US20180105808A1 - Methods and systems for barcoding nucleic acid molecules from individual cells or cell populations - Google Patents

Methods and systems for barcoding nucleic acid molecules from individual cells or cell populations Download PDF

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US20180105808A1
US20180105808A1 US15/825,740 US201715825740A US2018105808A1 US 20180105808 A1 US20180105808 A1 US 20180105808A1 US 201715825740 A US201715825740 A US 201715825740A US 2018105808 A1 US2018105808 A1 US 2018105808A1
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sequence
nucleic acid
cell
barcode
bead
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Tarjei Sigurd Mikkelsen
Phillip Belgrader
Xinying Zheng
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10X Genomics Inc
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10X Genomics Inc
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Priority to US15/825,740 priority Critical patent/US20180105808A1/en
Assigned to 10X GENOMICS, INC. reassignment 10X GENOMICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BELGRADER, PHILLIP, MIKKELSEN, TARJEI SIGURD, ZHENG, Xinying
Assigned to 10X GENOMICS, INC. reassignment 10X GENOMICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BELGRADER, PHILLIP, MIKKELSEN, TARJEI SIGURD, ZHENG, Xinying
Publication of US20180105808A1 publication Critical patent/US20180105808A1/en
Priority to US17/131,494 priority patent/US20210238581A1/en
Priority to US17/692,792 priority patent/US20220333191A1/en
Priority to US17/692,758 priority patent/US20220389503A1/en
Priority to US18/301,565 priority patent/US20240067953A1/en
Abandoned legal-status Critical Current

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Definitions

  • Nucleic acid sequencing can be used to obtain information in a wide variety of biomedical contexts, including diagnostics, prognostics, biotechnology, and forensic biology. Sequencing may involve methods including Maxam-Gilbert sequencing and chain-termination methods, or de novo sequencing methods including shotgun sequencing and bridge PCR, or next-generation methods including polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, HeliScope single molecule sequencing, SMRT® sequencing, and others. Nucleic acid sequencing technologies, including next-generation DNA sequencing, have been useful for genomic and proteomic analysis of cell populations.
  • Such cells include, but are not limited to, cancer cells, fetal cells, and immune cells involved in immune responses.
  • methods, compositions and systems for analyzing individual cells or a small population of cells including the analysis and attribution of nucleic acids from and to these individual cells or cell populations.
  • the present disclosure provides a method for nucleic acid sequencing, comprising (a) providing a plurality of droplets, wherein a droplet of the plurality of droplets comprises (i) a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, and (ii) a bead comprising a nucleic acid barcode molecule coupled thereto, wherein the nucleic acid barcode molecule comprises a barcode sequence; (b) using the RNA molecule and the nucleic acid barcode molecule to generate a barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, a sequence corresponding to the nucleic acid sequence of the RNA molecule and a complement of the barcode sequence; and (c) sequencing the barcoded nucleic acid molecule or a derivative thereof.
  • RNA ribonucleic acid
  • a bead comprising a nucleic acid barcode molecule coupled thereto, wherein the nucleic acid barcode
  • the RNA molecule is from a cell. In some embodiments, the droplet comprises the cell. In some embodiments, the method further comprises releasing the RNA molecule from the cell prior to (b).
  • the bead comprises a plurality of nucleic acid molecules coupled thereto, wherein the plurality of nucleic acid molecules comprises the nucleic acid barcode molecule.
  • each of the plurality of nucleic acid molecules comprises the barcode sequence. In some embodiments, each of the plurality of nucleic acid molecules comprises an additional barcode sequence that varies across the plurality of nucleic acid molecules.
  • the nucleic acid barcode molecule comprises a template switching sequence.
  • the method further comprises, prior to (c), subjecting the barcoded nucleic acid molecule or derivative thereof to nucleic acid amplification.
  • the nucleic acid amplification is performed subsequent to releasing the barcoded nucleic acid molecule or derivative thereof from the droplet.
