[go: up one dir, main page]

WO2024243298A1 - Procédés de sondage et d'amplification d'arn pour analyse de cellule unique sur des cellules fixes - Google Patents

Procédés de sondage et d'amplification d'arn pour analyse de cellule unique sur des cellules fixes Download PDF

Info

Publication number
WO2024243298A1
WO2024243298A1 PCT/US2024/030552 US2024030552W WO2024243298A1 WO 2024243298 A1 WO2024243298 A1 WO 2024243298A1 US 2024030552 W US2024030552 W US 2024030552W WO 2024243298 A1 WO2024243298 A1 WO 2024243298A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
nucleic acid
sequence
oligonucleotides
oligonucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/030552
Other languages
English (en)
Inventor
Hye-Won Song
Jody MARTIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Becton Dickinson and Co
Original Assignee
Becton Dickinson and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Becton Dickinson and Co filed Critical Becton Dickinson and Co
Priority to CN202480033772.7A priority Critical patent/CN121152885A/zh
Publication of WO2024243298A1 publication Critical patent/WO2024243298A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present disclosure relates generally to the field of molecular biology, for example determining gene expression using molecular barcoding.
  • the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality 7 of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary 7 to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality 7 of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target.
  • the method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.
  • the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality' of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target.
  • the method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.
  • the method comprises: contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and a predetermined spatial label.
  • probing oligonucleotides contacted with the same spatial location comprise the same spatial label sequence, and wherein probing oligonucleotides contacted with distinct spatial locations of the sample comprise different spatial label sequences.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality' of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality' of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target.
  • the method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a nucleic acid target to determine the copy number of the nucleic acid target at each spatial location of the sample.
  • barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality' of oligonucleotide barcodes comprises: providing a coupling oligonucleotide comprising a 5 ’ complement of the coupling sequence and a 3’ complement of a capture sequence; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5’ complement of the coupling sequence of the coupling oligonucleotide; hybridizing the 3’ complement of the capture sequence of the coupling oligonucleotide with a capture sequence of an oligonucleotide barcode of the plurality of oligonucleotide barcodes; and/or ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode.
  • the method can comprise: before ligating the extended probing oligonucleotide to the oligonucleotide barcode, filling a gap between the extended probing oligonucleotide and the hybridized oligonucleotide barcode with a DNA polymerase lacking at least one of 5’ to 3’ exonuclease activity and 3’ to 5’ exonuclease activity.
  • ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode is performed with a DNA ligase.
  • the coupling oligonucleotide is a single-stranded oligonucleotide.
  • the coupling oligonucleotide comprises at least 6 nucleotides. In some embodiments, the coupling sequence comprises at least 4 nucleotides. In some embodiments, the 5’ end of each probing oligonucleotide is phosphorylated. In some embodiments, the probing oligonucleotides are capable of entering a cell and/or a nucleus of the sample (e.g., a permeabilized cell and/or a permeabilized nucleus of the sample).
  • the method can comprise: after contacting the probing oligonucleotides with the sample, removing one or more probing oligonucleotides of the plurality of probing oligonucleotides that are not contacted with the sample, optionally removing the one or more probing oligonucleotides not contacted with the sample comprises: removing the one or more probing oligonucleotides that have not entered a cell of the sample.
  • the contacting step comprises contacting the sample with a device configured to deposit probing oligonucleotides (e.g., an ink jet device).
  • the device is a needle, a needle array, a tube, a suction device, an injection device, an electroporation device, a fluorescent activated cell sorter device, an ink jet device, a microfluidic device, or any combination thereof.
  • the device contacts distinct spatial locations of the sample at a specified rate.
  • the spatial label is 6-60 nucleotides in length.
  • said two or more spatial locations comprise at least about 3, about 4, about 5. about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, distinct spatial locations of the sample.
  • a spatial location of the sample corresponds to an area comprising no more than about 50 cells, about 45 cells, about 40 cells, about 35 cells, about 30 cells, about 25 cells, about 20 cells, about 15 cells, about 10 cells, about 9 cells, about 8 cells, about 7 cells, about 6 cells, about 5 cells, about 4 cells, about 3 cells, about 2 cells, or about 1 cell.
  • the method can comprise: contacting the sample with extension reagents. In some embodiments, at least a portion of the contacting step is performed in the presence in the presence of extension reagents. In some embodiments, the entire contacting step is performed in the presence in the presence of the extension reagents. In some embodiments, the contacting and extension steps are simultaneous. In some embodiments, the extension is performed in situ. In some embodiments, said extension comprises in situ reverse transcription. In some embodiments, cells of the sample remain intact during the extension step. In some embodiments, the extension reagents comprise reverse transcription reagents. In some embodiments, reverse transcription reagents comprise a reverse transcriptase and dNTPs.
  • the reverse transcriptase comprises a viral reverse transcriptase.
  • the viral reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase or a Moloney murine leukemia virus (MMLV) reverse transcriptase.
  • MLV murine leukemia virus
  • MMLV Moloney murine leukemia virus
  • the sample is physically divided or is intact during the contacting step.
  • the sample comprises a single cell.
  • the sample comprises a plurality of single cells.
  • the sample comprises a plurality of cells, and optionally the method comprises: disassociating the sample to generate a plurality of single cells, optionally said disassociating comprises chemical dissociating, enzy matic dissociating, and/or mechanical dissociating, optionally said disassociating employs one or more of collagenase, chymotrypsin, dispase, elastase, hyaluronidase, pancreatin, papain, and trypsin.
  • the method can comprise: prior to the barcoding step: partitioning the plurality of single cells to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of single cells; and in the partition comprising the single cell, contacting the extended probing oligonucleotide with the plurality of oligonucleotide barcodes.
  • a partition of the plurality of partitions comprises a single cell from the plurality of single cells
  • contacting the extended probing oligonucleotide with the plurality of oligonucleotide barcodes in the partition comprising the single cell, contacting the single cell with a lysis buffer at 15-65 °C to lyse the single cell.
  • the lysis buffer comprises an agent capable of dissociating protein-nucleic acid complexes.
  • each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a first universal sequence.
  • obtaining sequencing data comprises: amplifying the plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or complement thereof, and amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, thereby generating a plurality of amplified barcoded probing oligonucleotides, wherein obtaining sequencing data comprises obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides, or products thereof.
  • obtaining sequencing data comprises attaching the binding sites of sequencing primers and/or sequencing adaptors to the plurality of barcoded probing oligonucleotides, or products thereof.
  • the amplification primer(s) comprise a second universal sequence and/or wherein the first primer comprises a third universal sequence.
  • the first universal sequence, the second universal sequence, and/or the third universal sequence are the same.
  • the first universal sequence, the second universal sequence, and/or the third universal sequence are different.
  • the first universal sequence, the second universal sequence, and/or the third universal sequence comprise the binding sites of sequencing primers and/or sequencing adaptors, complementary' sequences thereof, and/or portions thereof.
  • the sequencing adaptors comprise a P5 sequence, a P7 sequence, complementary sequences thereof, and/or portions thereof.
  • the sequencing primers comprise a Read 1 sequencing primer, a Read 2 sequencing primer, complementary sequences thereof, and/or portions thereof.
  • the sample comprises a plurality of nucleic acid targets, such as, for example, a target panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350. about 375, about 400, about 425, about 450, about 475, or about 500, distinct nucleic acid targets.
  • two or more nucleic acid targets of the target panel are biomarkers.
  • the biomarkers are biomarkers for a disease or condition.
  • the disease or condition is a cancer, an infection, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a bacterial infection, or any combination thereof.
  • the contacting step comprises contacting the sample with a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality comprises a probe sequence configured hybridize a nucleic acid target of the plurality of nucleic acid targets.
  • determining the copy number of the nucleic acid target in the sample comprises determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of molecular labels with distinct sequences associated with the plurality of barcoded probing oligonucleotides, or products thereof, comprising a sequence of the each of the plurality of nucleic acid targets.
  • the method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with each of the plurality of nucleic acid targets to determine the copy number of each of the plurality of nucleic acid targets at each spatial location of the sample.
  • the amplification primer(s) comprise a panel of amplification primers configured to hybridize the plurality of nucleic acid targets, or complements thereof, such as, for example, a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80. about 90, about 100. about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500, distinct amplification primers.
  • the nucleic acid target comprises a nucleic acid molecule.
  • the nucleic acid molecule comprises ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation product, RNA comprising a poly(A) tail, a sample indexing oligonucleotide, a cellular component-binding reagent specific oligonucleotide, or any combination thereof.
  • the plurality of cells comprise one or more cell types.
  • said one or more cell types are selected from the group consisting of: brain cells, heart cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, and circulatory cells, or any combination thereof.
  • the sample comprises a biological sample, a clinical sample, an environmental sample, a biological fluid, a tissue, a tissue section derived from a subject, or any combination thereof.
  • the subject is a human, a mouse, a dog, a rat. or a vertebrate.
  • the method can comprise: determining genotype, phenotype, or one or more genetic mutations of the subject based on the spatial location of the nucleic acid targets in the sample.
  • the method can comprise: predicting susceptibility of the subject to one or more diseases, such as, for example, a cancer or a hereditary disease.
  • the method can comprise: determining cell types of the plurality of cells in the sample.
  • a drug is chosen based on predicted responsiveness of the cell types of the plurality of cells in the sample.
  • the method can comprise: imaging the sample, optionally imaging the sample before the contacting step and/or after the contacting step, optionally the imaging generates imaging data.
  • imaging the sample comprises staining the sample with a stain, wherein the stain is a fluorescent stain, a negative stain, an antibody stain, or any combination thereof.
  • staining comprises Immunocytochemistry (ICC), Immunohistochemistry (IHC), Immunofluorescence (IF), or any combination thereof.
  • imaging comprises microscopy, confocal microscopy, time-lapse imaging microscopy, fluorescence microscopy, multi-photon microscopy, quantitative phase microscopy, surface enhanced Raman spectroscopy, videography, manual visual analysis, automated visual analysis, or any combination thereof.
  • the method can comprise: associating the imaging data and sequencing data of one or more spatial locations of the sample.
  • the method can comprise: correlation analysis of the imaging data and the sequencing data of the spatial locations.
  • the correlation analysis identifies one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of therapeutic agents, and/or cellular targets of candidate therapeutic agents.
  • said imaging produces an image that is used to construct a map of a physical representation of said sample.
  • said map is two dimensional or three dimensional.
  • the method can comprise: mapping the nucleic acid targets and/or cellular component targets onto the map of the sample.
  • the method can comprise: mapping one or more single cells of the plurality of cells onto the map of the sample.
  • the sample has been contacted with one or more fixing agents and/or permeabilizing agents.
  • the sample comprises a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof.
  • the sample comprises a fresh tissue section, a frozen tissue section, a fixed tissue section, a formalin-fixed tissue section, a formalin-fixed paraffin-embedded (FFPE) tissue section, an acetone fixed tissue section, a paraformaldehyde (PF A) fixed tissue section, and/or a methanol fixed tissue section.
  • FFPE formalin-fixed paraffin-embedded
  • the sample comprises a nuclei suspension, such as, for example, a fixed nuclei suspension and/or a permeabilized nuclei suspension.
  • the sample comprises cell(s), such as, for example, fresh cell(s), frozen cell(s), fixed cell(s), formalin-fixed cell(s), formalin-fixed paraffin-embedded (FFPE) cell(s), acetone fixed cell(s), a paraformaldehyde (PF A) fixed cell(s), and/or a methanol fixed cell(s).
  • the method can comprise: permeabilizing the sample and/or fixing the sample.
  • fixing the sample comprises contacting the sample with a fixing agent.
  • the fixing agent comprises a non-cross-linking fixative (e.g., methanol). In some embodiments, the fixing agent comprises a cross-linking agent. In some embodiments, the crosslinking agent comprises a cleavable cross-linking agent.
  • the cleavable cross-linking agent comprises or is derived from dithiobis(succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), Bis
  • DSP dithiobis(succinimidyl propionate)
  • DST disuccinimidy
  • the cleavable cross-linking agent comprises a cleavable linkage selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, an acid labile linker, a heat sensitive linkage, an enzymatically cleavable linkage, and a combination thereof.
  • the cleavable cross-linking agent is a thiol-cleavable cross-linking agent or comprises a disulfide linker.
  • the fixing agent comprises paraformaldehyde (PFA), dithiobis(succinimidyl propionate (DSP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), CellCover, or a combination thereof.
  • PFA paraformaldehyde
  • DSPDP dithiobis(succinimidyl propionate
  • SPDP succinimidyl 3-(2-pyridyldithio)propionate
  • CellCover or a combination thereof.
  • fixing the sample and permeabilizing the sample are carried out simultaneously.
  • fixing and permeabilizing the sample is carried out in the presence of a dual function agent capable of fixing and permeabilizing the sample.
  • the dual functional agent is methanol.
  • permeabilizing the sample comprises contacting the sample with a permeabilizing agent.
  • the method can comprise: after contacting the plurality of probing oligonucleotides or the plurality of cellular component-binding reagents with the sample, removing the permeabilizing agent from the sample.
  • the permeabilizing agent is capable of (i) permeabilizing the cell membrane of the cell(s), (ii) making a cell membrane of the cell(s) permeable to the probing oligonucleotides or the cellular component-binding reagents, or both.
  • the permeabilizing agent comprises (i) a solvent, a detergent, or a surfactant; (ii) BD Cytoperm; (iii) a saponin or a derivative thereof; (iv) Triton X-100, (v) methanol or a derivative thereof, and/or (vi) digitonin or a derivative thereof.
  • the agent capable of dissociating protein-nucleic acid complexes comprises a broad-spectrum serine protease.
  • the broadspectrum serine protease is proteinase K.
  • the lysis buffer comprises an unfixing agent.
  • the unfixing agent comprises a thiol, hydoxylamine, periodate, a base, or any combination thereof.
  • the lysis buffer comprises DTT.
  • the method can comprise: reversing the fixation of the sample and/or single cells. In some embodiments, reversing the fixation of the sample and/or single cells comprises UV photocleaving, chemical treatment, heating, enzy me treatment, or any combination thereof.
  • the sample can comprise a plurality of cellular component targets, and the method further comprises: contacting a plurality of cellular component-binding reagents with the sample, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular componentbinding reagent is capable of specifically binding to at least one of the plurality of cellular component targets; barcoding the cellular component-binding reagent specific oligonucleotides to generate a plurality of barcoded cellular component-binding reagent specific oligonucleotides each comprising a sequence complementary' to at least a portion of the unique identifier sequence and a molecular label sequence: and obtaining sequencing data comprising a plurality’ of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleo
  • the method can comprise: after contacting the plurality 7 of cellular component-binding reagents with the sample, removing one or more cellular component-binding reagents of the plurality of cellular component-binding reagents that are not contacted with the sample, optionally removing the one or more cellular component-binding reagents not contacted with the sample comprises: removing the one or more cellular component-binding reagents not contacted with the respective at least one of the plurality of cellular component targets.
  • the cellular component target comprises an intracellular protein, a carbohydrate, a lipid, a protein, an extracellular protein, a cell-surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.
  • the cellular component-binding reagent specific oligonucleotide comprises a second molecular label, optionally at least ten of the plurality of cellular component-binding reagent specific oligonucleotides comprise different second molecular label sequences.
  • the second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides are different, and wherein the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides are identical. In some embodiments, the second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides are different, and wherein the unique identifier sequences of the at least two cellular componentbinding reagent specific oligonucleotides are different.
  • the number of unique molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample. In some embodiments, the number of unique second molecular label sequences associated with the unique identifier sequence for the cellular componentbinding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample.
  • the method can comprise: prior to contacting a plurality of cellular component-binding reagents with the sample and/or contacting the plurality of probing oligonucleotides with the sample, contacting the sample with a blocking reagent, one or more decoy oligonucleotides, and/or one or more blocking oligonucleotides.
  • contacting a plurality of cellular component-binding reagents with the sample is conducted in the presence of a blocking reagent.
  • the blocking reagent comprises a plurality of oligonucleotides complementary to at least a portion of the cellular componentbinding reagent specific oligonucleotides.
  • the blocking reagent comprises an antibody or a fragment thereof derived from a first species, and wherein the blocking reagent comprises sera derived from the first species.
  • the sample comprises one or more non-target nucleic acids, wherein the blocking reagent comprises a plurality’ of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acids.
  • each of the plurality of decoy oligonucleotides are capable of hybridizing to at least a portion of a non-target nucleic acid.
  • the decoy oligonucleotide comprises a sequence complementary to at least a portion of a non-target nucleic acid; comprises a sequence identical to or substantially similar to a sequence of the cellular component-binding reagents specific oligonucleotides, optionally the sequence is 3-40 nucleotides in length; has at most 50% sequence identity to the cellular component-binding reagent specific oligonucleotides; does not comprise a UMI; comprises a random sequence, and optionally the random sequence is about four, five, six, seven, eight, nine, ten.
  • eleven, twelve, thirteen, fourteen, or fifteen nucleotides in length does not comprise any sequence having more than four, five, six, or seven consecutive Ts or As; comprise at least one G or C in every four, five, six. or seven consecutive nucleotides; comprises one or more modified nucleotides; comprises a 5’ modification, and optionally the 5 ? modification comprises a 5' Amino Modifier C12 modification (5AmMC12); comprises a 3’ modification, and optionally the 3’ modification comprises a 3' dideoxy-C modification (ddC); and/or is 30 to 65 nucleotides in length.
  • 5AmMC12 5' Amino Modifier C12 modification
  • ddC dideoxy-C modification
  • the sample comprises one or more undesirable nucleic acid species
  • the method comprising: contacting a blocking oligonucleotide with the sample, wherein the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleic acid species; whereby the reverse transcription of the at least one of the one or more undesirable nucleic acid species is reduced by the blocking oligonucleotide.
  • the blocking oligonucleotide is contacted with the sample before the plurality of probing oligonucleotides is contacted with the sample; is contacted with the sample after the plurality of probing oligonucleotides is contacted with the sample; and/or is contacted with the sample when the plurality of probing oligonucleotides is contacted with the sample.
  • the method can comprise: providing blocking oligonucleotides that specifically bind to two or more undesirable nucleic acid species, optionally at least 10 or to at least 100 undesirable nucleic acid species, in the sample.
  • the blocking oligonucleotide is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, or a PNA/DNA chimera; specifically binds to within 100 nt, to within 50 nt, or to within 25 nt of the 3’ end of the one or more undesirable nucleic acid species; specifically binds to within 100 nt of the 5‘ end of the one or more undesirable nucleic acid species, or the blocking oligonucleotide specifically binds to within 100 nt of the middle of the one or more undesirable nucleic acid species; comprises or does not comprise non-natural nucleotides; has a Tm of at least 50 °C, of at least 60 °C, or of at least 70 °C; is unable to function as a primer for a reverse transcriptase or a polymerase; and/or is 8 n
