention

[0004] The present disclosure provides methods and systems for nucleic acid sequence detection in biological samples having a three-dimensional matrix. The present disclosure also provides methods and systems for sample processing for target analysis or detection in downstream applications, such as in situ sequencing.

[0005] In one aspect, the present disclosure provides a method for identifying a nucleic acid sequence in a biological sample. In one embodiment, the method comprises: (a) providing a biological sample comprising a ribonucleic acid (RNA) molecule hybridized to a deoxyribonucleic acid molecule (DNA) in a three-dimensional (3D) matrix, wherein the RNA molecule comprises the nucleic acid sequence; (b) using a reverse transcriptase to degrade or digest at least a portion of the RNA molecule hybridized to the DNA molecule, the DNA molecule comprising an additional nucleic acid sequence that is the reverse complement of the nucleic acid sequence; and (c) detecting the additional nucleic acid sequence in the biological sample, thereby identifying the nucleic acid sequence.

[0006] In some embodiments, the DNA molecule is a complementary deoxyribonucleic acid (cDNA) molecule. In some embodiments, the method further comprises, prior to (a), using another reverse transcriptase to reverse transcribe the RNA molecule to produce a DNA molecule hybridized with the RNA molecule in the biological sample. In some embodiments, the method further comprises, prior to (a), using a reverse transcriptase to reverse transcribe the RNA molecule to produce a DNA molecule hybridized with the RNA molecule in the biological sample.

[0007] In some embodiments, a DNA molecule is anchored to a 3D matrix. In some embodiments, a DNA molecule comprises a functional moiety, and wherein the DNA molecule is anchored to the 3D matrix via the functional moiety. In some embodiments, an RNA molecule is anchored to a 3D matrix. In some embodiments, an RNA molecule comprises a functional moiety, and wherein the RNA molecule is anchored to the 3D matrix via the functional moiety. In some embodiments, the method further comprises forming the 3D matrix using a matrix-forming material.

[0008] In some embodiments, (c) includes contacting the cDNA molecule with a probe. In some embodiments, the probe comprises a functional portion, wherein the probe is fixed to a 3D matrix by the functional portion. In some embodiments, the probe is a padlock probe, wherein the padlock probe comprises a 5' terminal region and a 3' terminal region complementary to the cDNA molecule. In some embodiments, the method also includes hybridizing the 5' terminal region and the 3' terminal region of the padlock probe with the cDNA molecule. In some embodiments, the method also includes cyclizing the padlock probe by linking the two ends of the padlock probe together to produce a cyclized padlock probe. In some embodiments, the two ends of the padlock probe are continuous. In some embodiments, the two ends of the padlock probe are separated by a gap region comprising at least one nucleotide. In some embodiments, the gap region comprises 2 to 500 nucleotides. In some embodiments, the method also includes filling the gap region by introducing at least one nucleotide in an extension reaction. In some embodiments, the method also includes filling the gap region by at least one other nucleotide or another oligonucleotide sequence. In some embodiments, the length of the other oligonucleotide sequence is 2 to 500 nucleotides.

[0009] In some embodiments, the method further comprises subjecting the circularized padlock probe to rolling circle amplification (RCA) to produce an amplified product of the sequence of the circularized padlock probe, the amplified product comprising a nucleic acid sequence corresponding to the nucleic acid sequence of the RNA molecule. In some embodiments, the method further comprises detecting the nucleic acid sequence of the amplified product, thereby identifying the nucleic acid sequence of the RNA molecule.

[0010] In some embodiments, the reverse transcriptase or another reverse transcriptase has RNA catalytic cleavage activity. In some embodiments, the reverse transcriptase or another reverse transcriptase has RNA catalytic cleavage activity of RNA / DNA duplex. In some embodiments, the reverse transcriptase or another reverse transcriptase is avian myeloblastosis virus (Avian myeloblastosis virus, AMV) reverse transcriptase, wild-type human immunodeficiency virus-1 (HIV-1) reverse transcriptase or Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase.

[0011] In some embodiments, the method further comprises, before (a), hybridizing a reverse transcription primer to an RNA molecule. In some embodiments, the reverse transcription primer can hybridize to the 5' end region of the padlock probe. In some embodiments, the reverse transcription primer comprises a functional portion, wherein the cDNA molecule is fixed to the 3D matrix through the functional portion. In some embodiments, the biological sample comprises a plurality of RNA molecules, wherein the plurality of RNA molecules have a relative 3D spatial relationship.

[0012] In some embodiments, (b) is performed under a first set of conditions and reverse transcription of the RNA molecule using an additional reverse transcriptase or using a reverse transcriptase is performed under a second set of conditions, wherein the first set of conditions is different from the second set of conditions. In some embodiments, the first set of conditions or the second set of conditions is selected from pH, temperature, cofactor concentration, and cation concentration. In some embodiments, the cofactor or cation includes Mg. 2+ 、Mn 2+ 、Na + , ATP, NADPH. In some embodiments, the second set of conditions inhibits the RNase activity of the reverse transcriptase. In some embodiments, the second set of conditions comprises an RNase inhibitor. In some embodiments, the RNase inhibitor is a small molecule inhibitor or a polypeptide.

[0013] In another aspect, the present disclosure provides a method for identifying a nucleic acid sequence in a biological sample. In one embodiment, the method comprises: (a) providing a biological sample comprising a ribonucleic acid (RNA) molecule hybridized to a deoxyribonucleic acid (DNA) molecule in a three-dimensional (3D) matrix, wherein the RNA molecule comprises the nucleic acid sequence; (b) degrading or digesting at least a portion of the RNA molecule hybridized to the DNA molecule using a deoxyribonucleic acid (DNA) binding protein that is not a reverse transcriptase or ribonuclease, the DNA molecule comprising an additional nucleic acid sequence that is a reverse complement of the nucleic acid sequence; and (c) detecting the additional nucleic acid sequence in the biological sample, thereby identifying the nucleic acid sequence.

[0014] In some embodiments, the DNA molecule is a complementary deoxyribonucleic acid (cDNA) molecule. In some embodiments, the method further comprises, before (a), reverse transcribing the RNA molecule using a reverse transcriptase to generate a DNA molecule hybridized with the RNA molecule in the biological sample.

[0015] In some embodiments, a DNA molecule is anchored to a 3D matrix. In some embodiments, a DNA molecule comprises a functional moiety, and wherein the DNA molecule is anchored to the 3D matrix via the functional moiety. In some embodiments, an RNA molecule is anchored to a 3D matrix. In some embodiments, an RNA molecule comprises a functional moiety, and wherein the RNA molecule is anchored to the 3D matrix via the functional moiety. In some embodiments, the method further comprises forming the 3D matrix using a matrix-forming material.

[0016] In some embodiments, (c) includes contacting the cDNA molecule with a probe. In some embodiments, the probe comprises a functional moiety, wherein the probe is fixed to a 3D matrix by the functional moiety. In some embodiments, the probe is a padlock probe, wherein the padlock probe comprises a 5' terminal region and a 3' terminal region complementary to the cDNA molecule, and the 5' terminal region and the 3' terminal region of the padlock probe are hybridized with the cDNA molecule. In some embodiments, the method also includes cyclizing the padlock probe by linking the two ends of the padlock probe together to produce a cyclized padlock probe. In some embodiments, the two ends of the padlock probe are continuous. In some embodiments, the two ends of the padlock probe are separated by a gap region comprising at least one nucleotide. In some embodiments, the gap region comprises 2 to 500 nucleotides. In some embodiments, the method also includes filling the gap region by introducing at least one nucleotide in an extension reaction. In some embodiments, the method also includes filling the gap region by at least one other nucleotide or other oligonucleotide sequence. In some embodiments, the length of the other oligonucleotide sequence is 2 to 500 nucleotides.

[0017] In some embodiments, the method further comprises subjecting the circularized padlock probe to rolling circle amplification (RCA) to produce an amplified product of the sequence of the circularized padlock probe, the amplified product comprising a nucleic acid sequence corresponding to the nucleic acid sequence of the RNA molecule. In some embodiments, the method further comprises detecting the nucleic acid sequence of the amplified product, thereby identifying the nucleic acid sequence of the RNA molecule.

[0018] In some embodiments, the DNA binding protein has RNA catalytic cleavage activity. In some embodiments, the DNA binding protein stabilizes DNA molecules. In some embodiments, the DNA binding protein increases the melting temperature of DNA molecules. In some embodiments, the DNA binding protein is Sso7d.

[0019] In some embodiments, the method further comprises, before (a), hybridizing a reverse transcription primer to an RNA molecule. In some embodiments, the reverse transcription primer can hybridize to the 5' end region of the padlock probe. In some embodiments, the reverse transcription primer comprises a functional portion, wherein the reverse transcription primer or the DNA molecule is fixed to the 3D matrix through the functional portion. In some embodiments, the biological sample comprises multiple RNA molecules, wherein the multiple RNA molecules have a relative 3D spatial relationship.

[0020] In another aspect, the present disclosure provides a method for identifying a nucleic acid sequence in a biological sample. In one embodiment, the method comprises: (a) providing a biological sample comprising a ribonucleic acid (RNA) molecule hybridized to a deoxyribonucleic acid molecule (DNA) in a three-dimensional (3D) matrix, wherein the RNA molecule comprises the nucleic acid sequence; (b) non-enzymatically degrading at least a portion of the RNA molecule hybridized to the DNA molecule, the DNA molecule comprising an additional nucleic acid sequence that is the reverse complement of the nucleic acid sequence; (c) contacting the DNA molecule with a probe; and (d) detecting the sequence of the probe or a derivative thereof, thereby identifying the nucleic acid sequence of the RNA molecule.

[0021] In some embodiments, the DNA molecule is a complementary deoxyribonucleic acid (cDNA) molecule. In some embodiments, the method further comprises, before (a), using a reverse transcriptase to reverse transcribe the RNA molecule to produce a DNA molecule hybridized with the RNA molecule in the biological sample. In some embodiments, the DNA molecule is fixed to a 3D matrix. In some embodiments, the DNA molecule comprises a functional portion, wherein the DNA molecule is fixed to the 3D matrix through the functional portion. In some embodiments, the RNA molecule is fixed to the 3D matrix. In some embodiments, the RNA molecule comprises a functional portion, wherein the RNA molecule is fixed to the 3D matrix through the functional portion.

[0022] In some embodiments, the probe is a padlock probe. In some embodiments, the probe comprises a functional portion, wherein the probe is fixed to a 3D matrix by the functional portion. In some embodiments, the method also includes using a matrix forming material to form a 3D matrix. In some embodiments, the padlock probe comprises a 5' terminal region and a 3' terminal region that are complementary to a DNA molecule. In some embodiments, the method also includes hybridizing the 5' terminal region and the 3' terminal region of the padlock probe to the DNA molecule.

[0023] In some embodiments, the method also comprises making the padlock probe cyclization by coupling the two ends of the padlock probe together, to produce the padlock probe of cyclization, and detect the nucleic acid sequence of cyclization padlock probe or its derivative, thereby identify the nucleic acid sequence of RNA molecule.In some embodiments, the two ends of padlock probe are continuous.In some embodiments, the two ends of padlock probe are separated by the gap region comprising at least one Nucleotide.In some embodiments, gap region comprises 2 to 500 Nucleotide.In some embodiments, the method also comprises filling gap region by introducing at least one Nucleotide in the extension reaction.In some embodiments, the method also comprises filling gap region by at least one other Nucleotide or other oligonucleotide sequence.In some embodiments, the length of other oligonucleotide sequence is 2 to 500 Nucleotide.

[0024] In some embodiments, (c) comprises subjecting the circularized padlock probe to rolling circle amplification (RCA) to produce an amplification product of the sequence of the circularized padlock probe, the amplification product comprising a nucleic acid sequence corresponding to the nucleic acid sequence of the RNA molecule. In some embodiments, (d) comprises detecting the nucleic acid sequence of the amplification product to identify the nucleic acid sequence of the RNA molecule.

[0025] In some embodiments, the method further comprises, prior to (a), hybridizing a reverse transcription primer to the RNA molecule. In some embodiments, the reverse transcription primer can hybridize to the 5' terminal region of the padlock probe. In some embodiments, the reverse transcription primer comprises a functional portion, wherein the DNA molecule is fixed to the 3D matrix via the functional portion. In some embodiments, (b) comprises chemically degrading the RNA molecule under conditions selected from the group consisting of: a pH of 6 to 14, a temperature of 10°C to 100°C, in the presence of heavy metal ions, in the presence of divalent cations, and any combination thereof.

[0026] In another aspect, the present disclosure provides a method for processing a biological sample. In one embodiment, the method comprises: (a) providing a biological sample comprising a ribonucleic acid (RNA) molecule in a three-dimensional (3D) matrix, wherein the RNA molecule comprises a nucleic acid sequence; (b) hybridizing a primer to the RNA molecule, the primer not comprising a functional moiety for immobilization to the matrix; and (c) reverse transcribing the RNA molecule using a reverse transcriptase by extending the primer to produce a complementary deoxyribonucleic acid (cDNA) molecule hybridized to the RNA molecule in the biological sample, the cDNA molecule comprising a functional moiety for immobilizing the cDNA molecule to the 3D matrix.

[0027] In some embodiments, the method further comprises degrading the RNA molecule hybridized to the cDNA molecule to provide a cDNA molecule immobilized to the 3D matrix via the functional moiety, wherein the cDNA molecule comprises an additional nucleic acid sequence that is a reverse complement of the nucleic acid sequence. In some embodiments, the RNA molecule comprises the functional moiety, wherein the RNA molecule is immobilized to the 3D matrix via the functional moiety.

[0028] In some embodiments, the degradation comprises degradation of the RNA molecule by a non-ribonuclease. In some embodiments, the non-ribonuclease is a reverse transcriptase or a DNA binding protein. In some embodiments, the degradation comprises degradation of the RNA molecule by a non-enzymatic reaction. In some embodiments, the non-enzymatic reaction is carried out under conditions selected from the group consisting of: a pH of 6 to 14, a temperature of 10° C. to 100° C., in the presence of heavy metal ions, in the presence of divalent cations, and any combination thereof.

[0029] In some embodiments, the method also includes contacting the cDNA molecule with the probe. In some embodiments, the probe includes a region that cannot hybridize with the cDNA molecule. In some embodiments, the probe is a padlock probe, wherein the padlock probe includes a 5' terminal region and a 3' terminal region that are complementary to the cDNA molecule, and the 5' terminal region and the 3' terminal region of the padlock probe are hybridized with the cDNA molecule. In some embodiments, the method also includes cyclizing the padlock probe by coupling the two ends of the padlock probe together to produce a cyclized padlock probe, and detecting the nucleic acid sequence of the cyclized padlock probe or its derivative, thereby identifying the nucleic acid sequence of the RNA molecule. In some embodiments, the two ends of the padlock probe are continuous. In some embodiments, the two ends of the padlock probe are separated by a gap region comprising at least one nucleotide. In some embodiments, the gap region comprises 2 to 500 nucleotides. In some embodiments, the method also includes filling the gap region by introducing at least one nucleotide in the extension reaction. In some embodiments, the method also includes filling the gap region by at least one other nucleotide or other oligonucleotide sequence. In some embodiments, the length of the other oligonucleotide sequence is 2 to 500 nucleotides. In some embodiments, the method further comprises subjecting the circularized padlock probe to rolling circle amplification (RCA) to produce an amplification product of the sequence of the circularized padlock probe, the amplification product comprising a nucleic acid sequence corresponding to the nucleic acid sequence of the RNA molecule. In some embodiments, the method further comprises detecting the nucleic acid sequence of the amplification product, thereby identifying the nucleic acid sequence of the RNA molecule. In some embodiments, the probe comprises a functional portion, wherein the probe is fixed to the 3D matrix by the functional portion. In some embodiments, the functional portion is directly conjugated to the probe. In some embodiments, the probe is hybridized with a tethered oligonucleotide comprising a functional portion. In some embodiments, the tethered oligonucleotide hybridizes with a region of the probe that is not hybridized with a cDNA molecule. In some embodiments, the method further comprises detecting the sequence of the probe or its derivatives, thereby identifying the nucleic acid sequence of the RNA molecule.