  • the nucleic acid amplification is polymerase chain reaction.
  • the RNA molecule is a messenger ribonucleic acid (mRNA) molecule.
  • the droplet comprises (i) an additional nucleic acid molecule comprising an additional nucleic acid sequence, and (ii) an additional nucleic acid barcode molecule comprising an additional barcode sequence, and wherein in (b) the additional nucleic acid molecule and the additional nucleic acid barcode molecule are used to generate an additional barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, the additional barcode sequence and an additional sequence corresponding to the additional nucleic acid sequence.
  • the additional nucleic acid barcode molecule is coupled to the bead. In some embodiments, the additional nucleic acid barcode molecule is coupled to an additional bead.
  • (b) is performed in the droplet.
  • the method further comprises releasing the barcoded nucleic acid molecule or a derivative thereof from the droplet.
  • the barcoded nucleic acid molecule further comprises, towards a 3′ end, a functional sequence for permitting the barcoded nucleic acid molecule or a derivative thereof to couple to a flow cell of a sequencer.
  • the sequence is a reverse complement of the nucleic acid sequence.
  • the method further comprises, prior to (c), using the barcoded nucleic acid molecule or a derivative thereof and a pair of primers to generate a subset of nucleic acids having a target nucleic acid sequence.
  • the target nucleic acid sequence comprises a T cell receptor variable region sequence, a B cell receptor variable region sequence, or an immunoglobulin variable region sequence.
  • the at least one of the pair of primers hybridizes to a constant region of a T cell receptor nucleic acid sequence, a constant region of a B cell receptor nucleic acid sequence, or a constant region of an immunoglobulin nucleic acid sequence.
  • the subset of nucleic acids or derivatives thereof are sequenced in (c).
  • the method further comprises releasing the nucleic acid barcode molecule from the bead.
  • the nucleic acid barcode molecule is released from the bead before the barcoded nucleic acid molecule is generated.
  • the nucleic acid barcode molecule is released from the bead while the barcoded nucleic acid molecule is generated.
  • the nucleic acid barcode molecule is released from the bead after the barcoded nucleic acid molecule is generated.
  • the bead is a gel bead.
  • the barcode sequence is a combinatorial assembly of a plurality of barcode segments.
  • the plurality of barcode segments comprises at least three segments.
  • the present disclosure provides a method for generating a labeled polynucleotide.
  • the method comprises (a) subjecting a reaction mixture to a first amplification reaction under conditions sufficient to generate a first amplification product, wherein the reaction mixture comprises a template polynucleotide and (i) a primer having a sequence towards a 3′ end that hybridizes to the template polynucleotide, and (ii) a template switching oligonucleotide that comprises a first predefined sequence towards a 5′ end; and (b) subjecting the first amplification product to a second amplification reaction in the presence of a barcoded oligonucleotide under conditions sufficient to generate a second amplification product, wherein the barcoded oligonucleotide comprises a sequence of at least a segment of the template switching oligonucleotide and at least a second predefined sequence, wherein (i) the second amplification reaction uses the first amplification product as
  • the second amplification reaction uses the first amplification product as a template and the barcoded oligonucleotide as a primer. In some embodiments, the second amplification reaction uses the barcoded oligonucleotide as a template and at least a portion of the first amplification product as a primer.
  • the present disclosure provides a method for generating a labeled polynucleotide comprising (a) providing a reaction mixture in a reaction volume, wherein the reaction mixture comprises (i) a template polynucleotide, (ii) a primer comprising a sequence towards a 3′ end of the primer that hybridizes to the template polynucleotide, and (iii) a template switching oligonucleotide; (b) in the reaction volume, subjecting the reaction mixture to a first reaction under conditions sufficient to generate a first nucleic acid product comprising the primer, a reverse complement of a sequence of the template polynucleotide, and a sequence complementary to at least a portion of the template switch oligonucleotide; and (c) subjecting the first nucleic acid product to a second reaction in the reaction volume, which second reaction comprises using (i) the first nucleic acid product as a template and a barcoded oligonucleotide as a
  • the template polynucleotide is obtained from a single cell.