  • the one or more undesirable nucleic acid species amounts to about 50%, to about 60%, to about 70%, or to about 80% of the nucleic acid content of the sample.
  • the undesirable nucleic acid species is selected from the group consisting of ribosomal RNA, mitochondrial RNA, genomic DNA, intronic sequence, high abundance sequence, and a combination thereof.
  • the one or more undesirable nucleic acid species are mRNA molecules and the blocking oligonucleotide specific binds to within 10 nt of the 3’ poly (A) tail of the one or more undesirable nucleic acid species.
  • each molecular label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides.
  • each capture sequence of the plurality of oligonucleotide barcodes comprises at least 4 nucleotides.
  • the plurality of oligonucleotide barcodes are associated with a solid support, and wherein a partition of the plurality of partitions comprises a single solid support.
  • the plurality of oligonucleotide barcodes each comprise a cell label.
  • each cell label of the plurality' of oligonucleotide barcodes comprises at least 6 nucleotides.
  • oligonucleotide barcodes of the plurality' of oligonucleotide barcodes associated with the same solid support comprise the same cell label. In some embodiments, oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with different solid supports comprise different cell labels. In some embodiments, the solid support comprises a synthetic particle, a planar surface, or a combination thereof. The method can comprise: associating a synthetic particle comprising the plurality of oligonucleotide barcodes with the cell in the partition. The method can comprise: lysing the cell after associating the synthetic particle with the cell.
  • lysing the cell comprises heating the cell, contacting the cell with a detergent, changing the pH of the cell, or any combination thereof.
  • the synthetic particle and the single cell are in the same partition, and optionally the partition is a well or a droplet.
  • at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is immobilized or partially immobilized on the synthetic particle, or at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is enclosed or partially enclosed in the synthetic particle.
  • the synthetic particle is disruptable (e.g., a disruptable hydrogel particle).
  • the synthetic particle comprises a bead.
  • the bead comprises a Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A/G conjugated bead, a protein L conjugated bead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or any combination thereof.
  • the synthetic particle comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof.
  • PDMS polydimethylsiloxane
  • polystyrene polystyrene
  • glass polypropylene
  • the support functional group and the linker functional group are associated with each other, and optionally the linker functional group and the support functional group are individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), and any combination thereof.
  • compositions e.g., kits.
  • the kit comprises: a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize a nucleic acid target, optionally the probing oligonucleotides comprise a predetermined spatial label; a coupling oligonucleotide comprising a 5’ complement of the coupling sequence and a 3’ complement of a capture sequence; a plurality of oligonucleotide barcodes, wherein the 3‘ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes is associated with a solid support, wherein the 5’ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a capture sequence; a first primer capable of hybridizing to the first universal sequence, optionally further
  • the plurality of probing oligonucleotides comprises a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality comprises a probe sequence configured hybridize a nucleic acid target of a plurality of nucleic acid targets, optionally a target panel of at least about 2, about 3. about 4, about 5, about 6. about 7, about 8, about 9, about 10, about 12.
  • nucleic acid targets about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500. distinct nucleic acid targets.
  • the amplification primer(s) comprise a panel of amplification primers configured to hybridize a plurality of nucleic acid targets, or complements thereof, optionally a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90. about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325. about 350, about 375, about 400, about 425, about 450, about 475, or about 500, distinct amplification primers.
  • FIG. 1 illustrates a non-limiting exemplary barcode.
  • FIG. 2 shows a non-limiting exemplary’ workflow of barcoding and digital counting.
  • FIG. 3 is a schematic illustration showing a non-limiting exemplary process for generating an indexed library of targets barcoded at the 3’-ends from a plurality' of targets.
  • FIGS. 4A-4D depict a non-limiting exemplary' schematic workflow for gene expression analysis of fixed cells.
  • mRNA messenger ribonucleotide acid
  • PCR digital polymerase chain reaction
  • PCR can have disadvantages such that each molecule replicates with a stochastic probability, and this probability varies by PCR cycle and gene sequence, resulting in amplification bias and inaccurate gene expression measurements.
  • Stochastic barcodes with unique molecular labels also referred to as molecular indexes (Mis)
  • Molecular indexes can be used to count the number of molecules and correct for amplification bias.
  • Stochastic barcoding such as the PreciseTM assay (Cellular Research, Inc.
  • the PreciseTM assay can utilize a non-depleting pool of stochastic barcodes with large number, for example 6561 to 65536, unique molecular label sequences on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs in a sample during the RT step.
  • a stochastic barcode can comprise a universal PCR priming site.
  • target gene molecules react randomly with stochastic barcodes. Each target molecule can hybridize to a stochastic barcode resulting to generate stochastically barcoded complementary' ribonucleotide acid (cDNA) molecules).
  • stochastically barcoded cDNA molecules from microwells of a microwell plate can be pooled into a single tube for PCR amplification and sequencing.
  • Raw sequencing data can be analyzed to produce the number of reads, the number of stochastic barcodes with unique molecular label sequences, and the numbers of mRNA molecules.
  • the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality' of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target.
  • the method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.
  • the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality 7 of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality’ of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target.
  • the method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality’ of barcoded probing oligonucleotides, or products thereof.
  • the method comprises: contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and a predetermined spatial label.
  • probing oligonucleotides contacted with the same spatial location comprise the same spatial label sequence, and wherein probing oligonucleotides contacted with distinct spatial locations of the sample comprise different spatial label sequences.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality’ of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides.
  • each oligonucleotide barcode of the plurality 7 of oligonucleotide barcodes comprises a molecular label
  • each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality 7 of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality 7 of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target.
  • the method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a nucleic acid target to determine the copy number of the nucleic acid target at each spatial location of the sample.
  • the term “adaptor” can mean a sequence to facilitate amplification or sequencing of associated nucleic acids.
  • the associated nucleic acids can comprise target nucleic acids.
  • the associated nucleic acids can comprise one or more of spatial labels, target labels, sample labels, indexing label, or barcode sequences (e.g., molecular labels).
  • the adaptors can be linear.
  • the adaptors can be pre-adenylated adaptors.
  • the adaptors can be double- or single-stranded.
  • One or more adaptor can be located on the 5’ or 3’ end of a nucleic acid. When the adaptors comprise known sequences on the 5’ and 3‘ ends, the known sequences can be the same or different sequences.
  • An adaptor located on the 5’ and/or 3’ ends of a polynucleotide can be capable of hybridizing to one or more oligonucleotides immobilized on a surface.
  • An adaptor can, in some embodiments, comprise a universal sequence.
  • a universal sequence can be a region of nucleotide sequence that is common to tw o or more nucleic acid molecules. The two or more nucleic acid molecules can also have regions of different sequence.
  • the 5’ adaptors can comprise identical and/or universal nucleic acid sequences and the 3’ adaptors can comprise identical and/or universal sequences.
  • a universal sequence that may be present in different members of a plurality 7 of nucleic acid molecules can allow the replication or amplification of multiple different sequences using a single universal primer that is complementary to the universal sequence.
  • at least one, two (e.g., a pair) or more universal sequences that may be present in different members of a collection of nucleic acid molecules can allow the replication or amplification of multiple different sequences using at least one, two (e.g.. a pair) or more single universal primers that are complementary to the universal sequences.
  • a universal primer includes a sequence that can hybridize to such a universal sequence.
  • the target nucleic acid sequence-bearing molecules may be modified to attach universal adaptors (e.g., non-target nucleic acid sequences) to one or both ends of the different target nucleic acid sequences.
  • the one or more universal primers attached to the target nucleic acid can provide sites for hybridization of universal primers.
  • the one or more universal primers attached to the target nucleic acid can be the same or different from each other.
  • association can mean that two or more species are identifiable as being co-located at a point in time.
  • An association can mean that two or more species are or were within a similar container.
  • An association can be an informatics association. For example, digital information regarding two or more species can be stored and can be used to determine that one or more of the species were co-located at a point in time.
  • An association can also be a physical association.
  • two or more associated species are “tethered”, “attached”, or “immobilized” to one another or to a common solid or semisolid surface.
  • An association may refer to covalent or non-covalent means for attaching labels to solid or semi-solid supports such as beads.
  • An association may be a covalent bond between a target and a label.
  • An association can comprise hybridization between two molecules (such as a target molecule and a label).
  • the term “complementary” can refer to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial.” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules.
  • a first nucleotide sequence can be said to be the “complement” of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence.
  • a first nucleotide sequence can be said to be the “reverse complement” of a second sequence, if the first nucleotide sequence is complementary to a sequence that is the reverse (i.e., the order of the nucleotides is reversed) of the second sequence.
  • a “complementary” sequence can refer to a “complement” or a “reverse complement” of a sequence. It is understood from the disclosure that if a molecule can hybridize to another molecule it may be complementary, or partially complementary’, to the molecule that is hybridizing.
  • digital counting can refer to a method for estimating a number of target molecules in a sample.
  • Digital counting can include the step of determining a number of unique labels that have been associated with targets in a sample. This methodology, which can be stochastic in nature, transforms the problem of counting molecules from one of locating and identifying identical molecules to a series of yes/no digital questions regarding detection of a set of predefined labels.
  • label can refer to nucleic acid codes associated with a target within a sample.
  • a label can be, for example, a nucleic acid label.
  • a label can be an entirely or partially amplifiable label.
  • a label can be entirely or partially sequencable label.
  • a label can be a portion of a native nucleic acid that is identifiable as distinct.
  • a label can be a known sequence.
  • a label can comprise a junction of nucleic acid sequences, for example a junction of a native and non-native sequence.
  • label can be used interchangeably with the terms, “index”, “tag,” or “label-tag.” Labels can convey information. For example, in various embodiments, labels can be used to determine an identity of a sample, a source of a sample, an identity of a cell, and/or a target.
  • non-depleting reservoirs can refer to a pool of barcodes (e.g., stochastic barcodes) made up of many different labels.
  • a non-depleting reservoir can comprise large numbers of different barcodes such that when the non-depleting reservoir is associated with a pool of targets each target is likely to be associated with a unique barcode.
  • the uniqueness of each labeled target molecule can be determined by the statistics of random choice, and depends on the number of copies of identical target molecules in the collection compared to the diversity of labels.
  • the size of the resulting set of labeled target molecules can be determined by the stochastic nature of the barcoding process, and analysis of the number of barcodes detected then allows calculation of the number of target molecules present in the original collection or sample.
  • the labeled target molecules are highly unique (i.e., there is a very low probability' that more than one target molecule will have been labeled with a given label).
  • nucleic acid refers to a polynucleotide sequence, or fragment thereof.
  • a nucleic acid can comprise nucleotides.
  • a nucleic acid can be exogenous or endogenous to a cell.
  • a nucleic acid can exist in a cell-free environment.
  • a nucleic acid can be a gene or fragment thereof.
  • a nucleic acid can be DNA.
  • a nucleic acid can be RNA.
  • a nucleic acid can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase).
  • analogs include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine. and wyosine.
  • Nucleic acid ; “polynucleotide, “target polynucleotide”, and “target nucleic acid” can be used interchangeably.
  • a nucleic acid can comprise one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g.. improved stability).
  • a nucleic acid can comprise a nucleic acid affinity tag.
  • a nucleoside can be a base-sugar combination. The base portion of the nucleoside can be a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides can be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to the 2’, the 3’, or the 5’ hydroxyl moiety of the sugar.
  • the phosphate groups can covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable.
  • linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound.
  • the phosphate groups can commonly be referred to as forming the intemucleoside backbone of the nucleic acid.
  • the linkage or backbone can be a 3’ to 5’ phosphodiester linkage.
  • a nucleic acid can comprise a modified backbone and/or modified intemucleoside linkages.
  • Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Suitable modified nucleic acid backbones containing a phosphorus atom therein can include, for example, phosphorothioates. chiral phosphorothioates, phosphorodithioates.
  • phosphotri esters aminoalkyl phosphotriesters, methyl and other alkyl phosphonate such as 3’-alkylene phosphonates, 5’- alkylene phosphonates, chiral phosphonates, phosphinates, phosphorami dates including 3’- amino phosphoramidate and aminoalkyl phosphoramidates. phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates.
  • thionoalkylphosphotriesters having normal 3 ’-5’ linkages, 2’ -5’ linked analogs, and those having inverted polarity wherein one or more intemucleotide linkages is a 3’ to 3’, a 5’ to 5’ or a 2’ to 2’ linkage.
  • a nucleic acid can comprise polynucleotide backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • These can include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
  • siloxane backbones siloxane backbones
  • sulfide, sulfoxide and sulfone backbones formacetyl and thioformacetyl backbones
  • a nucleic acid can comprise a nucleic acid mimetic.
  • the term “‘mimetic’ 7 can be intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the intemucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring can also be referred as being a sugar surrogate.
  • the heterocyclic base moiety or a modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid.
  • One such nucleic acid can be a peptide nucleic acid (PNA).
  • the sugar-backbone of a polynucleotide can be replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleotides can be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • the backbone in PNA compounds can comprise two or more linked aminoethylglycine units which gives PNA an amide containing backbone.
  • the heterocyclic base moieties can be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • a nucleic acid can comprise a morpholino backbone structure.
  • a nucleic acid can comprise a 6-membered morpholino ring in place of a ribose ring.
  • a phosphorodiamidate or other non-phosphodiester intemucleoside linkage can replace a phosphodiester linkage.
  • a nucleic acid can comprise linked morpholino units (e.g., morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring.
  • Linking groups can link the morpholino monomeric units in a morpholino nucleic acid.
  • Non-ionic morpholino-based oligomeric compounds can have less undesired interactions with cellular proteins.
  • Morpholinobased polynucleotides can be nonionic mimics of nucleic acids.
  • a variety of compounds within the morpholino class can be joined using different linking groups.
  • a further class of polynucleotide mimetic can be referred to as cyclohexenyl nucleic acids (CeNA).
  • the furanose ring normally present in a nucleic acid molecule can be replaced with a cyclohexenyl ring.
  • CeNA DMT protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry.
  • the incorporation of CeNA monomers into a nucleic acid chain can increase the stability of a DNA/RNA hybrid.
  • CeNA oligoadenylates can form complexes with nucleic acid complements with similar stability to the native complexes.
  • a further modification can include Locked Nucleic Acids (LNAs) in which the 2’-hydroxyl group is linked to the 4’ carbon atom of the sugar ring thereby forming a 2’-C, 4’-C-oxymethylene linkage thereby forming a bicyclic sugar moiety.
  • the linkage can be a methylene (-CH2). group bridging the 2 ? oxygen atom and the 4’ carbon atom wherein n is 1 or 2.
  • a nucleic acid may also include nucleobase (often referred to simply as “base”) modifications or substitutions.
  • nucleobases can include the purine bases, (e.g., adenine (A) and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine (C) and uracil (U)).
  • Modified nucleobases can include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin- 2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G- clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-amin
  • sample can refer to a composition comprising targets.
  • Suitable samples for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms.
  • sampling device can refer to a device which may take a section of a sample and/or place the section on a substrate.
  • a sample device can refer to, for example, a fluorescence activated cell sorting (FACS) machine, a cell sorter machine, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and/or a microtome.
  • FACS fluorescence activated cell sorting
  • solid support can refer to discrete solid or semisolid surfaces to which a plurality of barcodes (e.g., stochastic barcodes) may be attached.
  • a solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently).
  • a solid support may comprise a discrete particle that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like.
  • a bead can be non-spherical in shape.
  • a plurality of solid supports spaced in an array may not comprise a substrate.
  • a solid support may be used interchangeably with the term “bead.”
  • stochastic barcode can refer to a polynucleotide sequence comprising labels of the present disclosure.
  • a stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcoding.
  • Stochastic barcodes can be used to quantify targets within a sample.
  • Stochastic barcodes can be used to control for errors which may occur after a label is associated with a target.
  • a stochastic barcode can be used to assess amplification or sequencing errors.
  • a stochastic barcode associated with a target can be called a stochastic barcode-target or stochastic barcode-tag-target.
  • the term “gene-specific stochastic barcode” can refer to a polynucleotide sequence comprising labels and a target-binding region that is gene-specific.
  • a stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcoding.
  • Stochastic barcodes can be used to quantify targets within a sample.
  • Stochastic barcodes can be used to control for errors which may occur after a label is associated with a target.
  • a stochastic barcode can be used to assess amplification or sequencing errors.
  • a stochastic barcode associated with a target can be called a stochastic barcode-target or stochastic barcode- tag-target.
  • the term “stochastic barcoding” can refer to the random labeling (e.g., barcoding) of nucleic acids. Stochastic barcoding can utilize a recursive Poisson strategy to associate and quantify labels associated with targets. As used herein, the term “stochastic barcoding” can be used interchangeably with “stochastic labeling.”
  • target can refer to a composition which can be associated with a barcode (e.g., a stochastic barcode).
  • exemplary targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA. and the like. Targets can be single or double stranded.
  • targets can be proteins, peptides, or polypeptides.
  • targets are lipids.
  • target can be used interchangeably with “species.”
  • reverse transcriptases can refer to a group of enzymes having reverse transcriptase activity (i.e., that catalyze synthesis of DNA from an RNA template).
  • enzymes include, but are not limited to. retroviral reverse transcriptase, retrotransposon reverse transcriptase, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptase, and mutants, variants or derivatives thereof.
  • Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transciptases, and group II intron reverse transcriptases.
  • group II intron reverse transcriptases examples include the Lactococcus lactis LI.LtrB intron reverse transcriptase, the Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or the Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase.
  • Other classes of reverse transcriptases can include many classes of non-retroviral reverse transcriptases (i.e., retrons, group II introns, and diversity -generating retroelements among others).
  • universal adaptor primer refers to a nucleotide sequence that can be used to hybridize to barcodes (e.g., stochastic barcodes) to generate gene-specific barcodes.
  • a universal adaptor sequence can, for example, be a known sequence that is universal across all barcodes used in methods of the disclosure. For example, when multiple targets are being labeled using the methods disclosed herein, each of the target-specific sequences may be linked to the same universal adaptor sequence. In some embodiments, more than one universal adaptor sequences may be used in the methods disclosed herein.
  • a universal adaptor primer and its complement may be included in two oligonucleotides, one of which comprises a target-specific sequence and the other comprises a barcode.
  • a universal adaptor sequence may be part of an oligonucleotide comprising a target-specific sequence to generate a nucleotide sequence that is complementary to a target nucleic acid.