[0030] In some embodiments, (c) includes using a reverse transcriptase to introduce a nucleotide analog comprising a functional portion into the growing chain to produce a cDNA molecule comprising a nucleotide. In some embodiments, the nucleotide analog comprises amino-allyl dUTP, 5-TCO-PEG4-dUTP, C8-alkyne-dUTP, 5-azidomethyl-dUTP (5-Azidomethyl-dUTP), 5-vinyl-dUTP, 5-ethynyl dUTP, or a combination thereof. In some embodiments, the method further comprises, after (b), modifying the primer or cDNA molecule to comprise a functional portion. In some embodiments, the primer is modified to comprise a functional portion before the cDNA molecule is produced. In some embodiments, the primer comprises a region that cannot hybridize with an RNA molecule, wherein the region hybridizes to another tethered oligonucleotide comprising a functional portion.

[0031] In some embodiments, (c) includes connecting the functional moiety to the cDNA molecule by an enzymatic reaction or a non-enzymatic reaction. In some embodiments, the enzymatic reaction includes using an enzyme to connect a nucleotide or oligonucleotide having a functional moiety to the cDNA molecule. In some embodiments, the enzyme is a ligase, a polymerase, or a combination thereof. In some embodiments, the non-enzymatic reaction includes attaching a chemical reagent having a functional moiety to the cDNA molecule by alkylation or hydroxymercuration. In some embodiments, the cDNA molecule is also hybridized with a tethered oligonucleotide having a functional moiety. In some embodiments, the tethered oligonucleotide is hybridized with a primer. In some embodiments, the 3D matrix further comprises additional functional moieties, wherein the additional functional moieties react with the functional moieties of the cDNA molecule to immobilize the cDNA molecule.

[0032] Another aspect of the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, when executed by one or more computer processors, implements any of the methods above or elsewhere herein.

[0033] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto, wherein the computer memory comprises machine executable code that, when executed by the one or more computer processors, implements any of the methods described above or elsewhere herein.

[0034] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the detailed description below, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive.

[0035] Incorporation by reference

[0036] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent such incorporated by reference publications and patents or patent applications contradict the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and accompanying drawings (also referred to herein as "Figures") which illustrate illustrative embodiments utilizing the principles of the present invention, in which:

[0038] Figure 1 An example of a method for identifying a nucleic acid sequence in a biological sample is shown.

[0039] Figure 2 An example of a method for identifying a nucleic acid sequence in a biological sample is shown.

[0040] Figure 3 An example of a method for identifying a nucleic acid sequence in a biological sample is shown.

[0041] Figure 4 An example of a method for processing a biological sample is shown.

[0042] Figure 5 A computer system is shown that is programmed or otherwise configured to implement the methods provided herein.

[0043] Figure 6 Example images of tissue samples processed and imaged using the methods of the present disclosure are shown. DETAILED DESCRIPTION

[0044] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0045] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0046] As used herein, the term "amplification" generally refers to the production of one or more copies of a nucleic acid (or "amplification products"). The one or more copies can be produced by extending the nucleic acid. Such extension can be a single round of extension or multiple rounds of extension. Amplification products can be produced by polymerase chain reaction (PCR).

[0047] As used herein, the term "reverse transcription" generally refers to the production of deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template by the action of a reverse transcriptase. Reverse transcription PCR (or RT-PCR) refers to reverse transcription coupled with PCR.

[0048] As used herein, the term "nucleic acid" generally refers to nucleotides in a polymeric form of any length. Nucleic acid can include deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs) or their analogs. Nucleic acid can be an oligonucleotide or a polynucleotide. Nucleic acid can have any three-dimensional structure and can perform any function. Non-limiting examples of nucleic acid include coding or non-coding regions of DNA, RNA, genes or gene fragments, multiple loci (one locus) defined in linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid can include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after nucleic acid assembly. The nucleotide sequence of a nucleic acid can be interrupted by non-nucleotide components. Nucleic acids can be further modified after polymerization, for example by conjugation with functional moieties for immobilization.

[0049] As used herein, the term "subject" generally refers to an entity or medium that has or can have testable or detectable genetic information. A subject can be a person or individual. A subject can be a vertebrate, such as a mammal. Non-limiting examples of mammals include rodents, apes, and humans. A subject can be an animal, such as a farm animal. A subject can be a pet, such as a dog, cat, mouse, rat, or bird. Other examples of subjects include food, plants, soil, and water. A subject can exhibit a disease or symptoms of a disease. Alternatively, a subject can be asymptomatic.

[0050] Any suitable biological sample comprising nucleic acid can be obtained from a subject. Any suitable biological sample comprising nucleic acid can be used in the methods and systems described herein. The biological sample can be a solid substance (e.g., biological tissue) or can be a fluid (e.g., a biological fluid). Typically, a biological fluid can include any fluid associated with a living organism. Non-limiting examples of biological samples include blood (or blood components, such as white blood cells, red blood cells, platelets) obtained from any anatomical location of a subject (e.g., tissue, circulatory system, bone marrow), cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginal fluid, interstitial fluid from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, cavity fluid, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, eye fluids, sweat, mucus, earwax, oils, glandular secretions, spinal fluid, hair, nails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, umbilical cord blood, emphatic fluid, and / or other secretions or body tissues. The biological sample can be a cell-free sample. Such cell-free samples may include DNA and / or RNA.

[0051] Overview

[0052] Provided herein are methods and systems for sample processing in target analysis or detection. The methods and systems of the present disclosure can be used for various applications, such as in situ sequencing or sequence identification (e.g., sequencing or sequence identification in a sample (e.g., cell)). In these methods and systems, probes can be used for target capture and subsequently for analysis or detection of a sample. Such probes can be padlock probes. Padlock probes can be designed or configured to specifically bind to a target. In some cases, padlock probes can be designed to directly hybridize with a target. In other cases, padlock probes can be designed to indirectly bind to a target by hybridizing with a molecule derived from the target. For example, in some applications where ribonucleic acid (RNA) molecules are targets, complementary deoxyribonucleic acid (cDNA) molecules can be synthesized from an RNA target by reverse transcription, and padlock probes can be designed to bind to a cDNA molecule. By hybridizing with a cDNA molecule, the ends of the padlock probes are juxtaposed to connect. Connection can be direct or indirect. In other words, the ends of the padlock probes can be directly connected to each other, or they can be connected to an intermediate nucleic acid molecule or a nucleotide sequence. Thus, the terminal regions of the padlock probes can be complementary to adjacent or continuous regions in a cDNA molecule synthesized from an RNA target molecule, or they can be complementary to non-adjacent or non-contiguous regions of the cDNA. In cases where the padlock probes are complementary to non-adjacent or non-contiguous regions of the cDNA molecule, the "gap" between the two ends of the hybridized padlock probe can be filled with an intervening oligonucleotide molecule or nucleotide sequence for ligation.

[0053] After being added to the sample with target molecule, the end of padlock probe can hybridize with the complementary region in target molecule or its derivative (such as cDNA molecule). After hybridization, the end of padlock probe can be directly or indirectly connected to padlock probe by ligase and padlock probe is circularized. The padlock probe of circularization is amplified to produce an amplification product. For example, rolling circle amplification (RCA) can be carried out to the padlock probe of circularization, to produce DNA nanoball (i.e., rolony). The padlock probe of circularization can be triggered by the 3' end of cDNA (i.e., RCA is triggered by the target). DNA polymerase with 3'-5' exonuclease activity can be used. This can digest the cDNA chain along the 3'-5' direction to the position adjacent to the bound padlock probe. Alternatively, cDNA can have an appropriate length and can serve as a primer for amplification reaction mediated by DNA polymerase without the need for such digestion. As another alternative, other primers that can hybridize with padlock probe can be added to the sample for amplification reaction, rather than triggering RCA with cDNA molecules.

[0054] Amplified product (for example, rolony) can be used for the purpose of carrying out original position (for example in sample, for example intracellular) molecular detection by fluorescence in situ sequencing (FISSEQ) in biological sample (for example cell or tissue).Biological sample can comprise three-dimensional matrix (3D matrix).3D matrix can be formed by making biological sample stand fixative (such as formaldehyde).3D matrix can also be formed by matrix forming material (for example polymerizable monomer or cross-linkable polymer).Amplified product can be used as the amplification sequencing template of FISSEQ, wherein for example the sequence feature of amplified product can be detected in situ by fluorescence sequencing, and fluorescence sequencing includes but is not limited to synthetic sequencing (SBS), connection sequencing (SBL) or hybridization sequencing (SBH).Use multiple padlock probes, multiple target nucleic acids can be detected in a multiplex manner.

[0055] Compared with the method or system of the ligation reaction of the DNA-RNA "hybridization" duplex template formed between the RNA molecule and the DNA padlock probe, the method or system of the ligation reaction of the DNA-DNA duplex template formed between the cDNA molecule and the DNA padlock probe can be more effective. This may be due to the improved efficiency of the enzymatic ligation between the DNA-DNA duplex template compared with the DNA-RNA hybridization duplex template. Therefore, before hybridizing with the padlock probe, all or part of the target RNA molecule can first be converted into a cDNA molecule, for example, by reverse transcription. After generating the cDNA molecule, the RNA molecule can be degraded. The methods and systems provided herein use a variety of methods to degrade the RNA molecule. In some aspects, there is provided the use of a non-ribonuclease with ribonuclease activity to enzymatically digest RNA. In some other aspects, there is provided the chemical decomposition of RNA under conditions where cDNA remains substantially chemically stable. In some cases, the target RNA molecule can directly hybridize with the padlock probe without the need for reverse transcription in advance.

[0056] In addition, in some applications, the methods and systems for sample processing can retain the spatial information associated with each target molecule. Such spatial information can be stored in a biological sample having a 3D matrix. In order to retain the spatial information associated with each RNA molecule detected in the padlock probe assay, the cDNA molecule can be spatially fixed to the original position of the RNA molecule in the biological sample. In the present disclosure, a variety of methods for fixing cDNA molecules in a three-dimensional matrix are provided.

[0057] Target

[0058] Provided herein are methods and systems for sample processing in target analysis or detection. The target can be a target analyte in a biological sample. In some cases, the target can be a nucleic acid target. In some cases, the target can be a protein. In the case where the target is a protein, a binding agent that binds to the protein can be connected to a nucleic acid sequence, which can then be detected by the methods and systems provided herein. For example, the binding agent can be an antibody or antibody fragment conjugated to a nucleic acid barcode. The nucleic acid target can be ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The nucleic acid target can be a naturally occurring nucleic acid or a non-naturally occurring nucleic acid, such as a nucleic acid prepared using a synthetic method.

[0059] Whether naturally occurring or synthetic nucleic acid target can be present in three-dimensional (3D) matrix and be covalently linked to 3D matrix, thereby the relative position of each nucleic acid is fixed (for example immobilized) in 3D matrix.In this way, the 3D matrix of the covalently bound nucleic acid of any sequence can be provided.Each nucleic acid can have its own three-dimensional coordinate in host material, and each nucleic acid can represent information.In this way, a large amount of information can be stored in 3D matrix.Single information encoding nucleic acid target such as DNA or RNA can be amplified and sequenced in situ (that is, in matrix), thereby can store and read a large amount of information in suitable 3D matrix.Naturally occurring nucleic acid target can comprise endogenous DNA and RNA.Synthetic nucleic acid target can comprise primer, bar code, amplification product and probe.Synthetic nucleic acid target can be derived from endogenous nucleic acid molecule or comprise the sequence information of endogenous nucleic acid molecule.Synthetic nucleic acid target can be used for endogenous nucleic acid target being captured to 3D matrix, and can be subsequently sequenced or detected to identify sequence information and / or position (or space) information of endogenous nucleic acid molecule. For example, the synthetic nucleic acid target can be a primer with a polydeoxythymidine (dT) sequence, which can hybridize with endogenous mRNA molecules. The primer can be fixed to a 3D matrix and can be extended to include sequence information (e.g., sequence) of the mRNA molecule. The extended primer can then be captured by a padlock probe and amplified in situ for detection. In another example, the synthetic nucleic acid target can be a barcode conjugated to an antibody. The barcode can be captured by a padlock probe and amplified in situ for detection.

[0060] The nucleic acid target can be an endogenous nucleic acid in a biological sample, such as genomic DNA, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), small cytoplasmic RNA (scRNA), and small nuclear RNA (snRNA). The nucleic acid target can be a synthetic nucleic acid linked to a binding agent. The binding agent can bind to any biomolecule to be detected in a biological sample. For example, to detect a protein, the binding agent can be an antibody or portion thereof having a nucleic acid sequence linked thereto. As another example, to detect a protein, the binding agent can be an aptamer.

[0061] Nucleic acid target can be amplified to produce amplification product or amplicon in 3D matrix. Nucleic acid target can be amplified using nucleic acid amplification (such as polymerase chain reaction (PCR)). Nucleic acid target can be combined with probe, and probe can be subsequently amplified to produce amplification product or amplicon. In some cases, nucleic acid target is RNA target, and RNA target can be reverse transcribed to produce cDNA. Then cDNA can be amplified or contacted with probe (such as, padlock probe). Probe can hybridize with cDNA. In some cases, nucleic acid target is DNA target, and DNA target can be amplified or contacted with probe (such as, padlock probe). For example, DNA target can be directly amplified by amplification primer. For another example, padlock probe can contact with DNA target and hybridize with DNA target. Then padlock probe can be cyclized and amplified. Amplification product or amplicon can be connected to matrix, for example, by copolymerization or cross-linking. This can produce structurally stable and chemically stable nucleic acid 3D matrix. Nucleic acid 3D matrix can allow to extend information storage and readout cycle. The nucleic acid / amplicon matrix allows high-throughput sequencing of large arrays of samples in three dimensions.

[0062] Three-dimensional matrix

[0063] The present disclosure provides a three-dimensional (3D) matrix. The 3D matrix can contain a plurality of nucleic acids. The 3D matrix can contain a plurality of nucleic acids covalently or non-covalently attached.

[0064] In some cases, a matrix forming material can be used to form a 3D matrix. The matrix forming material can be a polymerizable monomer or polymer, or a cross-linkable polymer. The matrix forming material can be polyacrylamide, acrylamide monomer, cellulose, alginate, polyamide, agarose, dextran (dextran) or polyethylene glycol. The matrix forming material can use the method and method, reagent and condition dedicated to the matrix forming material to form a matrix by polymerization and / or cross-linking of the matrix forming material. The matrix forming material can form a polymer matrix. The matrix forming material can form a polyelectrolyte gel. The matrix forming material can form a hydrogel gel matrix.