  • the single cell is an immune cell.
  • the immune cell is a T-cell.
  • the immune cell is a B-cell.
  • the method further comprises lysing the single cell in the same reaction volume to obtain the template polynucleotide prior to generating the first amplification product in the first amplification reaction.
  • the template polynucleotide comprises a T-cell receptor gene or gene product. In some embodiments, the template polynucleotide comprises a B-cell receptor gene or gene product. In some embodiments, the template polynucleotide is among a plurality of template polynucleotides.
  • a concentration of the template switching oligonucleotide in the same reaction volume is at least two times that of a concentration of the barcoded oligonucleotide in the same reaction volume. In some embodiments, a concentration of the template switching oligonucleotide in the same reaction volume is at least five times that of a concentration of the barcoded oligonucleotide in the same reaction volume. In some embodiments, a concentration of the template switching oligonucleotide in the same reaction volume is at least ten times that of a concentration of the barcoded oligonucleotide in the same reaction volume.
  • a concentration of the template switching oligonucleotide in the same reaction volume is at least twenty times that of a concentration of the barcoded oligonucleotide in the same reaction volume. In some embodiments, a concentration of the template switching oligonucleotide in the same reaction volume is at least fifty times that of a concentration of the barcoded oligonucleotide in the same reaction volume. In some embodiments, a concentration of the template switching oligonucleotide in the same reaction volume is at least one hundred times that of a concentration of the barcoded oligonucleotide in the same reaction volume. In some embodiments, a concentration of the template switching oligonucleotide in the same reaction volume is at least two hundred times that of a concentration of the barcoded oligonucleotide in the same reaction volume.
  • the primer comprises a sequence towards a 5′ end that does not specifically hybridize to the template polynucleotide.
  • the first amplification reaction is facilitated using an enzyme comprising polymerase activity.
  • the enzyme is a DNA-dependent polymerase.
  • the enzyme is a reverse transcriptase.
  • the second amplification reaction is facilitated using an enzyme comprising polymerase activity.
  • the enzyme is a DNA-dependent polymerase.
  • the first amplification reaction comprises polymerase chain reaction. In some embodiments, the first amplification reaction comprises reverse transcription. In some embodiments, the second amplification reaction comprises polymerase chain reaction.
  • the first amplification reaction and the second amplification reaction are performed sequentially in the absence of an intervening purification step.
  • the template switching oligonucleotide is not available for primer extension during the second amplification reaction.
  • the method further comprises degrading the template switching oligonucleotide prior to the second amplification reaction.
  • the template switching oligonucleotide comprises ribonucleic acids (RNA). In some embodiments, the template switching oligonucleotide comprises at least 10% ribonucleic acids (RNA).
  • the method further comprises degrading the template switching oligonucleotide during the second amplification reaction.
  • the template switching oligonucleotide comprises ribonucleic acids (RNA). In some embodiments, the template switching oligonucleotide comprises at least 10% ribonucleic acids (RNA).
  • a first reaction rate of the second amplification reaction using the barcoded oligonucleotide is greater than a second reaction rate of the second amplification using the template switching oligonucleotide.
  • the first amplification product and the barcoded oligonucleotide has a higher melting temperature as compared to a melting temperature of the first amplification product and the template switching oligonucleotide.
  • a primer annealing temperature of the second amplification reaction is at least 0.5° C. greater than a primer annealing temperature of the first amplification reaction.
  • the template switching oligonucleotide comprises modified nucleotides. In some embodiments, the template switching oligonucleotide comprises at least 10% modified nucleotides. In some embodiments, the template switching oligonucleotide comprises modified nucleotides selected from unlocked nucleic acids (UNAs), locked nucleic acids (LNAs), and 5-hydroxybutynl-2′-deoxyuridine.