  • a second oligonucleotide comprising a barcode and a complementary sequence of the universal adaptor sequence may hybridize with the nucleotide sequence and generate a target-specific barcode (e.g.. a target-specific stochastic barcode).
  • a universal adaptor primer has a sequence that is different from a universal PCR primer used in the methods of this disclosure.
  • Barcodes such as stochastic barcoding, has been described in, for example, Fu et al., Proc Natl Acad Sci U.S.A., 2011 May 31,108(22):9026-31; U.S. Patent Application Publication No. US2011/0160078; Fan et al., Science, 2015 February 6, 347(6222): 1258367; US Patent Application Publication No. US2015/0299784; and PCT Application Publication No. W02015/031691; the content of each of these, including any supporting or supplemental information or material, is incorporated herein by reference in its entirety'.
  • the barcode disclosed herein can be a stochastic barcode which can be a polynucleotide sequence that may be used to stochastically label (e.g., barcode, tag) a target.
  • Barcodes can be referred to stochastic barcodes if the ratio of the number of different barcode sequences of the stochastic barcodes and the number of occurrence of any of the targets to be labeled can be, or be about, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, or a number or a range between any two of these values.
  • a target can be an mRNA species comprising mRNA molecules with identical or nearly identical sequences.
  • Barcodes can be referred to as stochastic barcodes if the ratio of the number of different barcode sequences of the stochastic barcodes and the number of occurrence of any of the targets to be labeled is at least, or is at most, 1 :1, 2: 1, 3: 1, 4: 1, 5:1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15:1. 16:1. 17: 1, 18: 1, 19: 1, 20: 1, 30: 1. 40: 1. 50: 1. 60: 1. 70: 1. 80: 1. 90: 1. or 100: 1. Barcode sequences of stochastic barcodes can be referred to as molecular labels.
  • a barcode for example a stochastic barcode, can comprise one or more labels.
  • Exemplary labels can include a universal label, a cell label, a barcode sequence (e.g., a molecular label), a sample label, a plate label, a spatial label, and/or a pre-spatial label.
  • FIG. 1 illustrates an exemplary barcode 104 with a spatial label.
  • the barcode 104 can comprise a 5 ’amine that may link the barcode to a solid support 105.
  • the barcode can comprise a universal label, a dimension label, a spatial label, a cell label, and/or a molecular label.
  • the order of different labels (including but not limited to the universal label, the dimension label, the spatial label, the cell label, and the molecule label) in the barcode can vary.
  • the universal label may be the 5’-most label
  • the molecular label may be the 3’-most label.
  • the spatial label, dimension label, and the cell label may be in any order.
  • the universal label, the spatial label, the dimension label, the cell label, and the molecular label are in any order.
  • the barcode can comprise a target-binding region.
  • the targetbinding region can interact with a target (e.g., target nucleic acid, RNA, mRNA, DNA) in a sample.
  • a target-binding region can comprise an oligo(dT) sequence which can interact with poly(A) tails of mRNAs.
  • the labels of the barcode e.g., universal label, dimension label, spatial label, cell label, and barcode sequence
  • the labels of the barcode may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides.
  • a label for example the cell label, can comprise a unique set of nucleic acid sub-sequences of defined length, e.g.. seven nucleotides each (equivalent to the number of bits used in some Hamming error correction codes), which can be designed to provide error correction capability.
  • the set of error correction sub-sequences comprise seven nucleotide sequences can be designed such that any pairwise combination of sequences in the set exhibits a defined ‘‘genetic distance” (or number of mismatched bases), for example, a set of error correction sub-sequences can be designed to exhibit a genetic distance of three nucleotides.
  • the length of the nucleic acid subsequences used for creating error correction codes can vary’, for example, they can be. or be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50, or a number or a range between any two of these values, nucleotides in length.
  • nucleic acid sub-sequences of other lengths can be used for creating error correction codes.
  • the barcode can comprise a target-binding region.
  • the target-binding region can interact with a target in a sample.
  • the target can be, or comprise, ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs, small interfering RNAs (siRNAs), RNA degradation products, RNAs each comprising a poly(A) tail, or any combination thereof.
  • RNAs ribonucleic acids
  • mRNAs messenger RNAs
  • microRNAs microRNAs
  • siRNAs small interfering RNAs
  • RNA degradation products RNAs each comprising a poly(A) tail, or any combination thereof.
  • the plurality of targets can include deoxyribonucleic acids (DNAs).
  • a target-binding region can comprise an oligo(dT) sequence which can interact with poly(A) tails of mRNAs.
  • One or more of the labels of the barcode e.g., the universal label, the dimension label, the spatial label, the cell label, and the barcode sequences (e.g., molecular label)
  • the spacer can be, for example, 1, 2, 3. 4, 5, 6, 7, 8, 9, 10. 11. 12, 13, 14, 15, 16, 17, 18, 19, or 20. or more nucleotides.
  • none of the labels of the barcode is separated by spacer.
  • a barcode can comprise one or more universal labels.
  • the one or more universal labels can be the same for all barcodes in the set of barcodes attached to a given solid support.
  • the one or more universal labels can be the same for all barcodes attached to a plurality of beads.
  • a universal label can comprise a nucleic acid sequence that is capable of hybridizing to a sequencing primer.
  • Sequencing primers can be used for sequencing barcodes comprising a universal label.
  • Sequencing primers e.g., universal sequencing primers
  • a universal label can comprise a nucleic acid sequence that is capable of hybridizing to a PCR primer.
  • the universal label can comprise a nucleic acid sequence that is capable of hybridizing to a sequencing primer and a PCR primer.
  • the nucleic acid sequence of the universal label that is capable of hybridizing to a sequencing or PCR primer can be referred to as a primer binding site.
  • a universal label can comprise a sequence that can be used to initiate transcription of the barcode.
  • a universal label can comprise a sequence that can be used for extension of the barcode or a region within the barcode.
  • a universal label can be, or be about. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length.
  • a universal label can comprise at least about 10 nucleotides.
  • a universal label can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.
  • a cleavable linker or modified nucleotide can be part of the universal label sequence to enable the barcode to be cleaved off from the support.
  • a barcode can comprise one or more dimension labels.
  • a dimension label can comprise a nucleic acid sequence that provides information about a dimension in which the labeling (e.g., stochastic labeling) occurred.
  • a dimension label can provide information about the time at which a target was barcoded.
  • a dimension label can be associated with a time of barcoding (e.g., stochastic barcoding) in a sample.
  • a dimension label can be activated at the time of labeling. Different dimension labels can be activated at different times.
  • the dimension label provides information about the order in which targets, groups of targets, and/or samples were barcoded. For example, a population of cells can be barcoded at the GO phase of the cell cycle.
  • the cells can be pulsed again with barcodes (e.g., stochastic barcodes) at the G1 phase of the cell cycle.
  • the cells can be pulsed again with barcodes at the S phase of the cell cycle, and so on.
  • Barcodes at each pulse e.g., each phase of the cell cycle
  • the dimension label provides information about which targets were labelled at which phase of the cell cycle.
  • Dimension labels can interrogate many different biological times. Exemplary' biological times can include, but are not limited to. the cell cycle, transcription (e.g., transcription initiation), and transcript degradation.
  • a sample e.g., a cell, a population of cells
  • a sample can be labeled before and/or after treatment with a drug and/or therapy.
  • the changes in the number of copies of distinct targets can be indicative of the sample’s response to the drug and/or therapy.
  • a dimension label can be activatable.
  • An activatable dimension label can be activated at a specific time point.
  • the activatable label can be, for example, constitutively activated (e.g., not turned off).
  • the activatable dimension label can be, for example, reversibly activated (e.g., the activatable dimension label can be turned on and turned off).
  • the dimension label can be, for example, reversibly activatable at least 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, or more times.
  • the dimension label can be reversibly activatable, for example, at least 1. 2, 3. 4, 5, 6. 7, 8, 9., 10 or more times.
  • the dimension label can be activated with fluorescence, light, a chemical event (e.g., cleavage, ligation of another molecule, addition of modifications (e.g., pegylated, sumoylated. acetylated, methylated, deacetylated, demethylated), a photochemical event (e.g., photocaging), and introduction of a non-natural nucleotide.
  • a chemical event e.g., cleavage, ligation of another molecule, addition of modifications (e.g., pegylated, sumoylated. acetylated, methylated, deacetylated, demethylated)
  • a photochemical event e.g., photocaging
  • the dimension label can, in some embodiments, be identical for all barcodes (e.g., stochastic barcodes) attached to a given solid support (e.g., a bead), but different for different solid supports (e.g., beads).
  • at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%, of barcodes on the same solid support can comprise the same dimension label.
  • at least 60% of barcodes on the same solid support can comprise the same dimension label.
  • at least 95% of barcodes on the same solid support can comprise the same dimension label.
  • a dimension label can be. or be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length.
  • a dimension label can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35. 40, 45, 50, 100, 200, or 300, nucleotides in length.
  • a dimension label can comprise between about 5 to about 200 nucleotides.
  • a dimension label can comprise between about 10 to about 150 nucleotides.
  • a dimension label can comprise between about 20 to about 125 nucleotides in length.
  • a barcode can comprise one or more spatial labels.
  • a spatial label can comprise a nucleic acid sequence that provides information about the spatial orientation of a target molecule which is associated with the barcode.
  • a spatial label can be associated with a coordinate in a sample.
  • the coordinate can be a fixed coordinate.
  • a coordinate can be fixed in reference to a substrate.
  • a spatial label can be in reference to a two or three-dimensional grid.
  • a coordinate can be fixed in reference to a landmark.
  • the landmark can be identifiable in space.
  • a landmark can be a structure which can be imaged.
  • a landmark can be a biological structure, for example an anatomical landmark.
  • a landmark can be a cellular landmark, for instance an organelle.
  • a landmark can be a non-natural landmark such as a structure with an identifiable identifier such as a color code, bar code, magnetic property, fluorescents, radioactivity, or a unique size or shape.
  • a spatial label can be associated with a physical partition (e.g., A well, a container, or a droplet). In some embodiments, multiple spatial labels are used together to encode one or more positions in space.
  • the spatial label can be identical for all barcodes attached to a given solid support (e.g., a bead), but different for different solid supports (e.g., beads).
  • the percentage of barcodes on the same solid support comprising the same spatial label can be, or be about, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or a range between any two of these values.
  • the percentage of barcodes on the same solid support comprising the same spatial label can be at least, or be at most. 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%.
  • at least 60% of barcodes on the same solid support can comprise the same spatial label.
  • at least 95% of barcodes on the same solid support can comprise the same spatial label.
  • a spatial label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length.
  • a spatial label can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200. or 300 nucleotides in length.
  • a spatial label can comprise between about 5 to about 200 nucleotides.
  • a spatial label can comprise between about 10 to about 150 nucleotides.
  • a spatial label can comprise between about 20 to about 125 nucleotides in length.
  • a barcode (e.g., a stochastic barcode) can comprise one or more cell labels.
  • a cell label can comprise a nucleic acid sequence that provides information for determining which target nucleic acid originated from which cell.
  • the cell label is identical for all barcodes attached to a given solid support (e.g., a bead), but different for different solid supports (e.g., beads).
  • the percentage of barcodes on the same solid support comprising the same cell label can be, or be about 60%, 70%. 80%. 85%. 90%. 95%. 97%. 99%. 100%, or a number or a range between any two of these values.
  • the percentage of barcodes on the same solid support comprising the same cell label can be, or be about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%.
  • at least 60% of barcodes on the same solid support can comprise the same cell label.
  • at least 95% of barcodes on the same solid support can comprise the same cell label.
  • a cell label can be, or be about, 1. 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length.
  • a cell label can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.
  • a cell label can comprise between about 5 to about 200 nucleotides.
  • a cell label can comprise between about 10 to about 150 nucleotides.
  • a cell label can comprise between about 20 to about 125 nucleotides in length.
  • a barcode can comprise one or more barcode sequences.
  • a barcode sequence can comprise a nucleic acid sequence that provides identifying information for the specific type of target nucleic acid species hybridized to the barcode.
  • a barcode sequence can comprise a nucleic acid sequence that provides a counter (e.g., that provides a rough approximation) for the specific occurrence of the target nucleic acid species hybridized to the barcode (e.g., target-binding region).
  • a diverse set of barcode sequences are attached to a given solid support (e.g., a bead).
  • a given solid support e.g., a bead
  • a plurality 7 of barcodes can comprise about 6561 barcodes sequences with distinct sequences.
  • a plurality 7 of barcodes can comprise about 65536 barcode sequences with distinct sequences.
  • the unique molecular label sequences can be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular label sequence is partially or entirely encompassed by a particle (e.g.. a hydrogel bead).
  • a barcode can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length.
  • a barcode can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15. 20. 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.
  • a barcode (e.g., a stochastic barcode) can comprise one or more molecular labels.
  • Molecular labels can include barcode sequences.
  • a molecular label can comprise a nucleic acid sequence that provides identifying information for the specific type of target nucleic acid species hybridized to the barcode.
  • a molecular label can comprise a nucleic acid sequence that provides a counter for the specific occurrence of the target nucleic acid species hybridized to the barcode (e.g., target-binding region).
  • a diverse set of molecular labels are attached to a given solid support (e.g., a bead).
  • a given solid support e.g., a bead
  • a plurality of barcodes can comprise about 6561 molecular labels with distinct sequences.
  • a plurality of barcodes can comprise about 65536 molecular labels with distinct sequences.
  • Barcodes with unique molecular label sequences can be attached to a given solid support (e.g., a head).
  • the ratio of the number of different molecular label sequences and the number of occurrence of any of the targets can be, or be about, 1: 1, 2:1, 3: 1, 4: 1, 5: 1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, or a number or a range between any two of these values.
  • a target can be an mRNA species comprising mRNA molecules with identical or nearly identical sequences.
  • the ratio of the number of different molecular label sequences and the number of occurrence of any of the targets is at least, or is at most, 1: 1, 2:1, 3: 1, 4:1, 5:1, 6: 1, 7: 1, 8:1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1. or 100: 1.
  • a molecular label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length.
  • a molecular label can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.
  • a barcode can comprise one or more target binding regions, such as capture probes.
  • a target-binding region can hybridize with a target of interest.
  • the target binding regions can comprise a nucleic acid sequence that hybridizes specifically to a target (e.g., target nucleic acid, target molecule, e.g., a cellular nucleic acid to be analyzed), for example to a specific gene sequence.
  • a target binding region can comprise a nucleic acid sequence that can attach (e.g., hybridize) to a specific location of a specific target nucleic acid.
  • the target binding region can comprise a nucleic acid sequence that is capable of specific hybridization to a restriction enzyme site overhang (e.g., an EcoRI sticky-end overhang).
  • the barcode can then ligate to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang.
  • a target binding region can comprise a non-specific target nucleic acid sequence.
  • a non-specific target nucleic acid sequence can refer to a sequence that can bind to multiple target nucleic acids, independent of the specific sequence of the target nucleic acid.
  • target binding region can comprise a random multimer sequence, a poly(dA) sequence, a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, or a combination thereof.
  • the target binding region can be an oligo(dT) sequence that hybridizes to the poly(A) tail on mRNA molecules.
  • a random multimer sequence can be, for example, a random dimer, trimer, quatramer, pentamer, hexamer, septamer, octamer, nonamer, decamer, or higher multimer sequence of any length.
  • the target binding region is the same for all barcodes attached to a given bead.
  • the target binding regions for the plurality of barcodes attached to a given bead can comprise two or more different target binding sequences.
  • a target binding region can be, or be about, 5, 10, 15, 20, 25, 30, 35, 40. 45, 50, or a number or a range between any two of these values, nucleotides in length.
  • a target binding region can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length.
  • an mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to generate a cDNA molecule with a poly(dC) tail.
  • a barcode can include a target binding region with a poly(dG) tail. Upon base pairing between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches template strands, from cellular RNA molecule to the barcode, and continues replication to the 5' end of the barcode. By doing so, the resulting cDNA molecule contains the sequence of the barcode (such as the molecular label) on the 3 ’ end of the cDNA molecule.
  • MMLV Moloney murine leukemia virus
  • a target-binding region can comprise an oligo(dT) which can hybridize with mRNAs comprising polyadenylated ends.
  • a target-binding region can be gene-specific.
  • a target-binding region can be configured to hybridize to a specific region of a target.
  • a target-binding region can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, or a number or a range between any two of these values, nucleotides in length.
  • a target-binding region can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30, nucleotides in length.
  • a target-binding region can be about 5-30 nucleotides in length.
  • a stochastic barcode (e.g., a stochastic barcode) can comprise one or more orientation properties which can be used to orient (e.g., align) the barcodes.
  • a barcode can comprise a moiety for isoelectric focusing. Different barcodes can comprise different isoelectric focusing points. When these barcodes are introduced to a sample, the sample can undergo isoelectric focusing in order to orient the barcodes into a known way. In this way, the orientation property 7 can be used to develop a known map of barcodes in a sample.
  • Exemplary 7 orientation properties can include, electrophoretic mobility (e.g., based on size of the barcode), isoelectric point, spin, conductivity, and/or self-assembly.
  • barcodes with an orientation property of self-assembly can self-assemble into a specific orientation (e g., nucleic acid nanostructure) upon activation.
  • a barcode (e.g., a stochastic barcode) can comprise one or more affinityproperties.
  • a spatial label can comprise an affinity 7 property.
  • An affinity property can include a chemical and/or biological moiety 7 that can facilitate binding of the barcode to another entity 7 (e.g., cell receptor).
  • an affinity 7 property can comprise an antibody, for example, an antibody specific for a specific moiety (e.g.. receptor) on a sample.
  • the antibody can guide the barcode to a specific cell type or molecule.
  • Targets at and/or near the specific cell type or molecule can be labeled (e.g., stochastically 7 labeled).
  • the affinity 7 property 7 can, in some embodiments, provide spatial information in addition to the nucleotide sequence of the spatial label because the antibody can guide the barcode to a specific location.
  • the antibody can be a therapeutic antibody, for example a monoclonal antibody or a polyclonal antibody.
  • the antibody can be humanized or chimeric.
  • the antibody can be a naked antibody or a fusion antibody.
  • the antibody can be a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, like an antibody 7 fragment.
  • immunoglobulin molecule e.g., an IgG antibody
  • immunologically active i.e., specifically binding
  • the antibody fragment can be, for example, a portion of an antibody such as F(ab')2, Fab', Fab, Fv, sFv and the like. In some embodiments, the antibody fragment can bind with the same antigen that is recognized by the full-length antibody.
  • the antibody fragment can include isolated fragments consisting of the variable regions of antibodies, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”).
  • Exemplary antibodies can include, but are not limited to, antibodies for cancer cells, antibodies for viruses, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.
  • a barcode can comprise one or more universal adaptor primers.
  • a gene-specific barcode such as a gene-specific stochastic barcode
  • a universal adaptor primer can refer to a nucleotide sequence that is universal across all barcodes.
  • a universal adaptor primer can be used for building gene-specific barcodes.
  • a universal adaptor primer can be, or be about, 1. 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18. 19. 20. 21. 22. 23. 24, 25, 26 27, 28, 29, 30, or a number or a range between any two of these nucleotides in length.
  • a universal adaptor primer can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30 nucleotides in length.
  • a universal adaptor primer can be from 5-30 nucleotides in length.
  • a barcode comprises more than one of a type of label (e.g, more than one cell label or more than one barcode sequence, such as one molecular label)
  • the labels may be interspersed with a linker label sequence.
  • a linker label sequence can be at least about 5, 10, 15, 20, 25, 30. 35. 40. 45, 50 or more nucleotides in length.