[0065] The matrix forming material can form a 3D matrix, and the 3D matrix comprises a plurality of nucleic acids, keeps the spatial relationship of nucleic acid simultaneously. In this respect, a plurality of nucleic acids can be fixed in the matrix material. A plurality of nucleic acids can be fixed in the matrix material by making nucleic acid and the matrix forming material copolymerization. A plurality of nucleic acids can also be fixed in the matrix material by making nucleic acid cross-linked on the matrix material or otherwise cross-linked with the matrix forming material. A plurality of nucleic acids also can be fixed in the matrix by covalent attachment or by the interaction of ligand-protein and matrix.

[0066] According to one aspect, the matrix can be porous, thereby allowing reagents to be introduced into the nucleic acid sites within the matrix to amplify nucleic acids. Porous matrices can be prepared according to various methods. For example, using a suitable acrylamide:bisacrylamide ratio to control the crosslink density, a polyacrylamide gel matrix can be copolymerized with acrylamide-modified streptavidin monomers and biotinylated DNA molecules. By adding additional crosslinking agents, such as functionalized polyethylene glycol, additional control of molecular sieve size and density can be achieved.

[0067] According to one aspect, the 3D matrix can be sufficiently optically transparent, or can have the optical properties suitable for standard sequencing chemistry and the deep three-dimensional imaging for high-throughput information reading. The example of sequencing chemistry utilizing fluorescence imaging includes ABISoLiD (Life Technologies), in which the sequencing primer on the template is connected to a fluorescently labeled octamer library with a cleavable terminator. After connection, four color channels (FITC, Cy3, Texas red and Cy5) can be used to image the template. The terminator can then be excised, leaving a free end to participate in the next connection-extension cycle. After determining all dinucleotide combinations, the image can be mapped to the color code space to determine the specific base call of each template. The workflow can be realized using automatic flow control technology and imaging equipment (i.e., SoLiD 5500W Genome Analyzer, ABI Life Technologies). Another example of sequencing platform uses synthetic sequencing, in which a DNA polymerase can be used to introduce a mononucleotide pool with a cleavable terminator. After imaging, the terminator can be cleaved, and the cycle can be repeated. The fluorescence image can then be analyzed to call bases for each DNA amplicon in the flow cell (HiSeq, Illumina).

[0068] In some aspects, a biological sample can be fixed in the presence of a matrix-forming material (e.g., a hydrogel subunit). "Fixing" a biological sample means exposing the biological sample (e.g., cells or tissue) to a fixative, causing the cellular components to become cross-linked with each other. "Hydrogel" or "hydrogel network" refers to a network of water-insoluble polymer chains, sometimes formed as a colloidal gel in which water is the dispersion medium. In other words, a hydrogel is a class of polymeric materials that can absorb large amounts of water without dissolving. Hydrogels can contain more than 99% water and can include natural or synthetic polymers, or a combination thereof. Due to their high water content, hydrogels can also have a flexibility very similar to that of natural tissue. "Hydrogel subunits" or "hydrogel precursors" refer to hydrophilic monomers, prepolymers, or polymers that can be cross-linked or "polymerized" to form a 3D hydrogel network. Without being bound by any scientific theory, fixing a biological sample in the presence of hydrogel subunits can cause components of the biological sample to cross-link with the hydrogel subunits, thereby fixing the molecular components in place, thereby preserving tissue architecture and cellular morphology.

[0069] In some cases, the biological sample (e.g., cell) can be permeabilized or otherwise made accessible to an environment external to the biological sample. In some cases, the biological sample can first be fixed and permeabilized, and then the matrix-forming material is added to the biological sample.

[0070] Any convenient fixative agent or "fixative" can be used to fix the biological sample in the absence or presence of a hydrogel subunit (e.g., formaldehyde, paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol, etc.). Typically, the fixative can be diluted in a buffer, such as saline, phosphate buffer (PB), phosphate buffered saline (PBS), citrate buffer, potassium phosphate buffer, etc. (typically at a concentration of about 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 10%), for example, 4% paraformaldehyde / 0.1M phosphate buffer; 2% paraformaldehyde / 0.2% picric acid / 0.1M phosphate buffer; 4% paraformaldehyde / 0.2% periodate / 1.2% lysine in 0.1M phosphate buffer; 4% paraformaldehyde / 0.05% glutaraldehyde in phosphate buffer, etc. The type of fixative used and the duration of exposure to the fixative depends on the sensitivity of the target molecules in the sample to denaturation by the fixative and can be readily determined using conventional histochemical or immunohistochemical techniques.

[0071] The fixative / hydrogel composition may comprise any hydrogel subunit, such as, but not limited to, poly(ethylene glycol) and its derivatives (e.g., PEG-diacrylate (PEG-DA), PEG-RGD), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate) and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like. Agents such as hydrophilic nanoparticles, such as polylactic acid (PLA), polyglycolic acid (PLG), poly(lactic-co-glycolic acid) (PLGA), polystyrene, poly(dimethylsiloxane) (PDMS), and the like, may be used to improve the permeability of the hydrogel while maintaining patternability. Materials such as block copolymers of PEG, degradable PEO, polylactic acid (PLA), and other similar materials may be used to enhance specific properties of the hydrogel. Cross-linking agents (eg, bisacrylamide, diazirine, etc.) and initiators (eg, azobisisobutyronitrile (AIBN), riboflavin, L-arginine, etc.) may be included to promote covalent bonding between interacting macromolecules in the subsequent polymerization step.

[0072] Biological samples (e.g., cells or tissues) may be permeabilized after fixation. Permeabilization may be performed to facilitate access to cytoplasm or intracellular molecules, components or structures of the cell. Permeabilization may allow reagents (e.g., phosphoselective antibodies, nucleic acid-conjugated antibodies, nucleic acid probes, primers, etc.) to enter the cell and reach a concentration within the cell that is higher than the concentration that would normally permeate the cell without such permeabilization. In some embodiments, the cell may be stored after permeabilization. In some cases, the cell may be contacted with one or more reagents to allow one or more reagents to permeate after permeabilization without any storage step and then analyzed. In some embodiments, the cell may be permeabilized in the presence of at least about 60%, 70%, 80%, 90% or more methanol (or ethanol) and incubated on ice for a period of time. The incubation period may be at least about 10, 15, 20, 25, 30, 35, 40, 50, 60 minutes or more.

[0073] In some embodiments, the permeability of cells can be carried out by any suitable method. Suitable permeability agent selection and optimization of incubation conditions and time can be carried out. Suitable methods include, but are not limited to, exposure to detergents (such as CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-β-D-maltoside, lauryl sulfate, glycodeoxycholic acid, n-lauroyl sarcosine, saponin and triton X-100) or organic alcohols (such as methanol and ethanol). Other permeability methods may include the use of certain peptides or toxins that make the membrane permeable. Permeability can also be carried out by adding organic alcohols to the cells.

[0074] By way of illustration and not limitation, permeabilization can also be achieved, for example, by the use of surfactants, detergents, phospholipids, phospholipid binding proteins, enzymes, viral membrane fusion proteins, etc.; by the use of osmotically active agents; by the use of chemical cross-linking agents; by physicochemical methods including electroporation, etc., or by other permeabilization methods.

[0075] Thus, for example, cells can be permeabilized using any of a variety of known techniques, such as exposure to one or more detergents (e.g., digitonin, Triton X-100, TM NP-40 TM , octylglucoside, etc. Certain transfection reagents, such as dioleoyl-3-trimethylammonium propane (DOTAP), can also be used. ATP can also be used to permeabilize intact cells. Low concentrations of chemicals used as fixatives (e.g., formaldehyde) can also be used to permeabilize intact cells.

[0076] Nucleic acid as described herein (for example, RNA molecule, cDNA molecule, primer or probe) may include a functional moiety. Nucleic acid can be connected to a 3D matrix by a functional moiety. The functional moiety can react with the reactive group on the 3D matrix by conjugation chemistry. In some cases, the functional moiety can be attached to the target by conjugation chemistry. In some cases, the functional moiety can be directly attached to the reactive group on the natural nucleic acid molecule. In some cases, the functional moiety can be indirectly connected to the target by an intermediate chemical or group. The conjugation strategy as described herein is not limited to nucleic acid targets, and can also be used for protein or small molecule targets. During nucleic acid synthesis or extension reaction, nucleotide analogs comprising a functional moiety can be introduced into the growing chain of nucleic acid (for example, cDNA molecule, probe or primer).

[0077] As used herein, the term "reactive group" or "functional moiety" refers to any part of the first reactant that can chemically react with another functional moiety or reactive group on the second reactant to form a covalent bond or an ionic bond. "Reactive group" and "functional moiety" are used interchangeably. For example, the reactive group of a monomer or polymer of a matrix forming material can chemically react with the functional moiety (or another reactive group) on a target substrate or a target to form a covalent bond or an ionic bond. The target substrate or target can then be fixed on the matrix by the key formed by the reactive group and the functional moiety. The example of suitable reactive group or functional moiety includes electrophile or nucleophile, which can react with the corresponding nucleophile or electrophile on the target substrate to form a covalent bond. Non-limiting examples of suitable electrophilic reactive groups can include, for example, esters (including activated esters (e.g., succinimidyl esters)), amides, acrylamides, acyl azides, acyl halides, acyl nitriles, aldehydes, ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, borates, carbodiimides, diazoalkanes, epoxides, haloacetamides, haloplatinates, halotriazines, imidoesters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonates, sulfonyl halides, etc. Non-limiting examples of suitable nucleophilic reactive groups can include, for example, amines, anilines, thiols, alcohols, phenols, hyrazines, hydroxylamines, carboxylic acids, diols, heterocyclic compounds, etc.

[0078] The present disclosure provides a method for in situ modification of nucleic acids to include a functional moiety. In some cases, the functional moiety may include a polymerizable group. In some cases, the functional moiety may include a free radical polymerizable group. In some cases, the functional moiety may include an amine, thiol, azide, alkyne, nitrone, alkene, tetrazine, tetrazole, acrylamide or other click-reactive groups. In some cases, the functional moiety may then be in situ attached to a 3D matrix. The functional moiety may further be used to retain the absolute or relative spatial relationship between two or more molecules in a sample.

[0079] The biological sample in the 3D matrix can be cleared of proteins and / or lipids that are not the target of interest. For example, the biological sample can be cleared of proteins by enzymatic proteolysis (also known as "deproteinization"). The clearance step can be performed before or after covalent immobilization of any target molecule or derivative thereof.

[0080] In some cases, a cleanup step is performed after the target nucleic acid molecule (e.g., RNA or DNA), primers (e.g., RT primers), derivatives of the target molecule (e.g., cDNA or amplicons), and probes (e.g., padlock probes) are covalently immobilized on the synthetic 3D matrix. Performing a cleanup step after immobilization allows any subsequent nucleic acid hybridization reactions to be performed after the sample has been substantially deproteinized (e.g., by enzymatic proteolysis ("protein cleanup")). This approach has the advantage of removing ribosomes and other proteins that bind to the RNA or nucleic acid target from the target molecule (while maintaining their spatial location), where protein components can hinder or inhibit primer binding, reverse transcription or padlock ligation, and amplification, thereby improving the sensitivity and quantitativeness of the test by reducing bias in probe capture events due to protein occupancy or protein crowding / proximity to the target nucleic acid.

[0081] The clearing step may include removing non-targets from the 3D matrix. The clearing step may include degrading the non-targets. The clearing step may include exposing the sample to an enzyme capable of degrading proteins (e.g., a protease). The clearing step may include exposing the sample to a detergent.

[0082] Protein can be removed from the sample using enzymes, denaturants, chelating agents, chemical reagents, etc., which can break down the protein into smaller components and / or amino acids. These smaller components can be more easily physically removed, and / or can be small enough or inert enough so that they do not significantly affect the background. Similarly, lipids can be removed from the sample using surfactants, etc. In some cases, one or more of these reagents can be used, for example, simultaneously or sequentially. Non-limiting examples of suitable enzymes include proteases, such as proteinase K, proteases or peptidases, or digestive enzymes, such as trypsin, pepsin or chymotrypsin. Non-limiting examples of suitable denaturants include guanidine hydrochloride, acetone, acetic acid, urea or lithium perchlorate. Non-limiting examples of chemical reagents that can denature proteins include solvents, such as phenol, chloroform, guanidine isocyanate, urea, formamide, etc. Non-limiting examples of surfactants include Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether), SDS (sodium dodecyl sulfate), Igepal CA-630, or poloxamer. Non-limiting examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate, or polyaspartic acid. In some embodiments, compounds such as these can be applied to a sample to remove proteins, lipids, and / or other components. For example, a buffer solution (e.g., containing tris(hydroxymethyl)aminomethane) can be applied to the sample and then removed.

[0083] In some cases, nucleic acids that are not the target of interest can also be removed. These non-target nucleic acids may not be captured and / or fixed to the 3D matrix, and therefore can be removed with enzymes to degrade nucleic acid molecules. Non-limiting examples of DNA enzymes that can be used to remove DNA include DNase I, dsDNase, various restriction enzymes, etc. Non-limiting examples of techniques for removing RNA include RNA enzymes, such as RNase A, RNase T, or RNase H; or chemical reagents, such as by alkaline hydrolysis (e.g., by increasing the pH value to greater than 10). Non-limiting examples of systems for removing sugar or extracellular matrix include enzymes, such as chitinase, heparinase, or other glycosylases. Non-limiting examples of systems for removing lipids include enzymes, such as lipases; chemical reagents, such as alcohols (e.g., methanol or ethanol); or detergents, such as Triton X-100 or sodium lauryl sulfate. In this way, the background of the sample can be removed, which can facilitate the analysis of nucleic acid probes or other targets (e.g., using fluorescence microscopy or other techniques described herein).

[0084] Solid support

[0085] Matrix can be used in combination with solid carrier.For example, matrix can be polymerized in such a way that a surface of matrix is attached to solid carrier (for example, glass surface, flow cell, slide, hole), and another surface of matrix is exposed or is sandwiched between two solid carriers.According to one aspect, matrix can be included in container.In some cases, biological sample can be fixed on solid carrier.

[0086] The solid supports of the present disclosure can be made into various shapes. In certain embodiments, the solid support is substantially planar. Examples of solid supports include plates, such as glass slides, multiwell plates, flow cells, cover slips, microchips, and the like; containers, such as microcentrifuge tubes, test tubes, and the like; tubes, sheets, pads, membranes, and the like. In addition, the solid support can be, for example, biological, non-biological, organic, inorganic, or a combination thereof.

[0087] As used herein, the term "solid surface" is intended to mean the surface of a solid support or substrate, and includes any material that can be used as a solid or semi-solid base to attach biological samples or other molecules (e.g., polynucleotides, amplicons, DNA spheres, other nucleic acids) and / or other polymers (including biopolymers). Example types of materials comprising solid surfaces include glass, modified glass, functionalized glass, inorganic glass, microspheres including inert and / or magnetic particles, plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, optical fibers or fiber bundles, various polymers other than those exemplified above, and porous plates. Specific types of exemplary plastics include acrylics, polystyrene, copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon. TM Specific types of exemplary silica-based materials include silicon and various forms of modified silicon.