  • UNAs unlocked nucleic acids
  • LNAs locked nucleic acids
  • 5-hydroxybutynl-2′-deoxyuridine 5-hydroxybutynl-2′-deoxyuridine.
  • the barcoded oligonucleotide comprises modified nucleotides. In some embodiments, the barcoded oligonucleotide comprises at least 10% modified nucleotides. In some embodiments, the barcoded oligonucleotide comprises modified nucleotides selected from locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), and 5-hydroxybutynl-2′-deoxyuridine.
  • LNAs locked nucleic acids
  • UNAs unlocked nucleic acids
  • 5-hydroxybutynl-2′-deoxyuridine 5-hydroxybutynl-2′-deoxyuridine.
  • the same reaction volume comprises an emulsion, a droplet, or a microwell.
  • the first defined sequence comprises at least one of an adaptor sequence, a barcode sequence, a unique molecular identifier sequence, a primer binding site, and a sequencing primer binding site.
  • the second defined sequence comprises at least one of an adaptor sequence, a barcode sequence, a unique molecular identifier sequence, a primer binding site, and a sequencing primer binding site.
  • the primer is among a plurality of primers.
  • the sequence towards the 3′ end of the primer comprises a random sequence.
  • the sequence towards the 3′ end of the primer comprises a gene specific sequence.
  • the sequence towards the 3′ end of the primer comprises a polyA sequence.
  • the template switching oligonucleotide is among a plurality of template switching oligonucleotides.
  • the barcoded oligonucleotide is among a plurality of barcoded oligonucleotides.
  • the method further comprises subjecting the second amplification product to sequencing.
  • the barcoded oligonucleotide is releasably coupled to a microcapsule. In some embodiments, the method further comprises releasing the barcoded oligonucleotide from the microcapsule. In some embodiments, the barcoded oligonucleotide is released from the microcapsule upon application of a stimulus. In some embodiments, the stimulus is at least one of a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, a photo stimulus, or any combination thereof. In some embodiments, the microcapsule is a degradable microcapsule and releasing the barcoded oligonucleotide comprises degrading the microcapsule.
  • the microcapsule comprises a polymer gel. In some embodiments, the polymer gel is a polyacrylamide. In some embodiments, the microcapsule comprises a bead. In some embodiments, the bead is a gel bead. In some embodiments, the microcapsule comprises a chemical cross-linker. In some embodiments, the chemical cross-linker is a disulfide bond.
  • the present disclosure provides a method comprising (a) providing a reaction volume comprising (i) a cell or cell derivative, and (ii) a bead comprising a barcoded oligonucleotide releasably coupled thereto, wherein the barcoded oligonucleotide is a template switching oligonucleotide; and (b) releasing the barcoded oligonucleotide from the bead to provide the barcoded oligonucleotide in the reaction volume at a concentration of at least about 0.20 ⁇ M; and (c) subjecting the reaction volume to an amplification reaction to generate an amplification product, wherein during the amplification reaction, the reaction volume comprises a template polynucleotide from the cell or cell derivative, the barcoded oligonucleotide and a primer having a sequence towards a 3′ end that hybridizes to the template polynucleotide, and wherein the amplification product has sequence
  • the present disclosure provides a method comprising (a) providing a reaction volume comprising a cell and a microcapsule comprising a barcoded oligonucleotide releasably coupled thereto, wherein the barcoded oligonucleotide is a template switching oligonucleotide; and (b) subjecting the reaction volume to dissociation conditions sufficient to release the barcoded oligonucleotide from the microcapsule, thereby providing the barcoded oligonucleotide in the reaction volume at a concentration of at least about 0.20 uM; and (c) subjecting the reaction volume to an amplification reaction to generate an amplification product, wherein during the amplification reaction, the reaction volume comprises a template polynucleotide from the cell, the barcoded oligonucleotide and a primer having a sequence towards a 3′ end that hybridizes to the template polynucleotide, and wherein the amplification product has sequence complement
  • the method further comprises subjecting the amplification product to sequencing.
  • the barcoded oligonucleotide does not hybridize to the template polynucleotide.