  • a linker label sequence can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. In some instances, a linker label sequence is 12 nucleotides in length.
  • a linker label sequence can be used to facilitate the synthesis of the barcode.
  • the linker label can comprise an error-correcting (e.g., Hamming) code.
  • Barcodes such as stochastic barcodes, disclosed herein can, in some embodiments, be associated with a solid support.
  • the solid support can be, for example, a synthetic particle.
  • some or all of the barcode sequences, such as molecular labels for stochastic barcodes (e.g., the first barcode sequences) of a plurality of barcodes (e.g., the first plurality of barcodes) on a solid support differ by at least one nucleotide.
  • the cell labels of the barcodes on the same solid support can be the same.
  • the cell labels of the barcodes on different solid supports can differ by at least one nucleotide.
  • first cell labels of a first plurality of barcodes on a first solid support can have the same sequence
  • second cell labels of a second plurality of barcodes on a second solid support can have the same sequence
  • the first cell labels of the first plurality of barcodes on the first solid support and the second cell labels of the second plurality of barcodes on the second solid support can differ by at least one nucleotide.
  • a cell label can be, for example, about 5-20 nucleotides long.
  • a barcode sequence can be, for example, about 5-20 nucleotides long.
  • the synthetic particle can be, for example, a bead.
  • the bead can be, for example, a silica gel bead, a controlled pore glass bead, a magnetic bead, a Dynabead, a Sephadex/Sepharose bead, a cellulose bead, a polystyrene bead, or any combination thereof.
  • the bead can comprise a material such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof.
  • PDMS polydimethylsiloxane
  • the bead can be a polymeric bead, for example a deformable bead or a gel bead, functionalized with barcodes or stochastic barcodes (such as gel beads from 10X Genomics (San Francisco, CA).
  • a gel bead can comprise a polymer based gels. Gel beads can be generated, for example, by encapsulating one or more polymeric precursors into droplets. Upon exposure of the polymeric precursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)), a gel bead may be generated.
  • an accelerator e.g., tetramethylethylenediamine (TEMED)
  • the particle can be disruptable (e.g., dissolvable, degradable).
  • the polymeric bead can dissolve, melt, or degrade, for example, under a desired condition.
  • the desired condition can include an environmental condition.
  • the desired condition may result in the polymeric bead dissolving, melting, or degrading in a controlled manner.
  • a gel bead may dissolve, melt, or degrade due to a chemical stimulus, a physical stimulus, a biological stimulus, a thermal stimulus, a magnetic stimulus, an electric stimulus, a light stimulus, or any combination thereof.
  • Analytes and/or reagents such as oligonucleotide barcodes, for example, may be coupled/immobilized to the interior surface of a gel bead (e g., the interior accessible via diffusion of an oligonucleotide barcode and/or materials used to generate an oligonucleotide barcode) and/or the outer surface of a gel bead or any other microcapsule described herein. Coupling/immobilization may be via any form of chemical bonding (e.g., covalent bond, ionic bond) or physical phenomena (e.g.. Van der Waals forces, dipole-dipole interactions, etc.).
  • chemical bonding e.g., covalent bond, ionic bond
  • physical phenomena e.g. Van der Waals forces, dipole-dipole interactions, etc.
  • coupling/immobilization of a reagent to a gel bead or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical cross-linker, including chemical cross-linkers described herein).
  • a labile moiety e.g., via a chemical cross-linker, including chemical cross-linkers described herein.
  • the labile moiety may be cleaved and the immobilized reagent set free.
  • the labile moiety is a disulfide bond.
  • oligonucleotide barcode is immobilized to a gel bead via a disulfide bond
  • exposure of the disulfide bond to a reducing agent can cleave the disulfide bond and free the oligonucleotide barcode from the bead.
  • the labile moiety may be included as part of a gel bead or microcapsule, as part of a chemical linker that links a reagent or analyte to a gel bead or microcapsule, and/or as part of a reagent or analyte.
  • a gel bead can comprise a wide range of different polymers including but not limited to: polymers, heat sensitive polymers, photosensitive polymers, magnetic polymers, pH sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and/or plastics.
  • Polymers may include but are not limited to materials such as poly (N -isopropylacrylamide) (PNIPAAm), poly (styrene sulfonate) (PSS), poly (allyl amine) (PAAm), poly(acrylic acid) (PAA), poly(ethylene imine) (PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrolle) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinyl pyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthaladehyde) (PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).
  • Numerous chemical stimuli can be used to trigger the disruption, dissolution, or degradation of the beads.
  • Examples of these chemical changes may include, but are not limited to pH-mediated changes to the bead wall, disintegration of the bead wall via chemical cleavage of crosslink bonds, triggered depolymerization of the bead wall, and bead wall switching reactions. Bulk changes may also be used to trigger disruption of the beads.
  • Bulk or physical changes to the microcapsule through various stimuli also offer many advantages in designing capsules to release reagents.
  • Bulk or physical changes occur on a macroscopic scale, in which bead rupture is the result of mechano-physical forces induced by a stimulus. These processes may include, but are not limited to pressure induced rupture, bead wall melting, or changes in the porosity' of the bead wall.
  • Bio stimuli may also be used to trigger disruption, dissolution, or degradation of beads.
  • biological triggers resemble chemical triggers, but many examples use biomolecules, or molecules commonly ⁇ found in living systems such as enzymes, peptides, saccharides, fatty' acids, nucleic acids and the like.
  • beads may comprise polymers with peptide cross-links that are sensitive to cleavage by specific proteases. More specifically, one example may comprise a microcapsule comprising GFLGK peptide cross links.
  • a biological trigger such as the protease Cathepsin B, the peptide cross links of the shell well are cleaved and the contents of the beads are released.
  • the proteases may be heat-activated.
  • beads comprise a shell wall comprising cellulose. Addition of the hydrolytic enzyme chitosan serves as biologic trigger for cleavage of cellulosic bonds, depolymerization of the shell wall, and release of its inner contents.
  • the beads may also be induced to release their contents upon the application of a thermal stimulus.
  • a change in temperature can cause a variety changes to the beads.
  • a change in heat may cause melting of a bead such that the bead wall disintegrates.
  • the heat may increase the internal pressure of the inner components of the bead such that the bead ruptures or explodes.
  • the heat may transform the bead into a shrunken dehydrated state.
  • the heat may also act upon heat-sensitive polymers within the wall of a bead to cause disruption of the bead.
  • a device of this disclosure may comprise magnetic beads for either purpose.
  • incorporation of Fe ⁇ O-i nanoparticles into polyelectrolyte containing beads triggers rupture in the presence of an oscillating magnetic field stimulus.
  • a bead may also be disrupted, dissolved, or degraded as the result of electrical stimulation. Similar to magnetic particles described in the previous section, electrically sensitive beads can allow for both triggered rupture of the beads as well as other functions such as alignment in an electric field, electrical conductivity or redox reactions. In one example, beads containing electrically sensitive material are aligned in an electric field such that release of inner reagents can be controlled. In other examples, electrical fields may induce redox reactions within the bead wall itself that may increase porosity.
  • a light stimulus may also be used to disrupt the beads.
  • Numerous light triggers are possible and may include systems that use various molecules such as nanoparticles and chromophores capable of absorbing photons of specific ranges of wavelengths.
  • metal oxide coatings can be used as capsule triggers.
  • UV irradiation of polyelectrolyte capsules coated with Si O2 may result in disintegration of the bead wall.
  • photo switchable materials such as azobenzene groups may be incorporated in the bead wall.
  • chemicals such as these undergo a reversible cis-to- trans isomerization upon absorption of photons.
  • incorporation of photon switches result in a bead wall that may disintegrate or become more porous upon the application of a light trigger.
  • barcoding e.g., stochastic barcoding
  • beads can be introduced onto the plurality of microwells of the microwell array at block 212.
  • Each microwell can comprise one bead.
  • the beads can comprise a plurality of barcodes.
  • a barcode can comprise a 5 ? amine region attached to a bead.
  • the barcode can comprise a universal label, a barcode sequence (e.g., a molecular label), a target-binding region, or any combination thereof.
  • the barcodes disclosed herein can be associated with (e.g., attached to) a solid support (e.g., a bead).
  • the barcodes associated with a solid support can each comprise a barcode sequence selected from a group comprising at least 100 or 1000 barcode sequences with unique sequences.
  • different barcodes associated with a solid support can comprise barcode with different sequences.
  • a percentage of barcodes associated with a solid support comprises the same cell label. For example, the percentage can be. or be about 60%, 70%, 80%. 85%. 90%. 95%, 97%, 99%, 100%, or a number or a range between any two of these values.
  • the percentage can be at least, or be at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%.
  • barcodes associated with a solid support can have the same cell label.
  • the barcodes associated with different solid supports can have different cell labels selected from a group comprising at least 100 or 1000 cell labels with unique sequences.
  • the barcodes disclosed herein can be associated to (e.g., attached to) a solid support (e.g., a bead).
  • barcoding the plurality of targets in the sample can be performed with a solid support including a plurality of synthetic particles associated with the plurality of barcodes.
  • the solid support can include a plurality of synthetic particles associated with the plurality of barcodes.
  • the spatial labels of the plurality of barcodes on different solid supports can differ by at least one nucleotide.
  • the solid support can, for example, include the plurality of barcodes in two dimensions or three dimensions.
  • the synthetic particles can be beads.
  • the beads can be silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex/Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof.
  • the solid support can include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof.
  • the solid supports can be free floating.
  • the solid supports can be embedded in a semi-solid or solid array.
  • the barcodes may not be associated with solid supports.
  • the barcodes can be individual nucleotides.
  • the barcodes can be associated with a substrate.
  • the terms “tethered,’' “attached,” and “immobilized,” are used interchangeably, and can refer to covalent or non-covalent means for attaching barcodes to a solid support. Any of a variety of different solid supports can be used as solid supports for attaching pre-synthesized barcodes or for in situ solid-phase synthesis of barcode.
  • the solid support is a bead.
  • the bead can comprise one or more types of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration which a nucleic acid can be immobilized (e.g.. covalently or non-covalently).
  • the bead can be, for example, composed of plastic, ceramic, metal, polymeric material, or any combination thereof.
  • a bead can be, or comprise, a discrete particle that is spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like.
  • a bead can be non-spherical in shape.
  • Beads can comprise a variety of materials including, but not limited to, paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (FesCh: magnetite) nanoparticles), ferromagnetic materials (e.g.. iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex. Sepharose, agarose, hydrogel, polymer, cellulose, nylon, or any combination thereof.
  • paramagnetic materials e.g., magnesium, molybdenum, lithium, and tantalum
  • superparamagnetic materials e.g., ferrite (FesCh: magnetite) nanoparticles
  • ferromagnetic materials e.g. iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds
  • the bead (e.g., the bead to which the labels are attached) is a hydrogel bead. In some embodiments, the bead comprises hydrogel.
  • Some embodiments disclosed herein include one or more particles (for example, beads).
  • Each of the particles can comprise a plurality of oligonucleotides (e.g., barcodes).
  • Each of the plurality of oligonucleotides can comprise a barcode sequence (e.g., a molecular label sequence), a cell label, and a target-binding region (e.g.. an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof).
  • the cell label sequence of each of the plurality of oligonucleotides can be the same.
  • the cell label sequences of oligonucleotides on different particles can be different such that the oligonucleotides on different particles can be identified.
  • the number of different cell label sequences can be different in different implementations. In some embodiments, the number of cell label sequences can be, or be about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 , 10 7 , 10 8 , 10 9 , a number or a range between any two of these values, or more.
  • the number of cell label sequences can be at least, or be at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 , 10 7 , 10 8 , or 10 9 .
  • no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more of the plurality of the particles include oligonucleotides with the same cell sequence.
  • the plurality of particles that include oligonucleotides with the same cell sequence can be at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, none of the plurality of the particles has the same cell label sequence.
  • the plurality of oligonucleotides on each particle can comprise different barcode sequences (e.g., molecular labels).
  • the number of barcode sequences can be, or be about 10, 100, 200. 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 , 10 7 , 10 8 , 10 9 , or a number or a range between any two of these values.
  • the number of barcode sequences can be at least, or be at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000. 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000. 10 6 , 10 7 , 10 8 , or 10 9 .
  • at least 100 of the plurality of oligonucleotides comprise different barcode sequences.
  • a single particle at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, a number or a range between any two of these values, or more of the plurality of oligonucleotides comprise different barcode sequences.
  • Some embodiments provide a plurality of the particles comprising barcodes.
  • the ratio of an occurrence (or a copy or a number) of a target to be labeled and the different barcode sequences can be at least 1: 1, 1:2, 1 :3, 1 :4, 1:5, 1 :6, 1:7, 1 :8, 1:9, 1: 10, 1:11, 1 :12, 1 :13, 1 : 14, 1: 15, 1: 16, 1: 17, 1: 18, 1: 19, 1:20, 1:30, 1 :40, 1 :50, 1 :60, 1:70, 1:80, 1 :90, or more.
  • each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both.
  • the particle can be, for example, a nanoparticle or microparticle.
  • the size of the beads can vary.
  • the diameter of the bead can range from 0. 1 micrometer to 50 micrometer.
  • the diameter of the bead can be, or be about, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometer, or a number or a range between any two of these values.
  • the diameter of the bead can be related to the diameter of the wells of the substrate.
  • the diameter of the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or a range between any two of these values, longer or shorter than the diameter of the well.
  • the diameter of the beads can be related to the diameter of a cell (e.g., a single cell entrapped by a well of the substrate).
  • the diameter of the bead can be at least, or be at most, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the well.
  • the diameter of the beads can be related to the diameter of a cell (e.g., a single cell entrapped by a well of the substrate).
  • the diameter of the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or a range between any two of these values, longer or shorter than the diameter of the cell.
  • the diameter of the beads can be at least, or be at most, 10%, 20%, 30%, 40%, 50%. 60%. 70%, 80%, 90%, 100%, 150%, 200%, 250%. or 300% longer or shorter than the diameter of the cell.
  • a bead can be attached to and/or embedded in a substrate.
  • a bead can be attached to and/or embedded in a gel, hydrogel, polymer and/or matrix.
  • the spatial position of a bead within a substrate e.g. gel, matrix, scaffold, or polymer
  • a substrate e.g. gel, matrix, scaffold, or polymer
  • beads can include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugated beads (e.g., anti-immunoglobulin microbeads), protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorochrome microbeads, and BcMagTM Carboxyl-Terminated Magnetic Beads.
  • streptavidin beads e.g., streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugated beads (e.g., anti-immunoglobulin microbeads), protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(
  • a bead can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make it fluorescent in one fluorescence optical channel or multiple optical channels.
  • a bead can be associated with iron oxide or chromium oxide to make it paramagnetic or ferromagnetic. Beads can be identifiable. For example, a bead can be imaged using a camera.
  • a bead can have a detectable code associated with the bead.
  • a bead can comprise a barcode.
  • a bead can change size, for example, due to swelling in an organic or inorganic solution.
  • a bead can be hydrophobic.
  • a bead can be hydrophilic.
  • a bead can be biocompatible.
  • a solid support (e.g., a bead) can be visualized.
  • the solid support can comprise a visualizing tag (e.g., fluorescent dye).
  • a solid support e.g., a bead
  • a solid support can comprise an insoluble, semi-soluble, or insoluble material.
  • a solid support can be referred to as “functionalized” when it includes a linker, a scaffold, a building block, or other reactive moiety attached thereto, whereas a solid support may be “nonfunctionalized” when it lack such a reactive moiety 7 attached thereto.
  • the solid support can be employed free in solution, such as in a microtiter well format; in a flow-through format, such as in a column; or in a dipstick.
  • the solid support can comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof.
  • a solid support can take the form of resins, gels, microspheres, or other geometric configurations.
  • a solid support can comprise silica chips, microparticles, nanoparticles, plates, arrays, capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold silver, aluminum, silicon and copper), glass supports, plastic supports, silicon supports, chips, filters, membranes, microwell plates, slides, plastic materials including multiwell plates or membranes (e.g., formed of polyethylene, polypropylene, polyamide, polyvinylidenedifluoride), and/or wafers, combs, pins or needles (e.g., arrays of pins suitable for combinatorial synthesis or analysis) or beads in an array of pits or nanoliter wells of flat surfaces such as wafers (e.g., silicon wafers), wafers with pits with or without filter bottom
  • the solid support can comprise a polymer matrix (e.g., gel. hydrogel).
  • the polymer matrix may be able to permeate intracellular space (e.g., around organelles).
  • the polymer matrix may able to be pumped throughout the circulatory system.
  • a substrate can refer to a type of solid support.
  • a substrate can refer to a solid support that can comprise barcodes or stochastic barcodes of the disclosure.
  • a substrate can, for example, comprise a plurality of microwells.
  • a substrate can be a well array comprising two or more microwells.
  • a microwell can comprise a small reaction chamber of defined volume.
  • a microwell can entrap one or more cells.
  • a microwell can entrap only one cell.
  • a microwell can entrap one or more solid supports.
  • a microwell can entrap only one solid support.
  • a microwell entraps a single cell and a single solid support (e.g., a bead).
  • a microwell can comprise barcode reagents of the disclosure.
  • the disclosure provides for methods for estimating the number of distinct targets at distinct locations in a physical sample (e.g., tissue, organ, tumor, cell).
  • the methods can comprise placing barcodes (e.g., stochastic barcodes) in close proximity with the sample, lysing the sample, associating distinct targets with the barcodes, amplifying the targets and/or digitally counting the targets.
  • the method can further comprise analyzing and/or visualizing the information obtained from the spatial labels on the barcodes.
  • a method comprises visualizing the plurality of targets in the sample. Mapping the plurality' of targets onto the map of the sample can include generating a two dimensional map or a three dimensional map of the sample.
  • the two dimensional map and the three dimensional map can be generated prior to or after barcoding (e.g., stochastically barcoding) the plurality of targets in the sample.
  • Visualizing the plurality' of targets in the sample can include mapping the plurality' of targets onto a map of the sample. Mapping the plurality of targets onto the map of the sample can include generating a two dimensional map or a three dimensional map of the sample.
  • the two dimensional map and the three dimensional map can be generated prior to or after barcoding the plurality of targets in the sample, in some embodiments, the two dimensional map and the three dimensional map can be generated before or after lysing the sample. Lysing the sample before or after generating the two dimensional map or the three dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.
  • barcoding the plurality' of targets comprises hybridizing a plurality of barcodes with a plurality of targets to create barcoded targets (e.g., stochastically barcoded targets).
  • Barcoding the plurality of targets can comprise generating an indexed library of the barcoded targets. Generating an indexed library' of the barcoded targets can be performed with a solid support comprising the plurality of barcodes (e.g., stochastic barcodes).
  • the disclosure provides for methods for contacting a sample (e.g., cells) to a substrate of the disclosure.
  • a sample comprising, for example, a cell, organ, or tissue thin section
  • barcodes e.g., stochastic barcodes
  • the cells can be contacted, for example, by gravity flow wherein the cells can settle and create a monolayer.
  • the sample can be a tissue thin section.
  • the thin section can be placed on the substrate.
  • the sample can be onedimensional (e.g., formsa planar surface).
  • the sample e.g., cells
  • the sample can be spread across the substrate, for example, by growing/culturing the cells on the substrate.
  • the targets When barcodes are in close proximity to targets, the targets can hybridize to the barcode.
  • the barcodes can be contacted at a non-depletable ratio such that each distinct target can associate with a distinct barcode of the disclosure.
  • the targets can be cross-linked to barcode.
  • the cells can be lysed to liberate the target molecules.
  • Cell lysis can be accomplished by any of a variety of means, for example, by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis, or optical lysis.