[0088] The shape of the solid surface can also vary depending on the application in the methods described herein. For example, a solid surface useful in the present disclosure can be planar, or contain concave or convex regions.

[0089] Amplification

[0090] Any type of nucleic acid amplification reaction can be used to carry out amplification reaction and produce amplified products in the method or system described herein. In addition, the amplification of nucleic acid can be linear, exponential or its combination. The non-limiting example of nucleic acid amplification method includes transcription (such as in vitro transcription), reverse transcription, primer extension, polymerase chain reaction, ligase chain reaction, helicase-dependent amplification, asymmetric amplification, rolling circle amplification and multiple displacement amplification (MDA). In some cases, the amplified product can be DNA. In the case of target RNA amplification, DNA can be obtained by reverse transcription of RNA, and subsequent DNA amplification can be used to produce amplified DNA products. In some cases, target RNA is reverse transcribed to produce cDNA by reverse transcriptase. In some cases, target DNA is transcribed to produce RNA by RNA polymerase. The amplified DNA product can indicate the presence of target RNA in biological sample. In the case of DNA amplification, any DNA amplification method can be adopted. Non-limiting examples of DNA amplification methods include polymerase chain reaction (PCR), variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap extension PCR, thermal asymmetric staggered PCR, touchdown PCR) and ligase chain reaction (LCR). In some cases, DNA amplification is linear. In some cases, DNA amplification is exponential. In some cases, DNA amplification can be achieved by nested PCR, which can improve the sensitivity of detecting the amplified DNA product.

[0091] The amplification of nucleic acid sequence can be carried out in matrix.The method for amplifying nucleic acid can include in situ rolling circle amplification.In some aspects, the method for amplifying nucleic acid can include using PCR, such as anchor PCR, RACE PCR or ligation chain reaction (LCR).Alternative amplification method includes but is not limited to self-sustaining sequence replication, transcription amplification system, Q-β replicase, recursive PCR or any other nucleic acid amplification method.

[0092] The nucleic acid in the 3D matrix can contact with reagent under the suitable reaction conditions that are enough to amplify nucleic acid.Matrix can be porous, to allow reagent to migrate in the matrix to contact nucleic acid. In some respects, nucleic acid can be selectively hybridized and increased by using ordinary method to make the amplification site of 3 ' end of amplification primer and nucleotide sequence.The length of amplification primer is 6 to 100, even up to 1,000 Nucleotide, but is generally 10 to 40 Nucleotide, although used oligonucleotide of different lengths. In some cases, the length of amplification primer can be at least about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or more Nucleotide. In some cases, the length of amplification primer can be at least about 20,30,40,50,60,70,80,90,100,200,300,400,500 or more Nucleotide. The amplification primer can hybridize to the nucleic acid probe that hybridizes to the DNA molecule, so that the amplification primer can be used to amplify the sequence of the nucleic acid probe. The amplification primer can be present in a solution to be added to the matrix, or it can be added during the formation of the matrix so as to be present in sufficient proximity to the nucleic acid to allow hybridization and amplification.

[0093] DNA polymerase can be used for amplification reaction. Any suitable DNA polymerase can be used, including commercially available DNA polymerases. DNA polymerase generally refers to an enzyme that can introduce nucleotides into a DNA chain in a template-bound manner. Non-limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment and variants thereof, modified products and derivatives thereof. Other enzymes can also be used in the amplification reaction, including but not limited to RNA polymerases (e.g., T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, etc.) and reverse transcriptases (e.g., avian myeloblastosis virus (AMV) reverse transcriptase, wild-type human immunodeficiency virus-1 (HIV-1) reverse transcriptase, or Moloney murine leukemia virus (M-MLV) reverse transcriptase).

[0094] Detection

[0095] The present disclosure provides sample processing methods and systems for nucleic acid detection. The sequence of a nucleic acid target can be identified. Various methods can be used for nucleic acid detection, including hybridization and sequencing. Nucleic acid detection can include imaging a biological sample or a 3D matrix as described herein.

[0096] The reporter can be connected to the nucleic acid, including the amplified product, by covalent or non-covalent interactions. Non-limiting examples of non-covalent interactions include ionic interactions, van der Waals forces, hydrophobic interactions, hydrogen bonding, and combinations thereof. The reporter can be combined with the initial reactant, and changes in the reporter level can be used to detect the amplified product. As the nucleic acid amplification proceeds, the reporter can be detectable (or undetectable). The reporter can be optically detectable. Optically active dyes (e.g., fluorescent dyes) can be used as reporters. Non-limiting examples of dyes include SYBR green, SYBR blue, DAPI, propidium iodide, Hoeste, SYBR gold, ethidium bromide, acridine, proflavine, acridine orange, acridine yellow, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyl, anthramycin, phenanthridine and acridine, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, Acridine Orange, 7-AAD, Actinomycin D, LDS751, Fluorogold (hydroxystilbamidine), SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5. Cy-7, Texas Red, Phar-Red, Allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, Ethidium Homodimer I, Ethidium Homodimer II, Ethidium Homodimer III, Ethidium Bromide, Umbelliferone, Eosin, Green Fluorescent Protein, Erythrosine, Coumarin, Methylcoumarin, Pyrene, Malachite Green, Stilbene, Lucifer Yellow yellow), Cascade Blue, dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes (such as those including europium and terbium), carboxytetrachlorofluorescein, 5- and / or 6-carboxyfluorescein (FAM), 5- (or 6-) iodoacetamidofluorescein, 5-{[[2(and 3)-5-(acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5- and / or 6-carboxyrhodamine (ROX), 7-aminomethylcoumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophore, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonate-4-amino-naphthalimide, phycobiliprotein, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or other fluorophores.

[0097] In some embodiments, the reporter can be a sequence-specific oligonucleotide probe that is optically active when hybridized to a nucleic acid target or a derivative thereof (e.g., an amplification product). The probe can be linked to any optically active reporter described herein (e.g., a dye) and can also include a quencher that blocks the optical activity of the associated dye. Non-limiting examples of probes that can be used as reporters include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

[0098] In some aspects, methods for determining the nucleic acid sequence of a target nucleic acid molecule include sequencing. In some aspects, sequencing by synthesis, sequencing by ligation, or sequencing by hybridization are used to determine the nucleic acid sequence of a target nucleic acid molecule. As disclosed herein, prior to sequencing, various amplification methods can be used to generate larger, particularly limited, nucleic acid samples. For example, amplification methods can generate targeted amplicon libraries.

[0099] In order to carry out ligation sequencing, the nucleic acid fragments of the label can be hybridized and identified to determine the sequence of the target nucleic acid molecule. In order to carry out synthesis sequencing (SBS), labeled nucleotides can be used to determine the sequence of the target nucleic acid molecule. The target nucleic acid molecule can be hybridized with a primer and cultivated in the presence of a polymerase and a labeled nucleotide containing a blocking group. The primer can be extended so that the labeled nucleotide is introduced. The presence of a blocking group allows the introduction of a single nucleotide. The presence of a label allows the identification of the introduced nucleotide. As used herein, the label can be any optically active dye described herein. A base can be added, or all four bases can be added simultaneously, particularly when each base is associated with a distinguishable label. After the nucleotide introduced is identified by its corresponding label, the label and blocking group can be removed, thereby allowing the introduction and identification of a subsequent round. Therefore, a cleavable linker can connect the label to the base. The example of a cleavable linker includes but is not limited to a peptide linker. In addition, a removable blocking group can be used so that multiple rounds of identification can be carried out, thereby allowing the identification of at least a portion of the target nucleic acid sequence. The compositions and methods disclosed herein can be used for this SBS method. In addition, the compositions and methods can be used to sequence from a solid support (e.g., an array or sample within a 3D matrix as described herein), wherein multiple sequences can be "read" simultaneously from multiple positions on the solid support because each nucleotide at each position can be identified based on its identifiable label. Example methods are described in US 2009 / 0088327, US 2010 / 0028885, and US 2009 / 0325172, each of which is incorporated herein by reference.

[0100] RNA degradation

[0101] The methods and systems described herein can use probes for target capture or detection. For example, the probe can be a padlock probe. In the padlock probe capture method, the ligation reaction of the DNA-DNA duplex template formed between the cDNA molecule and the DNA padlock probe is generally more effective than the ligation reaction of the DNA-RNA "hybridization" duplex template formed between the RNA molecule and the DNA padlock probe, because the enzymatic ligation efficiency between the DNA-DNA duplex template is higher than that of the DNA-RNA hybrid duplex template. Therefore, before capturing the target molecule by the padlock probe, all or part of the target RNA molecule can first be converted into a cDNA molecule by reverse transcription. In some cases, the padlock probe can be extended by DNA polymerization using a cDNA molecule as a template until the 3' and 5' ends of the padlock probe are close to each other so that a ligation reaction occurs.

[0102] After all or part of the target RNA molecule is converted into a cDNA molecule, the RNA molecule can be degraded. In some cases, RNA can be degraded using a non-ribonuclease having RNA catalytic cleavage activity. In other cases, RNA can be degraded by chemical decomposition under conditions where the cDNA can remain substantially chemically stable.

[0103] In some cases, the target RNA molecule can be captured directly by a DNA padlock probe (e.g., hybridized to a DNA padlock probe) without the need for a reverse transcription step. In this case, the target RNA molecule can be extended using the padlock probe as a template, or the padlock probe can be cyclized while hybridizing to the target RNA molecule. The target RNA molecule can then be degraded after the padlock probe is cyclized.

[0104] Reverse transcriptase

[0105] RNA molecules can be enzymatically digested by non-ribonuclease enzymes. According to certain embodiments, the enzymatic digestion of RNA molecules can be catalyzed by reverse transcriptase. Certain reverse transcriptases may have ribonuclease activity, including but not limited to RNase H activity. Certain reverse transcriptases may have the activity of specifically digesting RNA in RNA-DNA hybrid duplexes. For example, avian myeloblastosis virus (AMV) reverse transcriptase has inherent RNase H activity, which can degrade the RNA chain of RNA / DNA hybrids. Other reverse transcriptases that exhibit ribonuclease activity or RNA catalytic cleavage activity include but are not limited to wild-type HIV-1 and M-MLV reverse transcriptases.

[0106] Most reverse transcriptases do not have ribonuclease activity or RNA catalytic cleavage activity, thereby increasing the rate of full-length cDNA synthesis. For example, the most popular variant of M-MLV RT is the M-MLV RT RNase H point mutant, which has a single amino acid substitution that greatly reduces RNase H activity. However, for the purposes of padlock probe assays as described herein, the reverse transcription reaction can be specifically initiated using substantially complementary primers, or non-specifically initiated using a degenerate primer pool (primers approaching the padlock probe capture site along the RNA / cDNA molecule), in which case effective padlock probe capture can be performed without the need for full-length cDNA synthesis.

[0107] Compared to using a separate enzymatic reaction for RNA digestion, using a reverse transcriptase to degrade or digest RNA can save cost and time. The methods described herein can also reduce the number of components and / or steps in a padlock probe assay. In addition, using this strategy, the ribonuclease activity of the reverse transcriptase can be modulated by changing the composition of the reaction buffer to enable a multi-stage reaction that includes a first stage of efficient reverse transcription and a subsequent stage of efficient RNA digestion. Aspects of the reaction buffer composition that modulate ribonuclease activity can include ribonuclease inhibitors, including organic chemicals and polypeptides; cofactors, including metal ions, such as Mg; 2+ 、Mn 2+ 、Na 1+ , ATP, NADPH, etc. For example, the reverse transcription of HIV-1RTase can be carried out at low concentrations of Mg 2+ ions, although the intrinsic polymerase activity was greatly reduced by degradation of the RNA template due to RNase activity, and the subsequent increase in Mg 2+ The concentration increases the activity of ribonuclease, thereby releasing cDNA from the hybrid duplex.

[0108] DNA-binding proteins

[0109] According to another aspect of the present disclosure, RNA can be digested by another non-ribonuclease having ribonuclease activity or RNA catalytic cleavage activity. In a specific embodiment, a DNA binding protein having ribonuclease activity or RNA catalytic cleavage activity can be used for a dual purpose. One purpose can be to digest RNA molecules. Another purpose can be to stabilize single-stranded cDNA molecules, for example, by binding proteins to single-stranded DNA (ssDNA). DNA binding proteins can also promote hybridization between cDNA molecules and DNA padlock probe molecules. For example, the DNA binding protein Sso7d can be used to promote annealing of complementary DNA strands above the duplex melting point and can also be used to degrade RNA because Sso7 has ribonuclease activity.

[0110] Chemical decomposition

[0111] RNA can be chemically decomposed under conditions where cDNA remains substantially chemically stable. RNA hydrolysis can be a reaction in which the phosphodiester bond in the sugar-phosphate backbone of RNA is broken and the RNA molecule is cut. RNA can be susceptible to this base-catalyzed hydrolysis because the ribose in RNA has a hydroxyl group at the 2' position. Compared to DNA, which does not have this 2'OH group and is therefore not susceptible to base-catalyzed hydrolysis, this feature can make RNA chemically unstable. The chemical decomposition of RNA (e.g., by hydrolysis) can occur under a variety of conditions, under which DNA, including single-stranded cDNA, can remain substantially chemically stable. RNA can be heat-sensitive and susceptible to metal-catalyzed degradation. Under normal conditions, RNA hydrolysis can occur at a low frequency, but under certain conditions, such as at acidic pH, alkaline pH, high temperature, in the presence of divalent cations, and in the presence of heavy metal ions, RNA hydrolysis can be accelerated.

[0112] Buffer can be used to provide conditions for RNA chemical decomposition. For example, the buffer can include tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl) at a pH of at least about 7, 7.5, 8 or higher. The final concentration of tris (hydroxymethyl) aminomethane hydrochloride can be at least about 20, 30, 40, 50 mM or higher. The buffer can also include MgCl2 at a final concentration of at least about 15, 20, 25, 30, 35, 40, 45, 50 mM or higher. In some cases, the buffer includes 50 mM tris (hydroxymethyl) aminomethane hydrochloride and 20-50 mM MgCL2 at a pH of 7.5-8. For another example, the buffer can include sodium borate at a pH of at least about 7, 7.5, 8 or higher. The final concentration of sodium borate can be at least about 20, 30, 40, 50 mM or higher. The buffer may also contain MgCl2 at a final concentration of at least about 15, 20, 25, 30, 35, 40, 45, 50 mM or more. In some cases, the buffer comprises 50 mM sodium borate and 20-50 mM MgCl2 at a pH of 7.5-8. The divalent cation may be other than Mg. 2+ , and can be other types of divalent cations, such as Mn 2+ .