  • the template polynucleotide is an mRNA molecule.
  • the method further comprises subjecting the reaction volume to a second amplification reaction to generate an additional amplification product using the amplification product as a template.
  • the method further comprises subjecting the additional amplification product to sequencing.
  • the cell is a mammalian cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a B-cell. In some embodiments, the immune cell is a T-cell. In some embodiments, the cell is cancer cell. In some embodiments, the cancer cell is obtained from a tissue sample. In some embodiments, the cancer cell is obtained from a biological fluid. In some embodiments, the biological fluid comprises blood. In some embodiments, the biological fluid comprises lymph fluid.
  • the template polynucleotide comprises a T cell receptor gene sequence, a B cell receptor gene sequence, or an immunoglobulin gene sequence. In some embodiments, the template polynucleotide is a T cell receptor mRNA molecule, a B cell receptor mRNA molecule, or an immunoglobulin mRNA molecule.
  • the reaction volume further comprises an enzyme.
  • the enzyme is a DNA polymerase.
  • the enzyme is a reverse transcriptase.
  • the reaction volume further comprises at least one reagent for nucleic acid amplification.
  • the at least one reagent comprises dNTPs.
  • the at least one reagent comprises oligonucleotide primers.
  • the microcapsule comprises a polymer gel. In some embodiments, the polymer gel is a polyacrylamide. In some embodiments, the microcapsule comprises a bead. In some embodiments, the bead is a gel bead. In some embodiments, the microcapsule comprises a chemical cross-linker. In some embodiments, the chemical cross-linker is a disulfide bond. In some embodiments, the dissociation condition is at least one of a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, a photo stimulus, or any combination thereof.
  • the barcoded oligonucleotide comprises at least one of an adaptor sequence, a barcode sequence, unique molecular identifier sequence, a primer binding site, and a sequencing primer binding site.
  • the same reaction volume comprises an emulsion, a droplet, or a microwell.
  • the method further comprises performing a third reaction, wherein the third reaction specifically amplifies variable region cDNAs, wherein the variable region cDNA are derived from a T cell receptor cDNA, a B cell receptor cDNA, or an immunoglobulin cDNA.
  • the third reaction comprises use of a primer that specifically binds in the constant region of the T cell receptor cDNA, B cell receptor cDNA, or immunoglobulin cDNA, and extends through the variable region of the T cell receptor cDNA, B cell receptor cDNA, or immunoglobulin cDNA.
  • the third reaction results in an enrichment product that comprises at (a) least one of a T cell receptor variable region sequence, a B cell receptor variable region sequence, and an immunoglobulin variable region sequence, and (b) at least one of an adaptor sequence, a barcode sequence, a unique molecular identifier sequence, a primer binding site, and a sequencing primer binding site. In some embodiments, greater than about 25% of reads in a subsequent short-read sequencing reaction map to a T cell receptor, a B cell receptor, or an immunoglobulin gene.
  • the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, upon execution by one of more computer processors, implements a method for nucleic acid sequencing, comprising (a) providing a plurality of droplets, wherein a droplet of the plurality of droplets comprises (i) a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, and (ii) a bead comprising a nucleic acid barcode molecule coupled thereto, wherein the nucleic acid barcode molecule comprises a barcode sequence; (b) using the RNA molecule and the nucleic acid barcode molecule to generate a barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, a sequence corresponding to the nucleic acid sequence of the RNA molecule and a complement of the barcode sequence; and (c) sequencing the barcoded nucleic acid molecule or a derivative thereof.
  • RNA ribonucleic acid
  • the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, upon execution by one of more computer processors, implements a method for generating a labeled polynucleotide, the method comprising (a) subjecting a reaction mixture to a first reaction under conditions sufficient to generate a first nucleic acid product, wherein the reaction mixture comprises (i) a template polynucleotide, (ii) a primer having a sequence towards a 3′ end that hybridizes to the template polynucleotide, and (iii) a template switching oligonucleotide, wherein the first nucleic acid product comprises the primer, a reverse complement of a sequence of the template polynucleotide, and a sequence complementary to at least a portion of the template switch oligonucleotide; and (b) subjecting the first nucleic acid product to a second reaction in the presence of a barcoded oligonucleotide under
  • the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, upon execution by one of more computer processors, implements a method for generating a labeled polynucleotide.