  • Cells can be lysed by addition of a cell lysis buffer comprising a detergent (e.g., SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or digestive enzymes (e.g., proteinase K, pepsin, or trypsin), or any combination thereof.
  • a detergent e.g., SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP-40
  • an organic solvent e.g., methanol or acetone
  • digestive enzymes e.g., proteinase K
  • the sample can be lysed using a filter paper.
  • the filter paper can be soaked with a lysis buffer on top of the filter paper.
  • the filter paper can be applied to the sample with pressure which can facilitate lysis of the sample and hybridization of the targets of the sample to the substrate.
  • lysis can be performed by mechanical lysis, heat lysis, optical lysis, and/or chemical lysis.
  • Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin.
  • Lysis can be performed by the addition of a lysis buffer to the substrate.
  • a lysis buffer can comprise Tris HC1.
  • a lysis buffer can comprise at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris HC1.
  • a lysis buffer can comprise at most about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris HCL.
  • a lysis buffer can comprise about 0.1 M Tris HC1.
  • the pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the pH of the lysis buffer can be at most about 1, 2, 3, 4, 5, 6. 7, 8, 9,10, or more. In some embodiments, the pH of the lysis buffer is about 7.5.
  • the lysis buffer can comprise a salt (e.g., LiCl).
  • the concentration of salt in the lysis buffer can be at least about 0.1, 0.5, or 1 M or more.
  • the concentration of salt in the lysis buffer can be at most about 0.1, 0.5, or 1 M or more. In some embodiments, the concentration of salt in the lysis buffer is about 0.5M.
  • the lysis buffer can comprise a detergent (e.g., SDS, Li dodecyl sulfate, triton X, tween. NP-40).
  • concentration of the detergent in the lysis buffer can be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or more.
  • the concentration of the detergent in the lysis buffer can be at most about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%. 1%, 2%. 3%, 4%, 5%, 6%, or 7%, or more.
  • the concentration of the detergent in the lysis buffer is about 1% Li dodecyl sulfate.
  • the time used in the method for lysis can be dependent on the amount of detergent used. In some embodiments, the more detergent used, the less time needed for lysis.
  • the lysis buffer can comprise a chelating agent (e.g., EDTA, EGTA).
  • the concentration of a chelating agent in the lysis buffer can be at least about 1, 5, 10, 15, 20, 25, or 30 mM or more.
  • the concentration of a chelating agent in the lysis buffer can be at most about 1, 5, 10, 15, 20, 25, or 30mM or more. In some embodiments, the concentration of chelating agent in the lysis buffer is about 10 mM.
  • the lysis buffer can comprise a reducing reagent (e.g., beta-mercaptoethanol. DTT).
  • the concentration of the reducing reagent in the lysis buffer can be at least about 1, 5, 10, 15, or 20 mM or more.
  • the concentration of the reducing reagent in the lysis buffer can be at most about 1, 5, 10, 15, or 20 mM or more.
  • the concentration of reducing reagent in the lysis buffer is about 5 mM.
  • a lysis buffer can comprise about 0.1M TrisHCl, about pH 7.5, about 0.5M LiCl, about 1% lithium dodecyl sulfate, about lOmM EDTA, and about 5mM DTT.
  • Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or 30 °C. Lysis can be performed for about 1, 5, 10, 15, or 20 or more minutes.
  • a lysed cell can comprise at least about 100000, 200000, 300000, 400000, 500000, 600000, or 700000 or more target nucleic acid molecules.
  • a lysed cell can comprise at most about 100000, 200000, 300000, 400000, 500000, 600000, or 700000 or more target nucleic acid molecules.
  • the nucleic acid molecules can randomly associate with the barcodes of the co-localized solid support. Association can comprise hybridization of a barcode’s target recognition region to a complementary portion of the target nucleic acid molecule (e.g., oligo(dT) of the barcode can interact with a poly (A) tail of a target).
  • the assay conditions used for hybridization e.g., buffer pH, ionic strength, temperature, etc.
  • the nucleic acid molecules released from the lysed cells can associate with the plurality of probes on the substrate (e.g., hybridize with the probes on the substrate).
  • mRNA molecules can hybridize to the probes and be reverse transcribed.
  • the oligo(dT) portion of the oligonucleotide can act as a primer for first strand synthesis of the cDNA molecule.
  • mRNA molecules can hybridize to barcodes on beads.
  • single-stranded nucleotide fragments can hybridize to the target-binding regions of barcodes.
  • Attachment can further comprise ligation of a barcode’s target recognition region and a portion of the target nucleic acid molecule.
  • the target binding region can comprise a nucleic acid sequence that can be capable of specific hybridization to a restriction site overhang (e.g., an EcoRI sticky-end overhang).
  • the assay procedure can further comprise treating the target nucleic acids with a restriction enzyme (e.g., EcoRI) to create a restriction site overhang.
  • the barcode can then be ligated to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang.
  • a ligase e.g., T4 DNA ligase
  • T4 DNA ligase can be used to join the two fragments.
  • the labeled targets from a plurality of cells (or a plurality of samples) can be subsequently pooled, for example, into a tube.
  • the labeled targets can be pooled by, for example, retrieving the barcodes and/or the beads to which the targetbarcode molecules are attached.
  • the retrieval of solid support-based collections of attached target-barcode molecules can be implemented by use of magnetic beads and an externally-applied magnetic field. Once the target-barcode molecules have been pooled, all further processing can proceed in a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and/or nucleic acid extension reactions. Further processing reactions can be performed within the microwells, that is, without first pooling the labeled target nucleic acid molecules from a plurality of cells.
  • the disclosure provides for a method to create a target-barcode conjugate using reverse transcription (e.g., at block 224 of FIG. 2) or nucleic acid extension.
  • the target- barcode conjugate can comprise the barcode and a complementary sequence of all or a portion of the target nucleic acid (i.e., a barcoded cDNA molecule, such as a stochastically barcoded cDNA molecule).
  • Reverse transcription of the associated RNA molecule can occur by the addition of a reverse transcription primer along with the reverse transcriptase.
  • the reverse transcription primer can be an oligo(dT) primer, a random hexanucleotide primer, or a targetspecific oligonucleotide primer.
  • Oligo(dT) primers can be, or can be about, 12-18 nucleotides in length and bind to the endogenous poly (A) tail at the 3’ end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA at a variety of complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest.
  • reverse transcription of an mRNA molecule to a labeled-RNA molecule can occur by the addition of a reverse transcription primer.
  • the reverse transcription primer is an oligo(dT) primer, random hexanucleotide primer, or a target-specific oligonucleotide primer.
  • oligo(dT) primers are 12-18 nucleotides in length and bind to the endogenous poly (A) tail at the 3’ end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA at a variety of complementary sites.
  • Target-specific oligonucleotide primers ty pically selectively prime the mRNA of interest.
  • a target is a cDNA molecule.
  • an mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to generate a cDNA molecule with a poly(dC) tail.
  • a barcode can include a target binding region with a poly(dG) tail.
  • the reverse transcriptase switches template strands, from cellular RNA molecule to the barcode, and continues replication to the 5' end of the barcode.
  • the resulting cDNA molecule contains the sequence of the barcode (such as the molecular label) on the 3’ end of the cDNA molecule.
  • Reverse transcription can occur repeatedly to produce multiple labeled-cDNA molecules.
  • the methods disclosed herein can comprise conducting at least about 1, 2. 3, 4, 5. 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions.
  • the method can comprise conducting at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.
  • One or more nucleic acid amplification reactions can be performed to create multiple copies of the labeled target nucleic acid molecules.
  • Amplification can be performed in a multiplexed manner, wherein multiple target nucleic acid sequences are amplified simultaneously.
  • the amplification reaction can be used to add sequencing adaptors to the nucleic acid molecules.
  • the amplification reactions can comprise amplifying at least a portion of a sample label, if present.
  • the amplification reactions can comprise amplifying at least a portion of the cellular label and/or barcode sequence (e.g., a molecular label).
  • the amplification reactions can comprise amplifying at least a portion of a sample tag, a cell label, a spatial label, a barcode sequence (e.g., a molecular label), a target nucleic acid, or a combination thereof.
  • the amplification reactions can comprise amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a range or a number between any two of these values, of the plurality of nucleic acids.
  • the method can further comprise conducting one or more cDNA synthesis reactions to produce one or more cDNA copies of target-barcode molecules comprising a sample label, a cell label, a spatial label, and/or a barcode sequence (e.g., a molecular label).
  • a barcode sequence e.g., a molecular label
  • amplification can be performed using a polymerase chain reaction (PCR).
  • PCR can refer to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA.
  • PCR can encompass derivative forms of the reaction, including but not limited to, RT-PCR. real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR. and assembly PCR.
  • Amplification of the labeled nucleic acids can comprise non-PCR based methods.
  • non-PCR based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA). strand displacement amplification (SDA). real-time SDA, rolling circle amplification, or circle-to-circle amplification.
  • Non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify' DNA or RNA targets, a ligase chain reaction (LCR), and a Q(3 replicase (QP) method, use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using a restriction endonuclease, an amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to the extension reaction and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5’ exonuclease activity, rolling circle amplification, and ramification extension amplification (RAM).
  • the amplification does not produce circularized transcripts.
  • the methods disclosed herein further comprise conducting a polymerase chain reaction on the labeled nucleic acid (e.g., labeled-RNA, labeled- DNA, labeled-cDNA) to produce a labeled amplicon (e.g., a stochastically labeled amplicon).
  • the labeled amplicon can be double-stranded molecule.
  • the double-stranded molecule can comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or a RNA molecule hybridized to a DNA molecule.
  • One or both of the strands of the double-stranded molecule can comprise a sample label, a spatial label, a cell label, and/or a barcode sequence (e.g.. a molecular label).
  • the labeled amplicon can be a single-stranded molecule.
  • the singlestranded molecule can comprise DNA, RNA, or a combination thereof.
  • the nucleic acids of the disclosure can comprise synthetic or altered nucleic acids.
  • Amplification can comprise use of one or more non-natural nucleotides.
  • Nonnatural nucleotides can comprise photolabile or triggerable nucleotides.
  • Examples of non-natural nucleotides can include, but are not limited to, peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA).
  • PNA peptide nucleic acid
  • LNA morpholino and locked nucleic acid
  • GMA glycol nucleic acid
  • TAA threose nucleic acid
  • Non-natural nucleotides can be added to one or more cycles of an amplification reaction. The addition of the non-natural nucleotides can be used to identify products as specific cycles or time points in the amplification reaction.
  • Conducting the one or more amplification reactions can comprise the use of one or more primers.
  • the one or more primers can comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides.
  • the one or more primers can comprise at least 1, 2, 3, 4. 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides.
  • the one or more primers can comprise less than 12-15 nucleotides.
  • the one or more primers can anneal to at least a portion of the plurality of labeled targets (e.g., stochastically labeled targets).
  • the one or more primers can anneal to the 3’ end or 5’ end of the plurality' of labeled targets.
  • the one or more primers can anneal to an internal region of the plurality of labeled targets.
  • the internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3’ ends the plurality of labeled targets.
  • the one or more primers can comprise a fixed panel of primers.
  • the one or more primers can comprise at least one or more custom primers.
  • the one or more primers can comprise at least one or more control primers.
  • the one or more primers can comprise at least one or more gene-specific primers.
  • the one or more primers can comprise a universal primer.
  • the universal primer can anneal to a universal primer binding site.
  • the one or more custom primers can anneal to a first sample label, a second sample label, a spatial label, a cell label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof.
  • the one or more primers can comprise a universal primer and a custom primer.
  • the custom primer can be designed to amplify one or more targets.
  • the targets can comprise a subset of the total nucleic acids in one or more samples.
  • the targets can comprise a subset of the total labeled targets in one or more samples.
  • the one or more primers can comprise at least 96 or more custom primers.
  • the one or more primers can comprise at least 960 or more custom primers.
  • the one or more primers can comprise at least 9600 or more custom primers.
  • the one or more custom primers can anneal to two or more different labeled nucleic acids.
  • the two or more different labeled nucleic acids can correspond to one or more genes.
  • the first round PCR can amplify molecules attached to the bead using a gene specific primer and a primer against the universal Illumina sequencing primer 1 sequence.
  • the second round of PCR can amplify the first PCR products using a nested gene specific primer flanked by Illumina sequencing primer 2 sequence, and a primer against the universal Illumina sequencing primer 1 sequence.
  • the third round of PCR adds P5 and P7 and sample index to turn PCR products into an Illumina sequencing library. Sequencing using 150 bp x 2 sequencing can reveal the cell label and barcode sequence (e.g., molecular label) on read 1, the gene on read 2, and the sample index on index 1 read.
  • barcode sequence e.g., molecular label
  • nucleic acids can be removed from the substrate using chemical cleavage.
  • a chemical group or a modified base present in a nucleic acid can be used to facilitate its removal from a solid support.
  • an enzyme can be used to remove a nucleic acid from a substrate.
  • a nucleic acid can be removed from a substrate through a restriction endonuclease digestion.
  • treatment of a nucleic acid containing a dUTP or ddUTP with uracil-d-glycosylase (UDG) can be used to remove a nucleic acid from a substrate.
  • UDG uracil-d-glycosylase
  • a nucleic acid can be removed from a substrate using an enzyme that performs nucleotide excision, such as a base excision repair enzyme, such as an apurinic/apyrimidinic (AP) endonuclease.
  • a nucleic acid can be removed from a substrate using a photocleavable group and light.
  • a cleavable linker can be used to remove a nucleic acid from the substrate.
  • the cleavable linker can comprise at least one of biotin/ avidin, biotin/streptavidin, biotin/neutravidin, Ig-protein A, a photo-labile linker, acid or base labile linker group, or an aptamer.
  • the molecules can hybridize to the probes and be reverse transcribed and/or amplified.
  • the nucleic acid after the nucleic acid has been synthesized (e.g.. reverse transcribed), it can be amplified. Amplification can be performed in a multiplex manner, wherein multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adaptors to the nucleic acid.
  • amplification can be performed on the substrate, for example, with bridge amplification.
  • cDNAs can be homopolymer tailed in order to generate a compatible end for bridge amplification using oligo(dT) probes on the substrate.
  • the primer that is complementary to the 3’ end of the template nucleic acid can be the first primer of each pair that is covalently attached to the solid particle.
  • the template molecule can be annealed to the first primer and the first primer is elongated in the forward direction by addition of nucleotides to form a duplex molecule consisting of the template molecule and a newly formed DNA strand that is complementary to the template.
  • the duplex molecule can be denatured, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle through the first primer.
  • the complementary strand can hybridize to the second primer, which is complementary to a segment of the complementary strand at a location removed from the first primer. This hybridization can cause the complementary strand to form a bridge between the first and second primers secured to the first primer by a covalent bond and to the second primer by hybridization.
  • the second primer can be elongated in the reverse direction by the addition of nucleotides in the same reaction mixture, thereby converting the bridge to a double-stranded bridge.
  • the next cycle then begins, and the doublestranded bridge can be denatured to yield two single-stranded nucleic acid molecules, each having one end attached to the particle surface via the first and second primers, respectively, with the other end of each unattached.
  • each strand can hybridize to a further complementary primer, previously unused, on the same particle, to form new single-strand bridges.
  • the two previously unused primers that are now hybridized elongate to convert the two new bridges to double-strand bridges.
  • the amplification reactions can comprise amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of the plurality of nucleic acids.
  • Amplification of the labeled nucleic acids can comprise PCR-based methods or non-PCR based methods.
  • Amplification of the labeled nucleic acids can comprise exponential amplification of the labeled nucleic acids.
  • Amplification of the labeled nucleic acids can comprise linear amplification of the labeled nucleic acids.
  • Amplification can be performed by polymerase chain reaction (PCR).
  • PCR can refer to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA.
  • PCR can encompass derivative forms of the reaction, including but not limited to, RT- PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, suppression PCR.
  • amplification of the labeled nucleic acids comprises non-PCR based methods.
  • non-PCR based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-to-circle amplification.
  • MDA multiple displacement amplification
  • TMA transcription-mediated amplification
  • NASBA nucleic acid sequence-based amplification
  • SDA strand displacement amplification
  • real-time SDA rolling circle amplification
  • rolling circle amplification or circle-to-circle amplification.
  • Non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase- driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, a ligase chain reaction (LCR), a QP replicase (QP), use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using a restriction endonuclease, an amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to the extension reaction and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5’ exonuclease activity, rolling circle amplification, and/or ramification extension amplification (RAM).
  • LCR ligase chain reaction
  • QP QP replicase
  • amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cle
  • the methods disclosed herein further comprise conducting a nested polymerase chain reaction on the amplified amplicon (e.g., target).
  • the amplicon can be double-stranded molecule.
  • the double-stranded molecule can comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or a RNA molecule hybridized to a DNA molecule.
  • One or both of the strands of the double-stranded molecule can comprise a sample tag or molecular identifier label.
  • the amplicon can be a singlestranded molecule.
  • the single-stranded molecule can comprise DNA, RNA, or a combination thereof.
  • the nucleic acids of the present invention can comprise synthetic or altered nucleic acids.
  • the method comprises repeatedly amplifying the labeled nucleic acid to produce multiple amplicons.
  • the methods disclosed herein can comprise conducting at least about 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. or 20 amplification reactions.
  • the method comprises conducting at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.
  • Amplification can further comprise adding one or more control nucleic acids to one or more samples comprising a plurality of nucleic acids.
  • Amplification can further comprise adding one or more control nucleic acids to a plurality of nucleic acids.
  • the control nucleic acids can comprise a control label.
  • Amplification can comprise use of one or more non-natural nucleotides.
  • Nonnatural nucleotides can comprise photolabile and/or triggerable nucleotides.
  • Examples of nonnatural nucleotides include, but are not limited to, peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA).
  • PNA peptide nucleic acid
  • LNA morpholino and locked nucleic acid
  • GMA glycol nucleic acid
  • TAA threose nucleic acid
  • Non-natural nucleotides can be added to one or more cycles of an amplification reaction. The addition of the non-natural nucleotides can be used to identify products as specific cycles or time points in the amplification reaction.
  • Conducting the one or more amplification reactions can comprise the use of one or more primers.
  • the one or more primers can comprise one or more oligonucleotides.
  • the one or more oligonucleotides can comprise at least about 7-9 nucleotides.
  • the one or more oligonucleotides can comprise less than 12-15 nucleotides.
  • the one or more primers can anneal to at least a portion of the plurality of labeled nucleic acids.
  • the one or more primers can anneal to the 3’ end and/or 5’ end of the plurality of labeled nucleic acids.
  • the one or more primers can anneal to an internal region of the plurality of labeled nucleic acids.
  • the internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390. 400, 410, 420. 430, 440, 450. 460, 470, 480. 490, 500, 510. 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3' ends the plurality of labeled nucleic acids.
  • the one or more primers can comprise a fixed panel of primers.
  • the one or more primers can comprise at least one or more custom primers.
  • the one or more primers can comprise at least one or more control primers.
  • the one or more primers can comprise at least one or more housekeeping gene primers.
  • the one or more primers can comprise a universal primer.
  • the universal primer can anneal to a universal primer binding site.
  • the one or more custom primers can anneal to the first sample tag, the second sample tag, the molecular identifier label, the nucleic acid or a product thereof.
  • the one or more primers can comprise a universal primer and a custom primer.
  • the custom primer can be designed to amplify’ one or more target nucleic acids.
  • the target nucleic acids can comprise a subset of the total nucleic acids in one or more samples.
  • the primers are the probes attached to the array of the disclosure.
  • barcoding e.g., stochastically barcoding
  • the plurality’ of targets in the sample further comprises generating an indexed library of the barcoded targets (e.g., stochastically barcoded targets) or barcoded fragments of the targets.