[0113] The buffer for the chemical decomposition of RNA can also catalyze the click reaction between the click-reactive groups. In some cases, molecules (such as targets, primers, probes, or molecules derived from targets such as cDNA) can be tethered to a hydrogel matrix (such as click gel) functionalized with click-reactive groups by click reaction. For example, 5' azidomethyl-dUTP can be introduced into cDNA and then fixed on a hydrogel matrix functionalized by an alkyne group. Various click reactions can be used. The buffer described herein can both catalyze the functionalized fixed connection between the molecule and the matrix and hydrolyze the RNA molecule, thereby releasing DNA from the DNA-RNA hybrid duplex. Using a buffer with both chemical deposition and click reaction catalysis functions can improve workflow efficiency, reduce workflow time, and reduce the amount of assay reagents. For example, the buffer can be a Cu (I)-catalyzed alkyne-azide cycloaddition (abbreviated as CUAAC) click reaction catalysis buffer, which catalyzes alkyne-azide bonds in the click reaction. The buffer may comprise 1-25 mM copper (II) sulfate solution, 1-50 mM tris ((1-hydroxy-propyl-1H-1,2,3-triazol-4-yl) methyl) amine (abbreviated as THPTA) solution, 5-100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (abbreviated as HEPES) buffer, and 5-100 mM L-ascorbic acid. In some cases, the final concentration of copper (II) sulfate may be at least about 1, 5, 10, 15, 20, 25 mM or more. In some cases, the final concentration of THPTA may be at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mM or more. In some cases, the final concentration of HEPES can be at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM or more. In some cases, the final concentration of L-ascorbic acid can be at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM or more. Optionally, the buffer can be degassed or purged with argon to remove dissolved oxygen before use.

[0114] The RNA degradation methods described herein can save costs and time and improve the efficiency of releasing cDNA from hybrid duplexes. Chemical degradation of RNA is faster and more efficient than enzymatic digestion. In addition, chemical non-enzymatic reagents can be less expensive, easier to synthesize, easier to store, and have a longer shelf life than reagents used in enzymatic reactions.

[0115] Immobilization of nucleic acids in 3D matrices

[0116] In order to retain the spatial information related to each RNA molecule detected in the padlock probe assay, the cDNA molecule space in the biological sample (for example, cells and tissues) can be fixed to the original position of the RNA molecule. Similarly, in the case of directly capturing the target RNA or DNA molecule by a probe (for example, a padlock probe), the probe can be fixed to the original position of the target RNA or DNA molecule in space. The spatial origin of the target RNA or DNA molecule itself can usually be retained by forming a chemical or physical crosslink between the naturally occurring 3D matrix of the target RNA or DNA molecule and the biological sample biomolecule (for example, protein). Chemical or physical crosslinking can be formed by temperature, electromagnetic radiation (for example, microwaves) or chemicals such as formaldehyde and glutaraldehyde and other substances in the cell and tissue. Naturally occurring 3D matrix can be formed by crosslinking the endogenous or natural biomolecule (for example, protein and nucleic acid) in the cell or tissue. The spatial origin of the RNA molecule can also be retained for fixing the cDNA purpose by forming a chemical or physical crosslink between the RNA molecule and other natural or synthetic components, wherein the other natural or synthetic components are added to the sample to supplement or replace the natural cell component. For example, a synthetic 3D matrix can be formed in situ in whole cells and tissue samples to retain the spatial position of RNA molecules or DNA molecules. The synthetic 3D matrix can be a hydrogel matrix. For example, the 3D matrix can be composed of polyacrylamide or polyethylene glycol (PEG). During cDNA synthesis, the spatial position of cDNA can be retained by hydrogen bonding between RNA and cDNA in a hybrid duplex ("hybridization"). However, for example, the spatial position of the cDNA independent of the RNA molecule can be retained to avoid the loss of spatial position of cDNA after the hybrid RNA-DNA duplex is released.

[0117] The functional part can be bound to a cell or a cell component, or react with a cell or a cell component. The affinity binding group can be bound to a cell or a cell component. The functional part can include at least one nucleotide modified by biotin, an amine group, a lower alkylamine group, an acetyl group, DMTO, fluorescein, a thiol group or an acridine group. In the case of providing a synthetic 3D matrix for cDNA cross-linking, the sample can be immersed in a gel solution, which can form a gel matrix that can be attached to a cDNA molecule or a target RNA molecule after polymerization. As described herein, a method for fixing nucleic acid molecules in a matrix is provided.

[0118] Immobilization of cDNA molecules

[0119] Methods for immobilizing cDNA molecules that do not rely on hybridization of the cDNA molecule to RNA are provided. Generally, these advances can be divided into three categories: a) the in situ polymerized component of the cDNA can contain a functional moiety for immobilization; b) the functional moiety for immobilization can react with a pre-existing (at the time of reverse transcription) in situ matrix of non-cellular origin (i.e., synthetic or exogenous matrix); and c) the functional immobilization moiety can bind to the reverse transcription primer through hydrogen bonding via DNA hybridization.

[0120] According to the first aspect, the in situ polymerisation component of cDNA may comprise a functional moiety for immobilisation rather than a primer for initiating or priming reverse transcription.

[0121] The cDNA molecule can be functionalized during the reverse transcription reaction, for example, by adding a nucleotide triphosphate analog comprising a functional portion for fixation. Such nucleotide triphosphate analogs include, but are not limited to, amino-allyl dUTP, 5-TCO-PEG4-dUTP, C8-alkyne-dUTP, 5-azidomethyl-dUTP, 5-vinyl-dUTP, 5-ethynyl dUTP, and other nucleotide triphosphate analogs comprising a functional portion that fixes the cDNA by cross-linking or forming a chemical bond between the cDNA and a cellular or synthetic in situ matrix. In addition, the cellular or synthetic in situ matrix may contain or be made to contain a chemical portion (e.g., a reactive group) that can react with the functional portion in the cDNA by a functionalization reaction. For example, amino-allyl dUTP can be cross-linked to endogenous free amine groups in proteins and other biomolecules present in endogenous or exogenous cellular matrices, or in modified synthetic hydrogel matrices (e.g., amine-functionalized polyacrylamide hydrogels formed by copolymerization of polyacrylamide and N-(3-aminopropyl)-methacrylamide); similarly, nucleoside analogs containing azide functional moieties can be cross-linked to synthetic hydrogel matrices containing alkyne functional moieties (e.g., formed by copolymerization of acrylamide and propargyl acrylamide).

[0122] cDNA molecules can be functionalized using moieties for immobilization after reverse transcription. The mechanisms for post-synthesis cDNA functionalization can include a variety of biochemical and chemical methods. These methods include, but are not limited to: using ligation reactions to conjugate oligonucleotides with functional moieties for immobilization on cDNA molecules; using DNA polymerization reactions to add templated or non-templated bases to cDNA, such as by Taq polymerization in the A-tailing method or by using reactions mediated by DNA end repair mechanisms. Alternatively, chemical methods for DNA chemical functionalization can be used to conjugate functional moieties for immobilization. For example, Label-IT amine and Label-X are difunctionalizing agents that can react with nucleic acids through a nitrogen mustard alkylation mechanism to conjugate free amines or acryloyl groups to nucleic acids, which can be used for the purpose of immobilization on a matrix. Other chemical methods (including but not limited to DNA alkylation and hydroxymercuration) can provide mechanisms for functionalizing DNA.

[0123] The method for cDNA fixation provided herein has some advantages.For example, using the method provided herein, unmodified DNA oligonucleotide can be used as reverse transcription primer, thereby simplifies the manufacture of these reagents and reduces assay cost.In addition, by limiting the cross-linking functional part to the new polymeric component of cDNA, the residual reverse transcription primer that does not participate in the reverse transcription reaction can be easy to wash out from sample, and does not participate in the cDNA fixation mechanism, which can reduce background signal or improve the productive rate of downstream reactions.In addition, after reverse transcription, using a separate reaction to functionalize the cDNA with functional part can simplify experimental process.For example, DNA functionalization mechanism can be used for functionalizing cDNA and genomic DNA with functional part in a single reaction simultaneously, thereby promoting multi-omics in situ determination, such as fluorescence in situ sequencing (FISSEQ).

[0124] Functional moieties for fixing can react or crosslink with the pre-existing (when reverse transcription) in situ matrix of non-cellular origin (i.e., synthetic or exogenous) matrix. According to this aspect, a synthetic chemical matrix with a functional moiety for cDNA fixing can be formed in situ before reverse transcription. Suitable functional moieties include but are not limited to amine and click functional groups. After the synthesis of cDNA molecules, the functional moieties present in the cDNA can be crosslinked with the complementary functional moieties in the matrix. Fixing by using pre-formed in situ synthetic matrix can degrade, remove or chemically modify all or part of the natural biological matrix of the sample, thereby improving the reaction performance of reverse transcription or other reactions, including yield.

[0125] Primer modification

[0126] The reverse transcription primer itself may not carry a functional moiety for immobilization, but may be associated with one or more functional moieties for immobilizing the cDNA via hydrogen bonding. This method enables the use of unmodified reverse transcription primers, thereby simplifying and reducing the cost of assay manufacturing. For example, the primer may carry a functional domain, such as a common domain among multiple reverse transcription primers, which contains a tethered oligonucleotide hybridization site, and the tethered oligonucleotide may carry a functional moiety for cDNA immobilization. In addition, the tethered oligonucleotide may carry other features, such as partial complementarity with a domain responsible for binding RNA and initiating reverse transcription. The tethered oligonucleotide may carry additional sequences for enhancing the specificity of the reverse transcription primer-cDNA annealing reaction through a competitive hybridization mechanism.

[0127] In some other cases, the primer (e.g., a reverse transcription primer) may comprise a functional portion for immobilization to a 3D matrix. For example, the primer may not hybridize to a tethered oligonucleotide having a functional portion, but may be directly covalently linked to the functional portion for immobilization.

[0128] Modification of padlock probes

[0129] Padlock probes can include functional moieties for direct or indirect immobilization (e.g., via hybridized oligonucleotides) to a substrate in situ. For example, a tethered oligonucleotide that hybridizes to the backbone of a padlock probe (e.g., outside of the domain responsible for hybridization to a target cDNA molecule) can be used as a rolling circle amplification primer, thereby being used to tether the padlock probe molecule (and cDNA molecule) via DNA hybridization prior to rolling circle amplification, and subsequently to tether the rolling circle amplicon (i.e., rolony) after rolling circle amplification, thereby preserving spatial information associated with the original RNA molecule, the cognate cDNA molecule, the padlock probe, and the rolony.

[0130] In some cases, the efficiency or reaction yield of establishing a functional fixed connection between a target molecule, probe, cDNA, padlock probe or amplicon and a 3D matrix can be improved by the presence of more than one functional moiety in the molecule to be tethered. In the case of introducing a functional moiety into a cDNA during reverse transcription, this can be achieved by titrating the amount of functionally modified dNTPs in the mixture. In some embodiments, when a tethered oligonucleotide is used to indirectly immobilize a target or probe through hydrogen bonds provided by DNA hybridization, a tethered oligonucleotide with multiple functional moieties can be synthesized. For example, a tethered oligonucleotide with multiple functional moieties can be synthesized by introducing an internal amine during chemical DNA synthesis, followed by conversion (e.g., bulk conversion) of the amine to an azide, acryloyl or other functional moiety. This conversion can be achieved by an amine-reactive chemical group (e.g., NHS ester). The low efficiency of tethering a single molecule to a 3D matrix using a single tethering moiety can be due to the inherent rate of the immobilization reaction (i.e., the probability of acryloyl groups being incorporated into the in situ polymerized matrix, or the probability of azides entering into proximity with alkynes present in click gels), or due to a "non-ideal" in situ matrix synthesis process. For example, a "non-ideal" in situ matrix synthesis process can refer to a situation where a single molecule is conjugated to a polymer, but the polymer itself is not stably attached to the larger nascent 3D network architecture generated during 3D matrix formation, which only increases the molecular weight of the molecule but still allows the molecule and its conjugated polymer to diffuse from the 3D matrix. The presence of multiple immobilization moieties can mitigate these two mechanisms by increasing the probability of incorporation of a single molecule (in the form of the sum or product of the probabilities of each moiety to be incorporated into the reaction) and by providing the potential for attachment to multiple polymer chains in the 3D matrix. These multiple functional moieties can be closely adjacent, or alternatively, spaced at regular or irregular intervals within the molecule. For example, multiple functional moieties can be present continuously within a nucleic acid molecule. For example, multiple functional moieties can be separated by at least 1, 2, 3, 4, 5, 6, 7 or more nucleotides within the nucleic acid molecule.

[0131] Sample processing methods

[0132] In one aspect, the present disclosure provides a method for identifying a nucleic acid sequence in a biological sample. The method may include providing a biological sample comprising a ribonucleic acid (RNA) molecule hybridized with a deoxyribonucleic acid molecule (DNA) in a three-dimensional (3D) matrix. The RNA molecule may comprise the nucleic acid sequence. Then, a reverse transcriptase or a functional derivative thereof may be used to degrade or digest at least a portion of the RNA molecule hybridized with the DNA molecule. The DNA molecule may comprise another nucleic acid sequence that is a reverse complementary sequence of the nucleic acid sequence. Then, the other nucleic acid sequence in the biological sample may be detected to identify the nucleic acid sequence.

[0133] The DNA molecule can be a complementary deoxyribonucleic acid (cDNA) molecule. For example, a DNA molecule can be reverse transcribed from an RNA molecule. A cDNA molecule can be reverse transcribed from an RNA molecule and amplified by one or more rounds of amplification (e.g., by polymerase chain reaction (PCR)) to produce one or more copies of the cDNA molecule sequence.

[0134] In some cases, another reverse transcriptase or a functional derivative thereof can be used to reverse transcribe an RNA molecule to produce a DNA molecule that hybridizes with the RNA molecule in a biological sample. Alternatively, a reverse transcriptase or a functional derivative thereof (which is used to degrade or digest at least a portion of an RNA molecule) can be used to first reverse transcribe an RNA molecule to produce a DNA molecule that hybridizes with the RNA molecule in a biological sample.

[0135] DNA molecules can be fixed to a 3D matrix. For example, DNA molecules can be covalently fixed to a 3D matrix (e.g., by crosslinking, such as using disulfide bonds). DNA molecules can include functional moieties, and the DNA molecules can be fixed to the 3D matrix via the functional moieties. Examples of functional moieties include, but are not limited to, amines, acrylamides, alkynes, biotin, azides, and thiols. RNA molecules can be fixed to a 3D matrix. RNA molecules can include functional moieties, and the RNA molecules can be fixed to the 3D matrix via the functional moieties. For example, RNA molecules can be modified using LabelX, which can be used to covalently attach RNA molecules to a 3D matrix. The compound LabelX can be synthesized using NHS-ester chemistry from Acryloyl-X SE (6-((acryloyl)amino)hexanoic acid, succinimidyl ester) and Label-IT amine (MirusBio) and can react with RNA, for example, at the N7 guanine position. In addition to polymerizing acryl radicals into polyacrylamide, LabelX reagents can also develop other attachment chemistries. For example, NHS-ester-azide can be conjugated to Label-IT amine to create a new linker capable of tethering nucleic acids into a PEG-click hydrogel matrix.

[0136] In some cases, a 3D matrix can be formed using a matrix-forming material. The matrix-forming material can be a polymerizable monomer or polymer, or a cross-linkable polymer. The matrix-forming material can be polyacrylamide, acrylamide monomer, cellulose, alginate, polyamide, agarose, dextran or polyethylene glycol. The matrix-forming material can use methods and methods, reagents and conditions specific to the matrix-forming material to form a matrix through polymerization and / or cross-linking of the matrix-forming material. The matrix-forming material can form a polymer matrix. The matrix-forming material can form a polyelectrolyte gel. The matrix-forming material can form a hydrogel gel matrix.