  • the barcode sequences of different barcodes e.g., the molecular labels of different stochastic barcodes
  • Generating an indexed library of the barcoded targets includes generating a plurality of indexed polynucleotides from the plurality’ of targets in the sample.
  • the label region of the first indexed polynucleotide can differ from the label region of the second indexed polynucleotide by, by about, by at least, or by at most, 1, 2, 3, 4, 5, 6. 7, 8. 9, 10, 20, 30, 40, 50, or a number or a range between any two of these values, nucleotides.
  • generating an indexed library of the barcoded targets includes contacting a plurality of targets, for example mRNA molecules, with a plurality of oligonucleotides including a poly(T) region and a label region; and conducting a first strand synthesis using a reverse transcriptase to produce single-strand labeled cDNA molecules each comprising a cDNA region and a label region, wherein the plurality of targets includes at least two mRNA molecules of different sequences and the plurality of oligonucleotides includes at least two oligonucleotides of different sequences.
  • Generating an indexed library of the barcoded targets can further comprise amplifying the single-strand labeled cDNA molecules to produce double-strand labeled cDNA molecules: and conducting nested PCR on the double-strand labeled cDNA molecules to produce labeled amplicons.
  • the method can include generating an adaptor-labeled amplicon.
  • Barcoding can include using nucleic acid barcodes or tags to label individual nucleic acid (e.g., DNA or RNA) molecules. In some embodiments, it involves adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adaptors can be added for sequencing using, for example, next generation sequencing (NGS). The sequencing results can be used to determine cell labels, molecular labels, and sequences of nucleotide fragments of the one or more copies of the targets, for example at block 232 of FIG. 2.
  • NGS next generation sequencing
  • FIG. 3 is a schematic illustration showing a non-limiting exemplary process of generating an indexed library of the barcoded targets (e.g., stochastically barcoded targets), such as barcoded mRNAs or fragments thereof.
  • the reverse transcription process can encode each mRNA molecule with a unique molecular label sequence, a cell label sequence, and a universal PCR site.
  • RNA molecules 302 can be reverse transcribed to produce labeled cDNA molecules 304, including a cDNA region 306, by hybridization (e.g., stochastic hybridization) of a set of barcodes (e.g., stochastic barcodes) 310 to the poly(A) tail region 308 of the RNA molecules 302.
  • Each of the barcodes 310 can comprise a target-binding region, for example a poly(dT) region 312, a label region 314 (e.g., a barcode sequence or a molecule), and a universal PCR region 316.
  • the cell label sequence can include 3 to 20 nucleotides. In some embodiments, the molecular label sequence can include 3 to 20 nucleotides. In some embodiments, each of the plurality of stochastic barcodes further comprises one or more of a universal label and a cell label, wherein universal labels are the same for the plurality of stochastic barcodes on the solid support and cell labels are the same for the plurality 7 of stochastic barcodes on the solid support. In some embodiments, the universal label can include 3 to 20 nucleotides. In some embodiments, the cell label comprises 3 to 20 nucleotides.
  • the label region 314 can include a barcode sequence or a molecular label 318 and a cell label 320.
  • the label region 314 can include one or more of a universal label, a dimension label, and a cell label.
  • the barcode sequence or molecular label 318 can be, can be about, can be at least, or can be at most, 1, 2. 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length.
  • the cell label 320 can be, can be about, can be at least, or can be at most, 1, 2, 3, 4, 5, 6, 7, 8.
  • the universal label can be, can be about, can be at least, or can be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length.
  • Universal labels can be the same for the plurality of stochastic barcodes on the solid support and cell labels are the same for the plurality of stochastic barcodes on the solid support.
  • the dimension label can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length.
  • the label region 314 can comprise, comprise about, comprise at least, or comprise at most, 1, 2. 3, 4, 5. 6, 7, 8. 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500. 600, 700, 800. 900, 1000, or a number or a range between any of these values, different labels, such as a barcode sequence or a molecular label 318 and a cell label 320.
  • Each label can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length.
  • a set of barcodes or stochastic barcodes 310 can contain, contain about, contain at least, or can be at most, 10, 20, 40, 50, 70, 80, 90, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 2 °, or a number or a range between any of these values, barcodes or stochastic barcodes 310.
  • the set of barcodes or stochastic barcodes 310 can, for example, each contain a unique label region 314.
  • the labeled cDNA molecules 304 can be purified to remove excess barcodes or stochastic barcodes 310. Purification can comprise Ampure bead purification.
  • step 2 products from the reverse transcription process in step 1 can be pooled into 1 tube and PCR amplified with a 1 st PCR primer pool and a 1 st universal PCR primer. Pooling is possible because of the unique label region 314.
  • the labeled cDNA molecules 304 can be amplified to produce nested PCR labeled amplicons 322.
  • Amplification can comprise multiplex PCR amplification.
  • Amplification can comprise a multiplex PCR amplification with 96 multiplex primers in a single reaction volume.
  • multiplex PCR amplification can utilize, utilize about, utilize at least, or utilize at most, 10, 20, 40, 50, 70, 80, 90, 10 2 , 10 3 , 10 4 , IO 3 , 10 6 , 10 7 , 10 8 , IO 9 , IO 10 , IO 11 , 10 12 , IO 13 , IO 14 , 10 13 , IO 20 , or a number or a range between any of these values, multiplex primers in a single reaction volume.
  • Amplification can comprise using a 1 st PCR primer pool 324 comprising custom primers 326A-C targeting specific genes and a universal primer 328.
  • the custom primers 326 can hybridize to a region within the cDNA portion 306’ of the labeled cDNA molecule 304.
  • the universal primer 328 can hybridize to the universal PCR region 316 of the labeled cDNA molecule 304.
  • products from PCR amplification in step 2 can be amplified with a nested PCR primers pool and a 2 nd universal PCR primer.
  • Nested PCR can minimize PCR amplification bias.
  • the nested PCR labeled amplicons 322 can be further amplified by nested PCR.
  • the nested PCR can comprise multiplex PCR with nested PCR primers pool 330 of nested PCR primers 332a-c and a 2 nd universal PCR primer 328’ in a single reaction volume.
  • the nested PCR primer pool 328 can contain, contain about, contain at least, or contain at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or a range between any of these values, different nested PCR primers 330.
  • the nested PCR primers 332 can contain an adaptor 334 and hybridize to a region within the cDNA portion 306" of the labeled amplicon 322.
  • the universal primer 328’ can contain an adaptor 336 and hybridize to the universal PCR region 316 of the labeled amplicon 322.
  • step 3 produces adaptor-labeled amplicon 338.
  • nested PCR primers 332 and the 2 nd universal PCR primer 328’ may not contain the adaptors 334 and 336.
  • the adaptors 334 and 336 can instead be ligated to the products of nested PCR to produce adaptor-labeled amplicon 338.
  • PCR products from step 3 can be PCR amplified for sequencing using library amplification primers.
  • the adaptors 334 and 336 can be used to conduct one or more additional assays on the adaptor-labeled amplicon 338.
  • the adaptors 334 and 336 can be hybridized to primers 340 and 342.
  • the one or more primers 340 and 342 can be PCR amplification primers.
  • the one or more primers 340 and 342 can be sequencing primers.
  • the one or more adaptors 334 and 336 can be used for further amplification of the adaptor-labeled amplicons 338.
  • the one or more adaptors 334 and 336 can be used for sequencing the adaptor-labeled amplicon 338.
  • the primer 342 can contain a plate index 344 so that amplicons generated using the same set of barcodes or stochastic barcodes 310 can be sequenced in one sequencing reaction using next generation sequencing (NGS).
  • NGS next generation sequencing
  • RNA probes are provided which can bind to said mRNAs and in situ reverse transcription can generate short cDNA connected to the probes. These probes can be easily captured by barcoding particles (e.g., Rhapsody beads) and the use of specific targeted panel primers can improve specificity and eliminate potential non-specificity of probes.
  • Targeted probe panels and primer panels are provided herein, as well as in situ probe hybridization and RT kits.
  • RNA probing and amplification on single cell analysis systems e.g., Rhapsody
  • single cell analysis systems e.g., Rhapsody
  • Methods and compositions disclosed herein enable single cell transcriptome profiling of single cells from fixed samples via an RNA probing assay and single cell analysis systems (e.g., Rhapsody).
  • current single cell analysis workflows e.g.. Rhapsody
  • mRNAs are cross-linked and not available to capture by conventional mRNA capture methods (e.g., via poly A/dT capture).
  • mRNA sequence-specific probes can be used to bind to target mRNAs and can be extended by in situ reverse transcription to gain the sequence from the mRNA to gain another layer of specificity.
  • a high number target panel can be designed without limitation of the detection method.
  • cells can be washed to remove un-bound probes and loaded onto single cell analysis platforms (e.g., Rhapsody).
  • Probes with cDNA attached can be released from single cells and can captured by oligonucleotide barcodes associated with barcoding particles (e.g., Rhapsody beads) through a TSO capture oligo via a capture sequence added onto the probe.
  • a user can employ a targeted primer panel to amplify targeted genes from the beads alongside of cell label and UMI to analyze single cell targeted mRNA profiling via sequencing.
  • compositions and methods can open up the usage of archived formaline-fixed cells for single cell RNAseq analysis. Additionally, there are provided, in some embodiments, methods and compositions enabling spatial gene expression studies on FFPE fixed tissue sections. Samples which can be used in current single cell analysis systems (e.g., Rhapsody) are limited to live cells, fresh isolated nuclei or short-time preserved cells. The disclosed compositions and methods can enable the use of long-term stored formalin-fixed samples on single cell analysis systems (e.g., Rhapsody).
  • FIGS. 4A-4D depict a non-limiting exemplary' schematic workflow for gene expression analysis of fixed cells.
  • the workflow can comprise contacting a sample (e.g., a fixed sample comprising fixed cell(s)) with a plurality of probing oligonucleotides (step 400a).
  • Each of the probing oligonucleotides can comprise a coupling sequence and a probe sequence configured hybridize a nucleic acid target within the sample.
  • the 5’ end of each probing oligonucleotide can be phosphorylated.
  • the probing oligonucleotides can be capable of entering a cell and/or a nucleus of the sample (e.g., a permeabilized cell and/or a permeabilized nucleus of the sample).
  • the workflow can comprise after contacting the probing oligonucleotides with the sample, removing one or more probing oligonucleotides of the plurality of probing oligonucleotides that are not contacted with the sample. Removing the one or more probing oligonucleotides not contacted with the sample can comprise removing the one or more probing oligonucleotides that have not entered a cell of the sample.
  • the workflow can comprise extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary' to at least a portion of the nucleic acid target (step 400b).
  • the workflow can comprise contacting the sample with extension reagents.
  • the extension reagents can comprise reverse transcription reagents (e g., a reverse transcriptase and dNTPs).
  • the extension can be performed in situ and can comprise in situ reverse transcription.
  • cells of the sample remain intact during the extension step.
  • the sample can comprise a plurality of cells, and the workflow can comprises disassociating the sample to generate a plurality of single cells.
  • the workflow can comprise partitioning the plurality' of single cells to a plurality' of partitions.
  • the workflow can comprise contacting the plurality' of extended probing oligonucleotides with a barcoding particle (e.g., Rhapsody bead) (step 400c).
  • the barcoding particle e.g., bead
  • the barcoding particle can be associated with a plurality of oligonucleotide barcodes comprising one or more of a cell label (CL), molecular lable (UMI), a 5’ first universal sequence, and a 3 ? TSO (e.g.. capture sequence).
  • the workflow can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides.
  • Barcoding the plurality of extended probing oligonucleotides can comprise: providing a splint oligonucleotide (e.g., coupling oligonucleotide) comprising a 5’ complement of the coupling sequence and a 3’ complement of a capture sequence; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5‘ complement of the coupling sequence of the coupling oligonucleotide; hybridizing the 3’ complement of the capture sequence of the coupling oligonucleotide with a capture sequence of an oligonucleotide barcode of the plurality of oligonucleotide barcodes; and/or ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode.
  • a splint oligonucleotide e.g., coupling oligonucleotide
  • the workflow can comprise amplifying the plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or complement thereof, and amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, thereby generating a plurality of amplified barcoded probing oligonucleotides (step 400d).
  • the amplification primer(s) can comprise a second universal sequence (e.g., R2 primer sequence) and/or the first primer can comprise a third universal sequence.
  • the workflow can comprise obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides, or products thereof (step 400e).
  • Obtaining sequencing data can comprise attaching the binding sites of sequencing primers and/or sequencing adaptors to the plurality of barcoded probing oligonucleotides, or products thereof.
  • the workflow can comprise determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of amplified barcoded probing oligonucleotides, or products thereof.
  • the probing oligonucleotides comprise a predetermined spatial label, and the workflow comprises determining the spatial location and copy number of a nucleic acid target in the sample.
  • Some embodiments provide methods for labeling nucleic acid targets in a sample.
  • the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target.
  • the method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.
  • Some embodiments provide methods for determining the copy number of a nucleic acid target in a sample.
  • the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality' of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target.
  • the method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.
  • Some embodiments provide methods for determining the spatial location and copy number of a nucleic acid target in a sample.
  • the method comprises: contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and a predetermined spatial label.
  • probing oligonucleotides contacted with the same spatial location comprise the same spatial label sequence, and wherein probing oligonucleotides contacted with distinct spatial locations of the sample comprise different spatial label sequences.
  • the method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary’ to at least a portion of the nucleic acid target.
  • the method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.
  • the method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target.
  • the method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a nucleic acid target to determine the copy number of the nucleic acid target at each spatial location of the sample.
  • the method can comprise: contacting the sample with extension reagents. At least a portion of the contacting step can be performed in the presence in the presence of extension reagents. The entire contacting step can be performed in the presence in the presence of the extension reagents. The contacting and extension steps can be simultaneous.
  • the extension can be performed in situ. Said extension can comprise in situ reverse transcription. In some embodiments, cells of the sample remain intact during the extension step.
  • the extension reagents can comprise reverse transcription reagents.
  • Reverse transcription reagents can comprise a reverse transcriptase and dNTPs.
  • the reverse transcriptase can comprise a viral reverse transcriptase.
  • the viral reverse transcriptase can be a murine leukemia virus (MLV) reverse transcriptase or a Moloney murine leukemia virus (MMLV) reverse transcriptase.
  • Barcoding the plurality of extended probing oligonucleotides, or products thereof can comprise: providing a coupling oligonucleotide comprising a 5' complement of the coupling sequence and a 3’ complement of a capture sequence; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5’ complement of the coupling sequence of the coupling oligonucleotide; hybridizing the 3’ complement of the capture sequence of the coupling oligonucleotide with a capture sequence of an oligonucleotide barcode of the plurality of oligonucleotide barcodes; and/or ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode.
  • the method can comprise: before ligating the extended probing oligonucleotide to the oligonucleotide barcode, filling a gap between the extended probing oligonucleotide and the hybridized oligonucleotide barcode with a DNA polymerase lacking at least one of 5 ' to 3’ exonuclease activity and 3’ to 5’ exonuclease activity.
  • Ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode can be performed with a DNA ligase.
  • the coupling oligonucleotide can be a singlestranded oligonucleotide, a double-stranded oligonucleotide, or a mixture thereof.
  • the coupling oligonucleotide can comprise non-natural nucleotides.
  • the coupling oligonucleotide can comprise at least about 1, 2, 3, 4, 5. 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
  • the coupling sequence can comprise at least about 1, 2. 3, 4, 5. 6. 7, 8, 9. 10. 11. 12. 13, 14, 15, 16, 17, 18, 19,
  • each probing oligonucleotide can be phosphorylated.
  • the probing oligonucleotides can be capable of entering a cell and/or a nucleus of the sample (e.g., a permeabilized cell and/or a permeabilized nucleus of the sample).
  • the method can comprise: after contacting the probing oligonucleotides with the sample, removing one or more probing oligonucleotides of the plurality of probing oligonucleotides that are not contacted with the sample, optionally removing the one or more probing oligonucleotides not contacted with the sample comprises: removing the one or more probing oligonucleotides that have not entered a cell of the sample.
  • the contacting step can comprise contacting the sample with a device configured to deposit probing oligonucleotides (e.g., an ink jet device).
  • the device can be a needle, a needle array, a tube, a suction device, an injection device, an electroporation device, a fluorescent activated cell sorter device, an ink jet device, a microfluidic device, or any combination thereof.
  • the device contacts distinct spatial locations of the sample at a specified rate.
  • the spatial label can be at least about 1, 2, 3, 4, 5, 6, 7, 8. 9, 10, 11,
  • Said two or more spatial locations can comprise at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, distinct spatial locations of the sample.
  • a spatial location of the sample corresponds to an area comprising no more than about 50 cells, about 45 cells, about 40 cells, about 35 cells, about 30 cells, about 25 cells, about 20 cells, about 15 cells, about 10 cells, about 9 cells, about 8 cells, about 7 cells, about 6 cells, about 5 cells, about 4 cells, about 3 cells, about 2 cells, about 1 cell, or a number or a range between any two of these values.
  • Each oligonucleotide barcode of the plurality of oligonucleotide barcodes can comprise a first universal sequence.
  • obtaining sequencing data comprises: amplifying the plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or complement thereof, and amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, thereby generating a plurality of amplified barcoded probing oligonucleotides.
  • Obtaining sequencing data can comprise obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides, or products thereof.
  • Obtaining sequencing data can comprise attaching the binding sites of sequencing primers and/or sequencing adaptors to the plurality of barcoded probing oligonucleotides, or products thereof.
  • the amplification primer(s) can comprise a second universal sequence and/or the first primer can comprise a third universal sequence.
  • the first universal sequence, the second universal sequence, and/or the third universal sequence can be the same.
  • the first universal sequence, the second universal sequence, and/or the third universal sequence can be different.
  • the first universal sequence, the second universal sequence, and/or the third universal sequence can comprise the binding sites of sequencing primers and/or sequencing adaptors, complementary sequences thereof, and/or portions thereof.
  • the sequencing adaptors can comprise a P5 sequence, a P7 sequence, complementary' sequences thereof, and/or portions thereof.
  • the sequencing primers can comprise a Read 1 sequencing primer, a Read 2 sequencing primer, complementary sequences thereof, and/or portions thereof.
  • the sample can comprise a plurality of nucleic acid targets, such as, for example, a target panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12. about 14, about 16, about 18. about 20, about 30, about 40. about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, or a number or a range between any two of these values, distinct nucleic acid targets.
  • Two or more nucleic acid targets of the target panel can be biomarkers.
  • the biomarkers can be biomarkers for a disease or condition.
  • the disease or condition can be a cancer, an infection, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a bacterial infection, or any combination thereof.
  • the contacting step can comprise contacting the sample with a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality comprises a probe sequence configured hybridize a nucleic acid target of the plurality of nucleic acid targets.
  • Determining the copy number of the nucleic acid target in the sample can comprise determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of molecular labels with distinct sequences associated with the plurality of barcoded probing oligonucleotides, or products thereof, comprising a sequence of the each of the plurality of nucleic acid targets.
  • the method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with each of the plurality of nucleic acid targets to determine the copy number of each of the plurality of nucleic acid targets at each spatial location of the sample.
  • the amplification primer(s) can comprise a panel of amplification primers configured to hybridize the plurality of nucleic acid targets, or complements thereof, such as, for example, a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9. about 10, about 12, about 14, about 16, about 18, about 20. about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, or a number or a range between any two of these values, distinct amplification primers.
  • a panel of amplification primers configured to hybridize the plurality of nucleic acid targets, or complements thereof, such as, for example, a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9. about 10, about 12, about 14, about 16, about 18, about 20. about 30, about
  • panel shall be given its ordinary meaning and shall also refer to a set of nucleic acids designed to hybridize a set of target nucleic acid sequences of interest, or products thereof.
  • expression analysis of 200 genes can be performed using a panel of 200 different probing oligonucleotides (e.g., a panel of probing oligonucleotides comprising 200 pluralities of probing oligonucleotides) designed to bind the 200 transcripts of said genes.
  • the extension products can be barcoded as described herein and then amplified using a panel of 200 different amplification primers designed to bind a sequence of the 200 nucleic acid targets (or complements thereof).
  • the nucleic acid target can comprise a nucleic acid molecule (e.g., a ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation product, RNA comprising a poly(A) tail, a sample indexing oligonucleotide, a cellular component-binding reagent specific oligonucleotide, or any combination thereof).
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • siRNA small interfering RNA
  • RNA degradation product RNA comprising a poly(A) tail
  • RNA comprising a poly(A) tail
  • sample indexing oligonucleotide e.g., a sample indexing oligonucleotide, a cellular component-binding reagent specific oligonucleotide, or any combination thereof.
  • Each molecular label of the plurality of oligonucleotide barcodes can comprise at least 6 nucleotides.
  • Each capture sequence of the plurality of oligonucleotide barcodes can comprise at least 4 nucleotides.
  • the plurality of oligonucleotide barcodes can be associated with a solid support, and a partition of the plurality' of partitions can comprise a single solid support.
  • the plurality of oligonucleotide barcodes each can comprise a cell label.
  • Each cell label of the plurality of oligonucleotide barcodes can comprise at least 6 nucleotides.
  • Oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with the same solid support can comprise the same cell label. Oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with different solid supports can comprise different cell labels.
  • the solid support can comprise a synthetic particle, a planar surface, or a combination thereof.
  • the method can comprise: associating a synthetic particle comprising the plurality of oligonucleotide barcodes with the cell in the partition.
  • the method can comprise: lysing the cell after associating the synthetic particle with the cell. Lysing the cell can comprise heating the cell, contacting the cell with a detergent, changing the pH of the cell, or any combination thereof.
  • the synthetic particle and the single cell can be in the same partition, and the partition can be a well or a droplet. At least one oligonucleotide barcode of the plurality' of oligonucleotide barcodes can be immobilized or partially immobilized on the synthetic particle, and/or at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes can be enclosed or partially enclosed in the synthetic particle.
  • the synthetic particle can be disruptable (e.g., a disruptable hydrogel particle).
  • the synthetic particle can comprise a bead.
  • the bead can comprise a Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A/G conjugated bead, a protein L conjugated bead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or any combination thereof.
  • the synthetic particle can comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof.
  • PDMS polydimethylsiloxane
  • polystyrene glass
  • polypropylene agarose
  • gelatin hydrogel
  • paramagnetic ceramic
  • plastic glass
  • methylstyrene acrylic polymer
  • titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof methylstyrene
  • Each oligonucleotide barcode of the plurality of oligonucleotide barcodes can comprise a linker functional group.
  • the synthetic particle can comprise a solid support functional group.
  • the support functional group and the linker functional group can be associated with each other, and the linker functional group and the support functional group can be individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), and any combination thereof.
  • the sample can be physically divided or can be intact during the contacting step.
  • the sample can comprise a single cell.
  • the sample can comprise a plurality of single cells.
  • the sample can comprise a plurality of cells, and the method can comprises: disassociating the sample to generate a plurality of single cells.
  • Said disassociating can comprise chemical dissociating, enzymatic dissociating, and/or mechanical dissociating.
  • Said disassociating can employ one or more of collagenase, chymotrypsin, dispase, elastase, hyaluronidase, pancreatin, papain, and trypsin.
  • the method can comprise: prior to the barcoding step: partitioning the plurality of single cells to a plurality 7 of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality 7 of single cells; and in the partition comprising the single cell, contacting the extended probing oligonucleotide with the plurality of oligonucleotide barcodes.
  • the method comprises: in the partition comprising the single cell, contacting the single cell with a lysis buffer at 15-65 °C to lyse the single cell.
  • the lysis buffer can comprise an agent capable of dissociating protein-nucleic acid complexes.
  • the plurality of cells can comprise one or more cell types.
  • Said one or more cell types can be selected from the group consisting of: brain cells, heart cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary' cells, and circulatory cells, or any combination thereof.
  • the sample can comprise a biological sample, a clinical sample, an environmental sample, a biological fluid, a tissue, a tissue section derived from a subject, or any combination thereof.
  • the subject can be a human, a mouse, a dog, a rat, or a vertebrate.
  • the method can comprise: determining genoty pe, phenotype, or one or more genetic mutations of the subject based on the spatial location of the nucleic acid targets in the sample.
  • the method can comprise: predicting susceptibility of the subject to one or more diseases, such as, for example, a cancer or a hereditary disease.
  • the method can comprise: determining cell ty pes of the plurality 7 of cells in the sample.
  • a drug can be chosen based on predicted responsiveness of the cell ty pes of the plurality 7 of cells in the sample.
  • the method can comprise imaging the sample, optionally imaging the sample before the contacting step and/or after the contacting step, optionally the imaging generates imaging data.
  • Imaging the sample can comprise staining the sample with a stain, the stain can be a fluorescent stain, a negative stain, an antibody stain, or any combination thereof.
  • Staining can comprise Immunocytochemistry (ICC), Immunohistochemistry (IHC), Immunofluorescence (IF), or any combination thereof.
  • imaging can comprise microscopy, confocal microscopy, time-lapse imaging microscopy, fluorescence microscopy, multi-photon microscopy, quantitative phase microscopy, surface enhanced Raman spectroscopy, videography, manual visual analysis, automated visual analysis, or any combination thereof.
  • the method can comprise: associating the imaging data and sequencing data of one or more spatial locations of the sample.
  • the method can comprise: correlation analysis of the imaging data and the sequencing data of the spatial locations.
  • the correlation analysis can identify one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of therapeutic agents, and/or cellular targets of candidate therapeutic agents.
  • said imaging produces an image that is used to construct a map of a physical representation of said sample.
  • said map can be two dimensional or three dimensional.
  • the method can comprise: mapping the nucleic acid targets and/or cellular component targets onto the map of the sample.
  • the method can comprise: mapping one or more single cells of the plurality of cells onto the map of the sample.
  • Sequencing reads derived from the same single cell of the plurality of cells can comprise the same cell label. These sequence reads can also comprise the sequence of the spatial label. A user can associate a single cell of the sample with a spatial location of the sample based on the association of the cell label and the spatial label.
  • the sample can comprise a plurality of cellular component targets, and the method can comprises: contacting a plurality of cellular component-binding reagents with the sample, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular componentbinding reagent is capable of specifically binding to at least one of the plurality of cellular component targets; barcoding the cellular component-binding reagent specific oligonucleotides to generate a plurality of barcoded cellular component-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique identifier sequence and a molecular label sequence; and obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides
  • the cellular component target can comprise an intracellular protein, a carbohydrate, a lipid, a protein, an extracellular protein, a cell-surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.
  • the cellular component-binding reagent specific oligonucleotide can comprise a second molecular label, optionally at least ten of the plurality of cellular component-binding reagent specific oligonucleotides comprise different second molecular label sequences.
  • the second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides can be different, and the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides can be identical.
  • the second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides can be different, and the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides can be different.
  • the number of unique molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample. In some embodiments, the number of unique second molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample.
  • cellular component-binding reagent specific oligonucleotides are barcoded using the same plurality of oligonucleotides barcodes as used to barcode the extended probing oligonucleotides.
  • the solid support comprises two or more pluralities of oligonucleotide barcodes wherein each plurality comprises a distinct 3’ target-binding region or capture sequence.
  • the extended probing oligonucleotides are barcoded with a first plurality of oligonucleotides barcodes having a 3’ capture sequence configured to hybridize to a coupling oligonucleotide (e.g., TSO bait) and cellular component-binding reagent specific oligonucleotides are barcoded using a second plurality of oligonucleotide barcodes having a 3’ poly(dT) sequence.
  • the cellular component-binding reagent specific oligonucleotides comprise a poly(dA) sequence.
  • Sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof can each comprise a cell label sequence.
  • a user can associate a single cell of the sample with a spatial location of the sample based on the association of the cell label and the spatial label. Accordingly, a user can thereby determine the spatial location and copy number of a cellular component target in a sample based on the spatial label (and thereby spatial location) associated with said cell label in the sequencing data.
  • Embodiments of using cellular component binding reagents e.g., proteinbinding regents
  • oligonucleotides for example, oligo-conjugated antibodies (AbOs) and oligo-conjugated aptamers
  • oligonucleotides for example, oligo-conjugated antibodies (AbOs) and oligo-conjugated aptamers
  • sample tracking e.g., tracking sample origins
  • the systems, methods, compositions, and kits provided herein can, in some embodiments, be employed in concert with the systems, methods, compositions, and kits described in PCT Application Publication No. WO/2021/163374. the content of which is incorporated herein by reference in its entirety.
  • the systems, methods, compositions, and kits provided herein can, in some embodiments, be employed in concert with the systems, methods, compositions, and kits described in PCT Application Publication Nos. WO/2024/097719 and WO/2024/097718; the content of each of these applications is incorporated herein by reference in its entirety.
  • the sample can comprise a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof.
  • the sample can comprise a fresh tissue section, a frozen tissue section, a fixed tissue section, a formalin-fixed tissue section, a formalin-fixed paraffin- embedded (FFPE) tissue section, an acetone fixed tissue section, a paraformaldehyde (PFA) fixed tissue section, and/or a methanol fixed tissue section.
  • the sample can comprise a nuclei suspension, such as, for example, a fixed nuclei suspension and/or a permeabilized nuclei suspension.
  • the sample has been contacted with one or more fixing agents and/or permeabilizing agents.
  • the sample can comprise cell(s), such as.
  • the method can comprise: permeabilizing the sample and/or fixing the sample.
  • Fixing the sample can comprise contacting the sample with a fixing agent.
  • the fixing agent can comprise a non-cross-linking fixative (e.g., methanol).
  • the fixing agent can comprise a cross-linking agent.
  • the cross-linking agent can comprise a cleavable cross-linking agent.
  • the cleavable cross-linking agent can comprise or can be derived from dithiobis(succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST).
  • DSP dithiobis(succinimidyl propionate)
  • DST disuccinimidyl tartrate
  • BSOCOES Bis [2-(Succinimidooxycarbonyloxy)ethyl] Sulfone
  • EGS ethylene glycol bis(succinimidyl succinate)
  • DTBP dimethyl 3.3'- dithiobispropionimidate
  • SPDP succinimidyl 3-(2-pyridyldithio)propionate
  • SPDP succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate
  • LC-SPDP 4- succinimidyloxycarbonyl-alpha-methyl-a(2-pyridyldithio)tolu
  • the cleavable crosslinking agent can comprise a cleavable linkage selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, an acid labile linker, a heat sensitive linkage, an enzymatically cleavable linkage, and a combination thereof.
  • the cleavable cross- linking agent can be a thiol-cleavable cross-linking agent or can comprise a disulfide linker.
  • the fixing agent can comprise paraformaldehyde (PF A), dithiobis(succinimidyl propionate (DSP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), CellCover, or a combination thereof. Fixing the sample and permeabilizing the sample can be carried out simultaneously.
  • PF A paraformaldehyde
  • DSP dithiobis(succinimidyl propionate
  • SPDP succinimidyl 3-(2-pyridyldithio)propionate
  • CellCover or a combination thereof. Fixing the sample and permeabilizing the sample can be carried out simultaneously.
  • Fixing and permeabilizing the sample can be carried out in the presence of a dual function agent capable of fixing and permeabilizing the sample.
  • the dual functional agent can be methanol.
  • Permeabilizing the sample can comprise contacting the sample with a permeabilizing agent.
  • the method can comprise: after contacting the plurality of probing oligonucleotides or the plurality of cellular component-binding reagents with the sample, removing the permeabilizing agent from the sample.
  • the permeabilizing agent can be capable of (i) permeabilizing the cell membrane of the cell(s), (ii) making a cell membrane of the cell(s) permeable to the probing oligonucleotides or the cellular component-binding reagents, or both.
  • the permeabilizing agent can comprise (i) a solvent, a detergent, or a surfactant; (ii) BD Cytoperm; (iii) a saponin or a derivative thereof; (iv) Triton X-100, (v) methanol or a derivative thereof, and/or (vi) digitonin or a derivative thereof.
  • the agent capable of dissociating protein- nucleic acid complexes can comprise a broad-spectrum serine protease.
  • the broad-spectrum serine protease can be proteinase K.
  • the lysis buffer can comprise an unfixing agent.
  • the unfixing agent can comprise a thiol, hydoxylamine, periodate, a base, or any combination thereof.
  • the lysis buffer can comprise DTT.
  • the method can comprise: reversing the fixation of the sample and/or single cells. Reversing the fixation of the sample and/or single cells can comprise UV photocleaving, chemical treatment, heating, enzyme treatment, or any combination thereof.
  • the method can comprise: prior to contacting a plurality of cellular component-binding reagents with the sample and/or contacting the plurality of probing oligonucleotides with the sample, contacting the sample with a blocking reagent, one or more decoy oligonucleotides, and/or one or more blocking oligonucleotides.
  • a blocking reagent one or more decoy oligonucleotides
  • the methods and compositions provided herein can be employed in concert with the methods and compositions described in PCT Patent Application Number PCT/US22/75661, filed on August 30, 2022, entitled “RNA PRESERVATION AND RECOVERY FROM FIXED CELLS”, the content of which is incorporated herein by reference in its entirety.
  • the methods and compositions provided herein can be employed in concert with blocking reagents, such as those described in PCT Patent Application Number PCT/US22/75656, filed on August 30, 2022, entitled “USE OF DECOY POLYNUCLEOTIDES IN SINGLE CELL MULTIOMICS”, the content of which is incorporated herein by reference in its entirety.
  • Contacting a plurality of cellular component- binding reagents with the sample can be conducted in the presence of a blocking reagent.
  • the blocking reagent can comprise a plurality of oligonucleotides complementary to at least a portion of the cellular component-binding reagent specific oligonucleotides.
  • the blocking reagent can comprise an antibody or a fragment thereof derived from a first species, and the blocking reagent can comprise sera derived from the first species.
  • the sample can comprise one or more non-target nucleic acids, and the blocking reagent can comprise a plurality' of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acids. Each of the plurality of decoy oligonucleotides can be capable of hybridizing to at least a portion of a non-target nucleic acid.
  • the decoy oligonucleotide comprises a sequence complementary to at least a portion of a non-target nucleic acid; comprises a sequence identical to or substantially similar to a sequence of the cellular component-binding reagents specific oligonucleotides, optionally the sequence is 3-40 nucleotides in length; has at most 50% sequence identity to the cellular component-binding reagent specific oligonucleotides; does not comprise a UMI; comprises a random sequence, and optionally the random sequence is about four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen nucleotides in length; does not comprise any sequence having more than four, five, six, or seven consecutive Ts or As; comprise at least one G or C in every' four, five, six.
  • nucleotides comprises one or more modified nucleotides; comprises a 5’ modification, and optionally the 5’ modification comprises a 5’ Amino Modifier C12 modification (5AmMC12); comprises a 3’ modification, and optionally the 3’ modification comprises a 3' dideoxy-C modification (ddC); and/or is 30 to 65 nucleotides in length.
  • 5AmMC12 Amino Modifier C12 modification
  • ddC dideoxy-C modification
  • the sample can comprise one or more undesirable nucleic acid species
  • the method can comprise: contacting a blocking oligonucleotide with the sample, wherein the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleic acid species, and whereby the reverse transcription of the at least one of the one or more undesirable nucleic acid species is reduced by the blocking oligonucleotide.
  • the blocking oligonucleotide is contacted with the sample before the plurality of probing oligonucleotides is contacted with the sample; is contacted with the sample after the plurality of probing oligonucleotides is contacted with the sample; and/or is contacted with the sample when the plurality of probing oligonucleotides is contacted with the sample.
  • the method can comprise: providing blocking oligonucleotides that specifically bind to two or more undesirable nucleic acid species, optionally at least 10 or to at least 100 undesirable nucleic acid species, in the sample.
  • the blocking oligonucleotide is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, or a PNA/DNA chimera; specifically binds to within 100 nt, to within 50 nt, or to wi thin 25 nt of the 3’ end of the one or more undesirable nucleic acid species; specifically binds to within 100 nt of the 5’ end of the one or more undesirable nucleic acid species, or the blocking oligonucleotide specifically binds to within 100 nt of the middle of the one or more undesirable nucleic acid species; comprises or does not comprise non-natural nucleotides; has a Tm of at least 50 °C.
  • the one or more undesirable nucleic acid species can amount to about 50%, to about 60%, to about 70%, or to about 80% of the nucleic acid content of the sample.
  • the undesirable nucleic acid species can be selected from the group consisting of ribosomal RNA, mitochondrial RNA, genomic DNA, intronic sequence, high abundance sequence, and a combination thereof.
  • the one or more undesirable nucleic acid species can be mRNA molecules and the blocking oligonucleotide can specifically binds to within 10 nt of the 3' poly (A) tail of the one or more undesirable nucleic acid species.
  • compositions e.g., kits.
  • the kit comprises: a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize a nucleic acid target, optionally the probing oligonucleotides comprise a predetermined spatial label; a coupling oligonucleotide comprising a 5’ complement of the coupling sequence and a 3’ complement of a capture sequence; a plurality of oligonucleotide barcodes, wherein the 3‘ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes is associated with a solid support, wherein the 5’ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a capture sequence; a first primer capable of hybridizing to the first universal sequence,
  • the plurality of probing oligonucleotides can comprise a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality 7 comprises a probe sequence configured hybridize a nucleic acid target of a plurality of nucleic acid targets, optionally a target panel of at least about 2, about 3. about 4, about 5. about 6, about 7. about 8, about 9, about 10, about 12. about 14. about 16, about 18, about 20.
  • nucleic acid targets about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, or a number or a range between any two of these values, distinct nucleic acid targets.
  • the amplification primer(s) can comprise a panel of amplification primers configured to hybridize a plurality 7 of nucleic acid targets, or complements thereof, optionally a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12. about 14, about 16, about 18, about 20. about 30, about 40, about 50, about 60. about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, or a number or a range between any two of these values, distinct amplification primers.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des systèmes, des procédés, des compositions et des kits pour déterminer l'emplacement spatial et/ou le nombre de copies d'une cible d'acide nucléique dans un échantillon fixe. Selon certains modes de réalisation, la présente invention concerne des oligonucléotides de sondage contenant une séquence de couplage, une séquence de sonde conçue pour hybrider la cible d'acide nucléique et/ou un marqueur spatial prédéterminé. Le procédé peut comprendre la mise en contact de chacun d'au moins deux emplacements spatiaux d'un échantillon comprenant des copies d'une cible d'acide nucléique avec lesdits oligonucléotides de sondage. Le procédé peut comprendre l'extension in situ des oligonucléotides de sondage hybridés aux copies d'une cible d'acide nucléique pour générer une pluralité d'oligonucléotides de sondage étendus. Le procédé peut comprendre le codage à barres de la pluralité d'oligonucléotides de sondage étendus à l'aide d'une pluralité de codes-barres oligonucléotidiques pour générer une pluralité d'oligonucléotides de sondage à code-barres.
PCT/US2024/030552 2023-05-23 2024-05-22 Procédés de sondage et d'amplification d'arn pour analyse de cellule unique sur des cellules fixes Pending WO2024243298A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202480033772.7A CN121152885A (zh) 2023-05-23 2024-05-22 用于固定细胞上单细胞分析的rna探测和扩增方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363503856P 2023-05-23 2023-05-23
US63/503,856 2023-05-23