[0137] In some cases, the cDNA molecule can be contacted with the probe. The cDNA molecule can hybridize with the probe. The probe can include a functional portion. The probe can be fixed to the 3D matrix through the functional portion. The cDNA molecule can be indirectly fixed to the 3D matrix through the functional portion by hybridizing with the probe. The functional portion can bind to the cell or cellular component, or react with the cell or cellular component, or can be an affinity binding group capable of binding to the cell or cellular component. The functional portion can include at least one nucleotide modified by biotin, an amine group, a lower alkylamine group, an acetyl group, DMTO, fluorescein, a thiol group, or an acridine. The probe can be a padlock probe, wherein the padlock probe includes a 5' terminal region and a 3' terminal region that are complementary to the cDNA molecule. The 5' terminal region and 3' terminal region of the padlock probe can hybridize with the cDNA molecule. After hybridization with the cDNA molecule, the 5' and 3' ends can be juxtaposed. The two ends of the padlock probe can be continuous or separated by the gap region of the cDNA molecule. The gap region can have various lengths. In some embodiments, the gap region can comprise at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 nucleotides. In another embodiment, the gap region can comprise 2 to 10, 10 to 20, 20 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400 or 400 to 500 nucleotides. The padlock probe of cyclization can be generated by connecting the two ends of the padlock probe together. When the two ends are continuous, the two ends can directly connect. When the two ends are separated by the gap region, the gap region can be filled before connecting. The gap region can be filled by introducing one or more nucleotides in the extension reaction, for example, by extending from the 3' end in the two ends. The extension reaction can be carried out by polymerase. The extension reaction can use cDNA molecules as templates to carry out because the 5' end and 3' end of the padlock probe hybridize with the cDNA molecules, thereby the sequence information of the cDNA molecules is captured in the padlock probe. In some embodiments, the present invention provides the oligonucleotide sequence of the present invention.Gap area also can be filled by other Nucleotide or other oligonucleotide sequence and gap area hybridization.The length of other oligonucleotide sequence can be determined based on the length of gap area.For example, other oligonucleotide sequence can have identical length with gap area, make with after gap area hybridization, 5 ' end of other oligonucleotide sequence and 3 ' end of padlock probe are adjacent, and 3 ' end of other oligonucleotide sequence and 5 ' end of padlock probe are adjacent.For example, other oligonucleotide sequence can comprise 2 to 10,10 to 20,20 to 50,50 to 100,100 to 150,150 to 200,200 to 300,300 to 400 or 400 to 500 Nucleotide.Then can be by the end of other oligonucleotide sequence being connected with the end of padlock probe to produce cyclization padlock probe.

[0138] The padlock probe of circularization can further carry out rolling circle amplification (RCA) to produce the amplified product of the sequence of the padlock probe of circularization.Amplified product can comprise the nucleotide sequence corresponding to the nucleotide sequence of RNA molecule.Can detect the nucleotide sequence of amplified product, thereby identify the nucleotide sequence of RNA molecule.Different detection methods can be used for nucleic acid detection, comprise order-checking and hybridization.The example of sequencing method comprises sequencing by synthesis (SBS), connection order-checking (SBL) and hybridization order-checking (SBH).

[0139] The reverse transcriptase or its functional derivative may have RNA catalytic cleavage activity. For example, the reverse transcriptase or its functional derivative may have ribonuclease (RNase) activity. The reverse transcriptase or its functional derivative may cleave RNA of an RNA / DNA duplex. Another reverse transcriptase or its functional derivative may also have RNA catalytic cleavage activity. The reverse transcriptase or another reverse transcriptase may be avian myeloblastosis virus (AMV) reverse transcriptase, wild-type human immunodeficiency virus-1 (HIV-1) reverse transcriptase, or Moloney murine leukemia virus (M-MLV) reverse transcriptase.

[0140] In order to reverse transcribe RNA molecules, reverse transcription primers can be hybridized with RNA molecules.Reverse transcription primers can hybridize with the 5' terminal region of padlock probes.Reverse transcription primers may not hybridize with the 5' terminal region of padlock probes.When reverse transcription primers are not hybridized with the 5' terminal region of padlock probes, the region complementary to the 5' terminal region of the cDNA molecule generated can be at a distance of at least 1, at least 5, at least 10, at least 50, at least 100, at least 200, at least 500 or more nucleotides downstream of the reverse transcription primer. As used herein, the distance between the region A (for example, the region complementary to the 5' terminal region of the cDNA molecule) and the region B (for example, reverse transcription primers) of the same nucleic acid chain refers to the number of nucleotides between the 3' end of the 5' end of region A and region B, wherein region A is in the downstream of region B. As used herein, downstream refers to the direction from the 5' end of nucleic acid chain to the 3' end.

[0141] The reverse transcription primer may comprise a functional moiety. The reverse transcription primer or its extension product (e.g., a cDNA molecule) may be fixed to the 3D matrix via the functional moiety. Examples of functional moieties include, but are not limited to, amines, acrylamides, alkynes, biotin, azides, and thiols.

[0142] The biological sample may contain multiple target molecules. For example, the target molecules may be DNA molecules, RNA molecules, or protein molecules. The target molecules may have a relative 3D spatial relationship within the 3D matrix. In some cases, the biological sample may contain multiple RNA molecules. The multiple RNA molecules may have a relative 3D spatial relationship within the 3D matrix.

[0143] In some cases, the RNA molecule or portion thereof can be degraded or digested by a reverse transcriptase under a first set of conditions, and the RNA molecule can be reverse transcribed by the same reverse transcriptase or another reverse transcriptase under a second set of conditions. The first set of conditions can be different from the second set of conditions. The first set of conditions can be the same as the second set of conditions. The first set of conditions or the second set of conditions can be selected from pH, temperature, cofactor concentration, and cation concentration. Examples of cofactors or cations include, but are not limited to, Mg. 2+ 、Mn 2+ 、Na + , ATP and NADPH. The second set of conditions can inhibit the RNase activity of the reverse transcriptase used to reverse transcribe the RNA molecule. For example, the second set of conditions can include an RNase inhibitor. The RNase inhibitor can be a small molecule inhibitor or a polypeptide.

[0144] cDNA molecules may include functional moieties. cDNA molecules can be fixed to a 3D matrix via the functional moiety. The functional moiety can be covalently cross-linked to the matrix, copolymerized with the matrix, or otherwise non-covalently bound to the matrix. The functional moiety can react with a cross-linking agent. The functional moiety can be part of a ligand-ligand binding pair. DNTPs or dUTPs can be modified with functional groups, thereby allowing the functional moiety to be introduced into the DNA during amplification. Examples of functional moieties include, but are not limited to, amines, acrylamides, alkynes, biotin, azides, and thiols. In the case of cross-linking, the functional moiety can be cross-linked with modified dNTPs or dUTPs, or with both. Examples of cross-linking agent reactive groups include, but are not limited to, imido esters (DMP), succinimidyl esters (NHS), maleimides (Sulfo-SMCC), carbodiimides (DCC, EDC), and phenyl azide. Cross-linking agents within the scope of the present disclosure may include a spacer moiety. Such a spacer moiety may be functionalized. Such a spacer moiety may be chemically stable. Such spacer moieties may be of sufficient length to allow amplification of nucleic acids bound to the substrate.Examples of spacer moieties include, but are not limited to, polyethylene glycol, carbon spacers, photocleavable spacers, other spacers, and the like.

[0145] On the other hand, the present disclosure provides a method for identifying a nucleic acid sequence in a biological sample. The method may include providing a biological sample comprising an RNA molecule hybridized with a DNA in a 3D matrix. The RNA molecule may comprise the nucleic acid sequence. A DNA binding protein may then be used to degrade or digest at least a portion of the RNA molecule hybridized with the DNA molecule. The DNA binding protein may not be a reverse transcriptase, a ribonuclease, or both. The DNA molecule may comprise another nucleic acid sequence that is a reverse complement of the nucleic acid sequence. The other nucleic acid sequence in the biological sample may then be detected to identify the nucleic acid sequence. In some cases, a reverse transcriptase may be used to reverse transcribe the RNA molecule to produce a DNA molecule hybridized with the RNA molecule in the biological sample.

[0146] The DNA binding protein or its functional derivative may have RNA catalytic cleavage activity. For example, the DNA binding protein or its functional derivative may have ribonuclease (RNase) activity. The DNA binding protein or its functional derivative may cleave the RNA of an RNA / DNA duplex. The DNA binding protein may stabilize the DNA molecule. The DNA binding protein may stabilize the DNA of an RNA / DNA duplex. The DNA binding protein may increase the melting temperature of the DNA molecule. The DNA binding protein may stabilize the DNA molecule by increasing the melting temperature of the DNA molecule. In some cases, the DNA binding protein is Sso7d.

[0147] On the other hand, the present disclosure provides a method for identifying a nucleic acid sequence in a biological sample. The method may include providing a biological sample comprising an RNA molecule hybridized with a DNA molecule in a 3D matrix. The RNA molecule may comprise the nucleic acid sequence. Then, at least a portion of the RNA molecule hybridized with the DNA molecule may be subjected to non-enzymatic degradation. The DNA molecule may comprise another nucleic acid sequence that is a reverse complement of the nucleic acid sequence. Then, the DNA molecule may be contacted with a probe. When contacted with the probe, the DNA molecule may hybridize with the probe. Then, the sequence of the probe or its derivative may be detected to identify the nucleic acid sequence of the RNA molecule.

[0148] RNA molecules can be chemically degraded. RNA molecules can be degraded or hydrolyzed in the absence of enzymes. For example, RNA is susceptible to base-catalyzed hydrolysis because the ribose in RNA has a hydroxyl group at the 2' position. When the deprotonated 2'OH of ribose (as a nucleophile) attacks the adjacent phosphorus in the phosphodiester bond of the sugar-phosphate backbone of RNA, RNA hydrolysis can occur. As used herein, RNA hydrolysis refers to a reaction in which the phosphodiester bond of the sugar-phosphate backbone of RNA is broken, thereby cutting the RNA molecule. Inorganic and organic compounds can be used to degrade or cut RNA. In some cases, metal complexes can be used to degrade or cut RNA. In some cases, heavy metal ions can be used to degrade or cut RNA. The transesterification step in which 2', 3'-cyclic phosphates can be formed with RNA cleavage and the hydrolysis step in which 2', 3'-cyclic phosphates can be converted into phosphate monoesters can be catalyzed by inorganic or organic compounds.

[0149] Chemical degradation can be carried out under neutral to alkaline pH conditions. For example, the pH value can be at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 10.5, at least 11, at least 11.5, at least 12, at least 12.5, at least 13, at least 13.5, or 14. For another example, the pH can be 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, or 13 to 14. Chemical degradation can be carried out under acidic to neutral pH conditions. For example, the pH value can be 0, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, or at most 7. For another example, the pH can be 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, or 6 to 7. Chemical degradation can be carried out under temperature conditions where RNA is unstable. For example, temperature conditions suitable for RNA degradation may be at least 5°C, at least 10°C, at least 20°C, at least 30°C, at least 40°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, at least 90°C, at least 100°C or higher. For another example, temperature conditions suitable for RNA degradation may be 5°C to 10°C, 10°C to 20°C, 20°C to 30°C, 30 to 40°C, 40 to 50°C, 50 to 60°C, 60 to 70°C, 70 to 80°C, 80 to 90°C or 90 to 100°C. Chemical degradation can be carried out in the presence of heavy metal ions. The term heavy metal refers to any metallic chemical element having a relatively high density and which can be toxic or harmful at low concentrations. The heavy metal ion may be part of a metal complex. As used herein, a metal complex refers to a macrocyclic complex formed by the association of a central metal ion with a non-metallic ion or molecule. The metal ions may include copper, zinc, cobalt, nickel, palladium, lead, iridium, manganese, iron, molybdenum, vanadium, titanium, ruthenium, bismuth, cadmium, magnesium, rhodium, uranium, transition metals, yttrium and lanthanides. Non-metallic ions may comprise ligands, chelating agents or complexing agents. For large metal ions, such as lanthanides (III), ligands that provide six or more donor atoms may be used. The ligands for any metal ion within the scope of the present disclosure do not need to form thermodynamically stable complexes with the metal ion. It is sufficient that they are kinetically inert to the release of the metal ion. Therefore, if properly designed, macrocyclic ligands can form kinetically inert complexes with unstable metal ions. Moreover, tetraazamacrocyclic ligands strongly chelate transition metal ions and Zn (II). Chemical degradation can be achieved in the presence of divalent cations (e.g., Mg 2+ and Zn 2+ The chemical degradation may be carried out under conditions selected from the group consisting of a pH of 6 to 14, a pH of 0 to 6, a temperature of 10° C. to 100° C., in the presence of heavy metal ions, in the presence of divalent cations, and any combination thereof.

[0150] In another aspect, the present disclosure provides a method for processing a biological sample. The method may include providing a biological sample comprising an RNA molecule in a 3D matrix. The RNA molecule may comprise a nucleic acid sequence. A primer may then hybridize with the RNA molecule. The primer may be a reverse transcription primer. The primer may not include a functional moiety for anchoring to the 3D matrix. A reverse transcriptase may then be used to reverse transcribe the RNA molecule by extending the primer to produce a cDNA molecule in the biological sample that hybridizes with the RNA molecule. The cDNA molecule may include a functional moiety that anchors the cDNA molecule to the 3D matrix.

[0151] In some cases, the RNA molecule hybridized to the cDNA molecule can be degraded to provide the cDNA molecule immobilized to the 3D matrix via the functional moiety.The cDNA molecule can comprise an additional nucleic acid sequence that is the reverse complement of the nucleic acid sequence.

[0152] The RNA molecule may comprise a functional moiety. The RNA molecule may be immobilized to the 3D matrix via the functional moiety.

[0153] RNA molecules can be degraded using various methods. For example, RNA molecules can be degraded by non-ribonucleases. Non-ribonucleases can be reverse transcriptases or DNA-binding proteins. As another example, RNA molecules can be degraded by non-enzymatic reactions. Non-enzymatic reactions can be performed under conditions selected from the group consisting of a pH of 6 to 14, a temperature of 10° C. to 100° C., the presence of heavy metal ions, and any combination thereof.

[0154] The cDNA molecule can be contacted with a probe. The probe can include a region that cannot hybridize with the cDNA molecule. The region that cannot hybridize with the cDNA molecule can be an end region or an internal region between two end regions.

[0155] The probe can be a padlock probe, wherein the padlock probe comprises a 5' terminal region and a 3' terminal region that are complementary to the cDNA molecule. The 5' terminal region and the 3' terminal region of the padlock probe can hybridize with the cDNA molecule. The padlock probe can be cyclized by coupling the two ends of the padlock probe together to produce a cyclized padlock probe. For example, the two ends of the padlock probe can be linked together by a ligase. The nucleic acid sequence of the cyclized padlock probe or its derivative can be detected to identify the nucleic acid sequence of the RNA molecule. For example, the nucleic acid sequence of the cyclized padlock probe or its derivative can be detected by nucleic acid hybridization or sequencing.

[0156] After the two ends of the padlock probe are hybridized with the cDNA molecule, the two ends of the padlock probe can be continuous or separated by the gap region of the cDNA molecule. The gap region can have various lengths. For example, the gap region can comprise at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 nucleotides. For another example, the gap region can comprise 2 to 10, 10 to 20, 20 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400 or 400 to 500 nucleotides. The padlock probe of cyclization can be produced by linking the two ends of the padlock probe together. When the two ends are continuous, the two ends can directly connect. When the two ends are separated by the gap region, the gap region can be filled before connecting. One or more nucleotides can be introduced in the extension reaction, for example, the gap region can be filled by extending the 3' end from the two ends. The extension reaction can be carried out by polymerase. In some embodiments, the present invention provides the oligonucleotide sequence of the present invention.Also can fill the gap zone by other Nucleotide or other oligonucleotide sequence and gap zone hybridization.The length of other oligonucleotide sequence can be determined based on the length of gap zone.For example, other oligonucleotide sequence can have identical length with gap zone, make with after gap zone hybridization, 5 ' end of other oligonucleotide sequence and 3 ' end of padlock probe are adjacent, and 3 ' end of other oligonucleotide sequence and 5 ' end of padlock probe are adjacent.For example, other oligonucleotide sequence can comprise 2 to 10,10 to 20,20 to 50,50 to 100,100 to 150,150 to 200,200 to 300,300 to 400 or 400 to 500 Nucleotide.Then can produce the padlock probe of cyclization by the end of other oligonucleotide sequence being connected with the end of padlock probe.

[0157] The padlock probe of circularization can be further amplified to produce an amplified product of the sequence of the padlock probe of circularization.For example, the padlock probe of circularization can carry out rolling circle amplification (RCA).The amplified product can comprise the nucleotide sequence corresponding to the nucleotide sequence of RNA molecule.The nucleotide sequence of amplified product can be detected, thereby identifying the nucleotide sequence of RNA molecule.

[0158] The probe may comprise a functional moiety. The probe may be affixed to the 3D matrix via the functional moiety. The functional moiety may be directly conjugated to the probe. The functional moiety may be introduced into the probe during probe synthesis. For example, a nucleotide analog having a functional moiety or a precursor to the functional moiety may be introduced into the growing strand of the probe during synthesis. The functional moiety may be introduced into the probe via a tethering oligonucleotide. The probe may hybridize to a tethering oligonucleotide comprising the functional moiety. The tethering oligonucleotide may hybridize to a region of the probe that does not hybridize to the cDNA molecule. The sequence of the probe or its derivatives may be detected to identify the nucleic acid sequence of the RNA molecule.

[0159] The cDNA molecule may comprise a functional moiety. Reverse transcriptase can be used to introduce a nucleotide analog comprising a functional moiety into the growing chain to obtain a cDNA molecule comprising a nucleotide analog. For example, the nucleotide analog includes amino-allyl dUTP, 5-TCO-PEG4-dUTP, C8-alkyne-dUTP, 5-azidomethyl-dUTP, 5-vinyl-dUTP, 5-ethynyl dUTP, or a combination thereof.

[0160] After the cDNA molecule is generated, the primer or cDNA molecule can be modified to include a functional moiety. Before the cDNA molecule is generated, the primer can be modified to include a functional moiety. For example, the primer can be modified with a functional moiety through conjugation chemistry. The primer can include a region that is non-hybridizable to the RNA molecule. The region that is non-hybridizable to the RNA molecule can be coupled to a tethering oligonucleotide that includes a functional moiety. For example, the region can hybridize to a tethering oligonucleotide that includes a functional moiety.

[0161] The functional moiety can be attached to the cDNA molecule via an enzymatic reaction or a non-enzymatic reaction. An enzymatic reaction can include the use of an enzyme to attach a nucleotide or oligonucleotide having a functional moiety to the cDNA molecule. The enzyme can be a ligase, a polymerase, or a combination thereof. A non-enzymatic reaction can include the attachment of a chemical reagent having a functional moiety to the cDNA molecule via alkylation or hydroxymercuration. The cDNA molecule can be coupled to a tethered oligonucleotide having a functional moiety. For example, the cDNA molecule can be hybridized to a tethered oligonucleotide having a functional moiety. The tethered oligonucleotide can be hybridized to a primer.

[0162] The 3D matrix may further comprise additional functional moieties that can react with the functional moieties of the cDNA molecules, thereby immobilizing the cDNA molecules.

[0163] In another aspect, the disclosure features a kit comprising some or all of the reagents, enzymes, probes, and primers necessary to perform the methods described herein. The items comprising the kit can be provided in separate vials or mixed together where appropriate.

[0164] Figure 1An example of a method for identifying a nucleic acid sequence in a biological sample is shown. In a first operation 101, a biological sample comprising a ribonucleic acid (RNA) molecule hybridized with a deoxyribonucleic acid molecule (DNA) in a three-dimensional (3D) matrix can be provided. The RNA molecule can comprise a nucleic acid sequence to be identified. Then, in a second operation 102, a reverse transcriptase can be used to degrade or digest at least a portion of the RNA molecule hybridized with the DNA molecule. The DNA molecule can comprise another nucleic acid sequence that is a reverse complementary sequence of the nucleic acid sequence. Then, in a third operation 103, the other nucleic acid sequence in the biological sample can be detected to identify the nucleic acid sequence.

[0165] Figure 2 A method for identifying a nucleic acid sequence in a biological sample is shown. In a first operation 201, a biological sample comprising a ribonucleic acid (RNA) molecule hybridized with a deoxyribonucleic acid molecule (DNA) can be provided in a three-dimensional (3D) matrix. The RNA molecule can comprise a nucleic acid sequence to be identified. Then, in a second operation 202, a deoxyribonucleic acid (DNA) binding protein that is not a reverse transcriptase or ribonuclease can be used to degrade or digest at least a portion of the RNA molecule hybridized with the DNA molecule. The DNA molecule can comprise another nucleic acid sequence that is a reverse complementary sequence of the nucleic acid sequence. Then, in a third operation 203, the other nucleic acid sequence in the biological sample can be detected to identify the nucleic acid sequence.

[0166] Figure 3 An example of a method for identifying a nucleic acid sequence in a biological sample is shown. In a first operation 301, a biological sample comprising a ribonucleic acid (RNA) molecule hybridized with a deoxyribonucleic acid molecule (DNA) in a three-dimensional (3D) matrix can be provided. The RNA molecule can comprise a nucleic acid sequence to be identified. Then, in a second operation 302, non-enzymatic degradation can be performed on at least a portion of the RNA molecule hybridized with the DNA molecule. The DNA molecule can comprise another nucleic acid sequence that is a reverse complementary sequence of the nucleic acid sequence. Then, in a third operation 303, the cDNA molecule can be contacted with a probe. Then, in a fourth operation 304, the sequence of the probe or its derivative can be detected to identify the nucleic acid sequence of the RNA molecule.

[0167] Figure 4An example of a method for processing a biological sample is shown. In a first operation 401, a biological sample comprising a ribonucleic acid (RNA) molecule in a three-dimensional (3D) matrix can be provided. The RNA molecule can comprise a nucleic acid sequence. Then, in a second operation 402, a primer can be hybridized with the RNA molecule, the primer not comprising a functional portion for being fixed on the matrix. Then, in a third operation 403, a reverse transcriptase is used to reverse transcribe the RNA molecule by extending the primer to produce a complementary deoxyribonucleic acid (cDNA) molecule that hybridizes with the RNA molecule in the biological sample. The cDNA molecule can comprise a functional portion that fixes the cDNA molecule to the 3D matrix.

[0168] In various embodiments, target DNA or RNA molecule can hybridize with primer or probe in the presence of hybridization reaction enhancer.Compared with another hybridization reaction carried out between target nucleic acid molecule and probe in the absence of hybridization reaction enhancer, the hybridization reaction enhancer can improve the speed of hybridization reaction between target nucleic acid molecule and the probe with sequence complementarity with the target sequence of the target molecule.The example of hybridization reaction enhancer can be dextran sulfate.However, enzyme can be strongly inhibited by dextran sulfate, and as a high-charge, high-molecular-weight polymer, it is difficult to wash dextran sulfate to the extent that downstream enzymatic reaction (such as reverse transcription, connection, DNA polymerization) is not strongly inhibited from the sample.However, in the absence of hybridization reaction enhancer, the kinetics of in situ hybridization can be several orders of magnitude slower.Therefore, in the method described herein, the hybridization reaction enhancer that can not strongly inhibit enzymatic reaction can be used.The hybridization reaction enhancer can be a high-molecular-weight, high-valence charged polymer.For example, the hybridization reaction enhancer can be a polymer, such as polyacrylic acid, polyvinyl sulfonic acid and alginate.The hybridization reaction enhancer can be a polymer similar to dextran sulfate.

[0169] In some cases, the intermolecular organization of the hybridization reaction enhancer can be a factor in determining its effectiveness as a hybridization reaction enhancer. For example, dextran sulfate can help network formation (e.g., high local concentration of probes) during hybridization, thereby accelerating the annealing process. The G-block of alginate can participate in the interaction with divalent cations (e.g., Ca 2+ ) intermolecular crosslinking to form a hydrogel. Except under exogenous chemical crosslinking reaction and in the presence of chitosan (both of which are not present in typical nucleic acid hybridization reactions), dextran sulfate may not form a hydrogel. In some cases, the hybridization reaction enhancer may self-bind during the formation of the hydrogel. Alternatively, the hybridization reaction enhancer may not self-bind during the formation of the hydrogel. The difference in self-binding ability in the formation of the hydrogel can explain the difference between dextran sulfate and alginate in improving the kinetics of nucleic acid DNA hybridization reactions.

[0170] For example, polyacrylic acid and polyvinyl sulfonic acid both effectively act as hybridization enhancers, whereas alginate does not. This may be due to intermolecular organization, which reduces its effectiveness in aggregating DNA. In some cases, polyacrylic acid can inhibit enzymatic reactions, but polyvinyl sulfonic acid may exhibit less inhibition. For example, one mechanism of inhibition may be through chelation of essential metals or charged cofactors, such as Mg. 2+ , Ca 2+ 、Mn 2+ 、Na + In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.In some embodiments, the present invention relates to the enzyme that comprises the cation of the present invention.

[0171] In some embodiments, the properties or activity of the hybridization reaction enhancer can be controlled (e.g., inactivated). For example, certain polymers that act as hybridization reaction enhancers may contain charged groups, which can then be cleaved or neutralized. This method can convert the polymer into a neutral polymer, such as PEG, which can improve the efficiency of the enzymatic reaction. In some embodiments, the polymer that acts as a hybridization reaction enhancer can be specifically degraded into small monomers and can be easily removed from the sample, for example, by washing it out from the sample. The chemical process for passivation or degradation of the hybridization reaction enhancer may need to be orthogonal to the nucleic acid, that is, not degrading the nucleic acid or making it incompatible with the nucleic acid. Some of these functional groups include α-hydroxy acids, which can be cleaved by sodium periodate; β-keto acids, which can be thermally cleaved; thiophosphate bonds, which can be cleaved by silver ions; disulfide bonds, which can be cleaved into thiols by reduction; and other types of chemical bonds that can be cleaved by light or chemical treatment.

[0172] Examples of polyions or polyelectrolytes for enzyme compatibility enhancement of nucleic acid hybridization kinetics include condensation products of Cys(Lys)nCys; polymers such as PEG, PVA, or PAA, which can be subsequently modified with cleavable linkers to include chemical groups that impart ionic charge; or polymers formed from monomers that include cleavable bonds so that the polymer can be degraded after acting as a hybridization enhancer. As an alternative to ionic charge, these polymers can include nonionic groups that hydrate in solution, which can increase the rate of nucleic acid hybridization by molecular aggregation and / or chelating water.

[0173] In some cases, it may be impossible to control the hybridization reaction enhancer. For example, the hybridization reaction enhancer may not include a cleavable functional group that inactivates or degrades to a monomer. In these cases, the hybridization reaction enhancer can be removed from the sample after it acts as a hybridization reaction enhancer to enhance nucleic acid hybridization. For example, the hybridization reaction enhancer can be washed out from the sample or 3D matrix. A washing step can be performed after the primer or probe is hybridized with the target nucleic acid molecule and before any subsequent enzymatic reaction (for example, reverse transcription, connection, and amplification).

[0174] The present disclosure provides a method for enhancing probe hybridization, wherein the probe is used to capture RNA, cDNA and DNA species onto a 3D matrix for detection by FISSEQ. A hybridization reaction enhancer can be added to the hybridization buffer. In some embodiments, a hybridization buffer containing high salt, such as SSC (sodium chloride sodium citrate buffer) can be used. In some embodiments, a hybridization buffer containing a blocker can be used. The blocker can reduce the non-specific binding of the probe to the off-target sequence and / or by preventing electrostatic interactions with other components of the sample (e.g., yeast tRNA, salmon sperm, detergents (e.g., Triton-X, Tween 20, SPAN), peptides (e.g., BSA) and other reagents (e.g., Ficoll)). In some embodiments, the hybridization buffer includes a reagent that changes the annealing properties (e.g., melting temperature) of the DNA. The example of the reagent that can change the annealing temperature includes formamide. The average molecular weight of the hybridization reaction enhancer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 kDa or larger. In some embodiments, the weight of the hybridization reaction enhancer per volume in the reaction can be at least about 1%, 5%, 10%, 15%, 20% or larger.

[0175] Hybridization reaction enhancers can be polyions, polyelectrolytes, hydrophilic or hydrated polymers. Hybridization reaction enhancers can comprise a polymer backbone and one or more hydrating groups. The hydrating groups can be ionic, electrolyte or hydrophilic. In some embodiments, the hydrating groups can be specifically inactivated, for example, by imparting a neutral charge to the ionic groups or by weakly hydrating the strong hydrating groups. The inactivated chemical substances can be substantially unreactive with RNA, DNA, proteins and / or other types of biomolecules. The inactivated polymers are compatible with enzymatic reactions.

[0176] The hybridization reaction enhancer may comprise a cleavable bond between the polymer backbone and the hydration group. The cleavable bond may comprise an α-hydroxy acid, which may be cleaved by sodium periodate. The cleavable bond may comprise a β-keto acid, which may be thermally cleaved. The cleavable bond may comprise a phosphorothioate bond, which may be cleaved by silver ions. The cleavable bond may comprise a disulfide bond, which may be cleaved into thiols by reduction. Other types of chemical bonds may be cleaved by light or chemical treatment. In some cases, the hybridization reaction enhancer may comprise a cleavable bond along the polymer backbone, wherein the cleavable bond may be any type of cleavable bond described herein.

[0177] The methods provided herein can include the use of hybridization reaction enhancers in sample processing for target RNA or DNA detection. In some cases, a plurality of probes can be hybridized in situ using a hybridization buffer containing one of the hybridization reaction enhancers described herein.

[0178] The methods described herein may include in situ hybridization of multiple probes using a hybridization buffer containing one of the hybridization reaction enhancers described herein. In some embodiments, the method also includes triggering the cracking of the cleavable group of the hybridization reaction enhancer to inactivate the hybridization reaction enhancer. In some other embodiments, the method also includes removing the hybridization reaction enhancer from the sample or 3D matrix without inactivating the hybridization reaction enhancer.

[0179] Computer system

[0180] The present disclosure provides computer systems programmed to perform the methods of the present disclosure. Figure 5 A computer system 501 is shown that is programmed or otherwise configured for processing a biological sample and / or identifying a nucleic acid sequence in a biological sample. The computer system 501 can regulate various aspects of the components and / or devices of the present disclosure used for detecting nucleic acid sequences in a biological sample and / or sample processing, such as light sources, detectors (e.g., photodetectors), devices or components for releasing reagents, devices or components for providing reaction (e.g., hybridization, sequencing, enzymatic reaction) conditions, etc. The computer system 501 can be an electronic device of a user or a computer system remotely located relative to an electronic device. The electronic device can be a mobile electronic device.