Publications (1)

Publication Number Publication Date
WO2024243298A1 true WO2024243298A1 (fr) 2024-11-28

Family

ID=91586044

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/030552 Pending WO2024243298A1 (fr) 2023-05-23 2024-05-22 Procédés de sondage et d'amplification d'arn pour analyse de cellule unique sur des cellules fixes

Country Status (2)

Country Link
CN (1) CN121152885A (fr)
WO (1) WO2024243298A1 (fr)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110160078A1 (en) 2009-12-15 2011-06-30 Affymetrix, Inc. Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels
WO2015031691A1 (fr) 2013-08-28 2015-03-05 Cellular Research, Inc. Analyse massivement parallèle de cellules uniques
US20180088112A1 (en) 2016-09-26 2018-03-29 Cellular Research, Inc. Measurement of protein expression using reagents with barcoded oligonucleotide sequences
US20180346970A1 (en) 2017-06-05 2018-12-06 Cellular Research, Inc. Sample indexing for single cells
WO2020037065A1 (fr) 2018-08-17 2020-02-20 Cellular Research, Inc. Codage à barres par aptamères
WO2021163374A2 (fr) 2020-02-12 2021-08-19 Becton, Dickinson And Company Abseq intracellulaire
US20210371909A1 (en) * 2020-06-02 2021-12-02 Becton, Dickinson And Company Oligonucleotides and beads for 5 prime gene expression assay
WO2023034872A1 (fr) * 2021-09-01 2023-03-09 Becton, Dickinson And Company Multiomique spatiale utilisant la transcription inverse in situ
WO2024097719A1 (fr) 2022-11-02 2024-05-10 Becton, Dickinson And Company Application pour bloqueur d'acide nucléique peptidique (pna)
WO2024097718A1 (fr) 2022-11-02 2024-05-10 Becton, Dickinson And Company Modification d'extrémité à médiation par polymérase d'abseq

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110160078A1 (en) 2009-12-15 2011-06-30 Affymetrix, Inc. Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels
WO2015031691A1 (fr) 2013-08-28 2015-03-05 Cellular Research, Inc. Analyse massivement parallèle de cellules uniques
US20150299784A1 (en) 2013-08-28 2015-10-22 Cellular Research, Inc. Massively parallel single cell analysis
US20180088112A1 (en) 2016-09-26 2018-03-29 Cellular Research, Inc. Measurement of protein expression using reagents with barcoded oligonucleotide sequences
US20180346970A1 (en) 2017-06-05 2018-12-06 Cellular Research, Inc. Sample indexing for single cells
WO2020037065A1 (fr) 2018-08-17 2020-02-20 Cellular Research, Inc. Codage à barres par aptamères
WO2021163374A2 (fr) 2020-02-12 2021-08-19 Becton, Dickinson And Company Abseq intracellulaire
US20210371909A1 (en) * 2020-06-02 2021-12-02 Becton, Dickinson And Company Oligonucleotides and beads for 5 prime gene expression assay
WO2023034872A1 (fr) * 2021-09-01 2023-03-09 Becton, Dickinson And Company Multiomique spatiale utilisant la transcription inverse in situ
WO2024097719A1 (fr) 2022-11-02 2024-05-10 Becton, Dickinson And Company Application pour bloqueur d'acide nucléique peptidique (pna)
WO2024097718A1 (fr) 2022-11-02 2024-05-10 Becton, Dickinson And Company Modification d'extrémité à médiation par polymérase d'abseq

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
FAN ET AL., SCIENCE, vol. 347, no. 6222, 6 February 2015 (2015-02-06), pages 1258367
FILIP VAN NIEUWERBURGH ET AL: "Quantitative Bias in Illumina TruSeq and a Novel Post Amplification Barcoding Strategy for Multiplexed DNA and Small RNA Deep Sequencing", PLOS ONE, vol. 6, no. 10, 1 January 2011 (2011-01-01), pages e26969, XP055157339, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0026969 *
FU ET AL., PROC NATL ACAD SCI U.S.A., vol. 108, no. 22, 31 May 2011 (2011-05-31), pages 9026 - 31
KURSCHAT WOLFRAM C ET AL: "Optimizing splinted ligation of highly structured small RNAs", RNA, COLD SPRING HARBOR LABORATORY PRESS, US, vol. 11, no. 12, 1 December 2005 (2005-12-01), pages 1909 - 1914, XP002467859, ISSN: 1355-8382, DOI: 10.1261/RNA.2170705 *
SAMBROOK ET AL.: "A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS, article "Molecular Cloning"
SINGH RAJESH R.: "Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology", DIAGNOSTICS, vol. 12, no. 7, 24 June 2022 (2022-06-24), pages 1539, XP093093035, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9318977/pdf/diagnostics-12-01539.pdf> DOI: 10.3390/diagnostics12071539 *
SINGLETON ET AL.: "Dictionary of Microbiology and Molecular Biology", 1994, J. WILEY & SONS

Also Published As

Publication number Publication date
CN121152885A (zh) 2025-12-16

Similar Documents

Publication Publication Date Title
US20220348904A1 (en) Molecular barcoding on opposite transcript ends
US12391940B2 (en) Single cell assay for transposase-accessible chromatin
US11932901B2 (en) Target enrichment using nucleic acid probes for scRNAseq
US20230109336A1 (en) Selective extension in single cell whole transcriptome analysis
US12421548B2 (en) High throughput multiomics sample analysis
EP4242324B1 (fr) Codage à barres par aptamères
US20220010362A1 (en) cDNA SPIKE-IN CONTROL FOR SINGLE CELL ANALYSIS
US20200263169A1 (en) Hybrid targeted and whole transcriptome amplification
US20250109427A1 (en) Preparing nucleic acid for further analysis of their sequence
WO2024097719A1 (fr) Application pour bloqueur d&#39;acide nucléique peptidique (pna)
EP4612322A1 (fr) Modification d&#39;extrémité à médiation par polymérase d&#39;abseq
WO2024243298A1 (fr) Procédés de sondage et d&#39;amplification d&#39;arn pour analyse de cellule unique sur des cellules fixes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24734576

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: CN2024800337727

Country of ref document: CN