[0181] Computer system 501 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 505, which can be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 501 also includes memory or memory locations 510 (e.g., random access memory, read-only memory, flash memory), electronic storage 515 (e.g., a hard disk), a communication interface 520 (e.g., a network adapter) for communicating with one or more other systems, and peripherals 525, such as cache memory, other memory, data storage, and / or electronic display adapters. Memory 510, storage 515, interface 520, and peripherals 525 communicate with CPU 505 via a communication bus (solid lines), such as a motherboard. Storage 1115 can be a data storage unit (or data repository) for storing data. Computer system 501 can be operatively coupled to a computer network ("network") 530 via communication interface 520. Network 530 can be the Internet, an internetwork and / or an extranet, or an intranet and / or an extranet in communication with the Internet. In some cases, network 530 is a telecommunications and / or data network. Network 530 may include one or more computer servers, which may enable distributed computing, such as cloud computing. In some cases, network 530 may implement a peer-to-peer network with the help of computer system 501, which may enable devices coupled to computer system 501 to act as clients or servers.

[0182] The CPU 505 can execute a series of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a memory location such as the memory 510. The instructions can be directed to the CPU 505, which can then program or otherwise configure the CPU 505 to implement the methods of the present disclosure. Examples of operations performed by the CPU 505 can include fetching, decoding, executing, and writing back.

[0183] CPU 505 may be part of a circuit such as an integrated circuit. One or more other components of system 501 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0184] Storage unit 515 can store files such as drivers, libraries, and saved programs. Storage unit 515 can store user data such as user preferences and user programs. In some cases, computer system 501 can include one or more additional data storage units located external to computer system 501, such as on a remote server that communicates with computer system 501 via an intranet or the Internet.

[0185] The computer system 501 can communicate with one or more remote computer systems via the network 530. For example, the computer system 501 can communicate with a remote computer system of a user (e.g., a user performing sample processing or nucleic acid sequence detection of the present disclosure). Examples of remote computer systems include personal computers (e.g., portable PCs), tablet PCs, or tablet computers (e.g., iPad, Galaxy Tab), phones, smartphones (e.g. iPhone, Android-enabled devices, ) or personal digital assistant. Users can access computer system 501 through network 530.

[0186] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location (e.g., memory 510 or electronic storage unit 515) of computer system 501. The machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by processor 505. In some cases, the code may be retrieved from storage unit 515 and stored in memory 510 for ready access by processor 505. In some cases, electronic storage unit 515 may be eliminated and the machine executable instructions may be stored in memory 510.

[0187] The code may be precompiled and configured for a machine with a processor suitable for executing the code, or may be compiled during runtime. The code may be provided in a programming language that may be selected so that the code can be executed in a precompiled or as-compiled manner.

[0188] Various aspects of the systems and methods provided herein (e.g., computer system 501) can be embodied in programming. Various aspects of the technology can be viewed as "products" or "articles" typically carried on machine-readable media or in the form of machine (or processor) executable code and / or associated data embodied in the form of machine-readable media. Machine executable code can be stored in an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media may include any or all tangible memories of a computer, processor, etc., or related modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage for software programming at any time. All or part of the software can sometimes be communicated via the Internet or various other telecommunications networks. For example, such communication can enable software to be loaded from one computer or processor to another, such as from a management server or host to a computer platform of an application server. Therefore, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as those used on physical interfaces between local devices via wired and optical landline networks and via various air-links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered media that carry the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0189] Thus, a machine-readable medium such as computer executable code may take a variety of forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, and the like, such as may be used to implement the databases shown in the accompanying drawings. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that make up a bus within a computer system. Carrier transmission media may take the form of electrical or electromagnetic signals or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example, a floppy disk, a diskette, a hard disk, magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punched card tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave that transports data or instructions, a cable or link that transports such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0190] The computer system 501 may include or communicate with an electronic display 535, which includes a user interface (UI) 540 for providing, for example, protocols for performing the sample processing methods and / or nucleic acid sequence detection methods described herein. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0191] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented when executed by the central processing unit 505. For example, the algorithms can be executed to detect nucleic acid sequences using the methods and systems disclosed in the present disclosure. Optionally, the algorithms can be executed to control or implement the operation of components (e.g., light sources, detectors, reagent streams, etc.) of the systems described herein to perform detection of nucleic acid sequences.

[0192] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not intended to be interpreted in a restrictive sense. Without departing from the present invention, those skilled in the art will now appreciate many variations, changes, and substitutions. In addition, it will be understood that all aspects of the present invention are not limited to the specific description, configuration, or relative proportions set forth herein, which depend on various conditions and variables. It will be understood that various alternatives to the embodiments of the present invention described herein can be used to implement the present invention. Therefore, it is contemplated that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. It is intended that the scope of the present invention be defined by the appended claims, and that methods and structures and their equivalents within the scope of these claims be encompassed thereby.

[0193] Example

[0194] Example 1 - Fixation, amplification and imaging of intracellular DNA / RNA molecules

[0195] Human iPS cells or human primary fibroblasts were grown on 1.5 coverslips. They were fixed with 4% formaldehyde in PBS for 15 min, followed by three washes with 70% ethanol. A reverse transcription mixture containing 1 μM random hexamer or 0.1 μM polydT(18)V primer with an additional adapter sequence (TCTCGGGAACGCTGAAGA), 250 μM dNTPs, 40 μM aminoallyl dUTP (Anaspec), 20 U RNase inhibitor, and 100 U MMuLV reverse transcriptase (enzymatic) was added to the fixed cells and incubated overnight at 37°C. The samples were then washed with PBS and cross-linked with 100 μM BS(PEG)9 (Thermo-Fisher Scientific) in PBS for 1 hour, followed by treatment with 1 M Tris for 15 min. A cyclization mixture containing 25U CircLigase (Epicentre), 1mM MnCl and 1M betaine was added and the sample was incubated at 60°C for 2 hours. RNA was degraded at 37°C for 1 hour using avian myeloblastosis virus (AMV) reverse transcriptase. Alternatively, RNA was degraded using a solution with a pH of 8 to 10 or by heating the sample at 100°C for approximately 20 minutes. RCA primers were then hybridized to the sample at 60°C for 15 minutes and washed. For rolling circle amplification, 100U phi29 DNA polymerase (enzymatic), 250μM dNTPs and 40μM aminoallyl dNTPs were added to the sample and incubated overnight at 30°C. The sample was then washed with PBS and cross-linked with 100μM BS(PEG)9 in PBS for 1 hour and then treated with 1M Tris for 15 minutes. For DNA amplicon detection, 1 μM fluorescently labeled oligonucleotides were diluted in 2× SSC and hybridized to the matrix containing DNA amplicons at 60°C and washed. Imaging was performed using a Leica SP5 scanning confocal microscope using a 10×, 20×, or 63× objective in four color channels (FITC, Cy3, Texas Red, and Cy5). Image stacks containing up to 50 optical sections were then visualized using Imaris Bitplane software to perform three-dimensional reconstruction of the DNA amplicons within the sample matrix.

[0196] Method described herein allows technicians to fix, amplify and image single DNA / RNA molecules in three-dimensional space, without interfering with structure. DNA / RNA can be amplified in situ. DNA / RNA can be copolymerized in situ in host material, and single amplicon can be interrogated (interrogate) / hybridized with fluorescent oligonucleotide, and imaged. When observing with much higher magnification, single amplicon can be imaged using confocal microscope. This allows technicians to find different DNA / RNA molecules, how to distinguish them between different cell types and form, and how they are presented in developing tissue and change over time. Similar conception can be used for many other samples in natural and synthetic materials, as long as they can be copolymerized and / or encapsulated by DNA amplicon.

[0197] According to a specific aspect, in a single mammalian cell, 20 to 500KmRNA molecules can be distributed throughout the cytoplasm. Cells can be fixed and permeabilized. Then dUTP can be used instead of dTTP, or in addition to dTTP, dUTP is also used to convert cellular RNA into cDNA molecules. The cDNA molecules comprising the modified dUMP residues can then be cross-linked and cyclized to form a three-dimensional quasi-polymer of circular cDNA molecules in a single cell. Rolling circle amplification can then be used to amplify the cDNA network into a DNA amplicon network. Then, the cell-based DNA amplicon network stores information about the identity, position, variation / mutation of each transcript. Cell-based DNA amplicon matrix can be read using ligation sequencing (i.e., ABI Solid), synthetic sequencing (i.e., Illumina) or any other proprietary or open sequencing chemistry. Sequencing can be whole genome sequencing or targeted sequencing. Sequencing can be carried out by massively parallel array sequencing (e.g., Illumina) or single molecule sequencing (e.g., Pacific Biosciences or Oxford Nanopore in California). Given the three-dimensional nature of the DNA amplicon network, confocal or multiphoton microscopy can be used to sequence individual amplicons throughout the entire thickness of the amplicon network, allowing researchers to visualize the cDNA distribution of transcripts between the apical and basal surfaces of the cell. Given the tight packing density, researchers can selectively read different subpopulations sequentially, thereby reducing the density of information read at any given time and extending it over time to achieve better spatial resolution.

[0198] Example II - Sample Processing and Target Detection within a 3D Matrix

[0199] Mouse brain tissue samples were dissected onto slides. They were fixed with 4% formaldehyde in PBS for 20 minutes, quenched with 100 mM glycine for 15 minutes, and rinsed with 1X PBS for 5 minutes. Samples were then gradually dehydrated in ethanol and incubated overnight in 100% ethanol. Samples were gradually rehydrated to 1X PBST and permeabilized in 0.2% Triton X-100. A 1 μM tetherable reverse transcription primer was hybridized overnight at 37°C in a buffer containing 2X SSC and 10% dextran sulfate. Samples were washed at 37°C for 20 minutes to remove excess oligonucleotides embedded in the polyacrylamide matrix, and proteins were cleared overnight at 37°C using 16 U / mL protease in 4% SDS buffer at pH 7.3. To remove residual hybridization enhancers and proteases, samples were washed in 1X PBST for several hours, changing the buffer every 30 minutes. A reverse transcription mixture containing 1.25 mM dNTPs, 1 U / μL RNase inhibitor (enzymatic), and 10 U / μL EnzScript reverse transcriptase (enzymatic) was added to the clarified fraction and incubated overnight at 37°C. ssRNA and RNA that formed duplexes with the cDNA product were removed by borate buffer-mediated chemical hydrolysis at 55°C for 2 hours, and then a DNA-locked oligonucleotide probe was hybridized to the ss-cDNA during an overnight incubation at 37°C in a buffer containing 2X SSC and 10% dextran sulfate. The sample was washed for 20 minutes at 37°C to remove unbound DNA-locked oligonucleotide probe, and then further washed in 1X PBST for several hours, changing the buffer every 30 minutes to remove residual hybridization enhancers. DNA-locked oligonucleotide probes were ligated using 30 U / μL T7 ligase (enzymatic) at room temperature for 60 min, followed by rolling circle amplification at 30°C overnight in the presence of 0.5 U / μL phi29 DNA polymerase (enzymatic), 625 μM dNTPs, and 0.025 μM compaction oligonucleotides. For DNA amplicon detection, 1 μM fluorescently labeled oligonucleotides were diluted in 2× SSC and hybridized to the matrix containing DNA amplicons at 60°C and washed. Imaging was performed using a ReadCoor automated fluorescence in situ sequencing instrument. 3D image data were processed to identify individual amplicons and visualized using a web browser-based software tool to perform three-dimensional reconstruction of the DNA amplicons within the matrix. Figure 6 An exemplary image of a mouse brain tissue sample 600 processed and imaged using the above-described method is shown. Cell nuclei (e.g., Figure 6 601) are shown as larger bright spots on the entire image. Figure 6The sequencing readout of the fluorescent signal of 602 is displayed as a smaller and darker spot on the entire image. The image shows the sequencing readout of one sequencing cycle. Multiple sequencing cycles are performed on the sample to determine the base sequence of each amplicon.

Claims

1. A method for processing a biological sample, comprising: (a) providing a biological sample comprising ribonucleic acid (RNA) molecules, wherein the RNA molecules comprise nucleic acid sequences; (b) hybridizing a primer to the RNA molecule, wherein the primer comprises a region that is non-hybridizable to the RNA molecule, the region being hybridized to a tethered oligonucleotide comprising a functional moiety; (c) reverse transcribing the RNA molecule using a reverse transcriptase by extending the primer to produce a complementary deoxyribonucleic acid (cDNA) molecule that hybridizes with the RNA molecule in the biological sample, the cDNA molecule comprising an additional nucleic acid sequence that is the reverse complement of the nucleic acid sequence; (d) immobilizing the cDNA molecule to a synthetic three-dimensional (3D) matrix using the functional moiety, The primer does not include a functional portion for immobilization to the 3D matrix.

2. The method of claim 1, further comprising degrading the RNA molecule hybridized to the cDNA molecule.

3. The method of claim 2, wherein degrading comprises degrading the RNA molecule by a non-ribonuclease. The method according to claim 3 , wherein the non-ribonuclease is a reverse transcriptase or a DNA binding protein.

5. The method of claim 2, wherein degrading comprises degrading the RNA molecule by a non-enzymatic reaction.

6. The method according to claim 5, wherein the non-enzymatic reaction is carried out under conditions selected from the group consisting of: a pH of 6 to 14, a temperature of 10°C to 100°C, in the presence of heavy metal ions, in the presence of divalent cations, and any combination thereof.

7. The method of claim 1, further comprising contacting the cDNA molecule with a probe and detecting the sequence of the probe, thereby identifying the nucleic acid sequence of the RNA molecule.

8. The method of claim 7, wherein the probe is a padlock probe, wherein the padlock probe comprises a 5' terminal region and a 3' terminal region that are complementary to the cDNA molecule, and further comprising hybridizing the 5' terminal region and the 3' terminal region of the padlock probe to the cDNA molecule.

9. The method of claim 8, further comprising circularizing the padlock probe by ligating both ends of the padlock probe together to produce a circularized padlock probe, and detecting the nucleic acid sequence of the circularized padlock probe or a derivative thereof, thereby identifying the nucleic acid sequence of the RNA molecule.

10. The method of claim 9, wherein the two ends of the padlock probe are continuous. The method according to claim 9 , wherein the two ends of the padlock probe are separated by a gap region comprising at least one nucleotide.

12. The method of claim 11, wherein the gap region comprises 2 to 500 nucleotides.

13. The method of claim 11, further comprising filling the gap region by introducing at least one nucleotide in an extension reaction.

14. The method of claim 9, further comprising subjecting the circularized padlock probe to rolling circle amplification (RCA) to produce an amplification product from the circularized padlock probe, the amplification product comprising a nucleic acid sequence corresponding to the nucleic acid sequence of the RNA molecule, and detecting the nucleic acid sequence of the amplification product to thereby identify the nucleic acid sequence of the RNA molecule.

15. The method of claim 7, wherein the probe comprises a functional moiety, wherein the probe is immobilized to the synthetic 3D matrix via the functional moiety.

16. The method of claim 1, wherein the primer is hybridized to the tethered oligonucleotide prior to generating the cDNA molecule.

17. The method of claim 1, wherein the synthetic 3D matrix is a synthetic hydrogel matrix.

Images