  • the method comprises (a) subjecting a reaction mixture to a first amplification reaction under conditions sufficient to generate a first amplification product, wherein the reaction mixture comprises a template polynucleotide and (i) a primer having a sequence towards a 3′ end that hybridizes to the template polynucleotide, and (ii) a template switching oligonucleotide that comprises a first predefined sequence towards a 5′ end; and (b) subjecting the first amplification product to a second amplification reaction in the presence of a barcoded oligonucleotide under conditions sufficient to generate a second amplification product, wherein the barcoded oligonucleotide comprises a sequence of at least a segment of the template switching oligonu
  • FIG. 1 schematically illustrates a microfluidic channel structure for partitioning individual or small groups of cells.
  • FIG. 2 schematically illustrates a microfluidic channel structure for co-partitioning cells and microcapsules (e.g., beads) comprising additional reagents.
  • FIG. 3 schematically illustrates an example process for amplification and barcoding of cell's nucleic acids.
  • FIG. 4 provides a schematic illustration of use of barcoding of cell's nucleic acids in attributing sequence data to individual cells or groups of cells for use in their characterization.
  • FIG. 5 provides a schematic illustration of cells associated with labeled cell-binding ligands.
  • FIG. 6 provides a schematic illustration of an example workflow for performing RNA analysis using the methods described herein.
  • FIG. 7 provides a schematic illustration of an example barcoded oligonucleotide structure for use in analysis of ribonucleic (RNA) using the methods described herein.
  • FIG. 8 provides an image of individual cells co-partitioned along with individual barcode bearing beads.
  • FIGS. 9A-9E provide schematic illustration of example barcoded oligonucleotide structures for use in analysis of RNA and example operations for performing RNA analysis.
  • FIG. 9A discloses SEQ ID NOS 167 and 167, respectively, in order of appearance.
  • FIG. 9B discloses SEQ ID NOS 167, 167 and 167, respectively, in order of appearance.
  • FIG. 9C discloses SEQ ID NOS 167 and 167, respectively, in order of appearance.
  • FIG. 9D discloses SEQ ID NOS 167 and 167, respectively, in order of appearance.
  • FIG. 9E discloses SEQ ID NOS 167 and 167, respectively, in order of appearance.
  • FIG. 10 provides schematic illustration of example barcoded oligonucleotide structure for use in example analysis of RNA and use of a sequence for in vitro transcription.
  • FIG. 10 discloses SEQ ID NOS 167 and 167, respectively, in order of appearance.
  • FIG. 11 provides schematic illustration of an example barcoded oligonucleotide structure for use in analysis of RNA and example operations for performing RNA analysis.
  • FIG. 11 discloses SEQ ID NOS 168-169 and 168-169, respectively, in order of appearance.
  • FIGS. 12A-12B provide schematic illustrations of example barcoded oligonucleotide structure for use in analysis of RNA.
  • FIGS. 13A-13C provide illustrations of example yields from template switch reverse transcription and PCR in partitions.
  • FIGS. 14A-14B provide illustrations of example yields from reverse transcription and complementary deoxyribonucleic acid (cDNA) amplification in partitions with various cell numbers.
  • cDNA complementary deoxyribonucleic acid
  • FIG. 15 provides an illustration of example yields from cDNA synthesis and real-time quantitative PCR at various input cell concentrations and also the effect of varying primer concentration on yield at a fixed cell input concentration.
  • FIG. 16 provides an illustration of example yields from in vitro transcription.
  • FIG. 17 shows an example computer control system that is programmed or otherwise configured to implement methods provided herein.
  • FIG. 18 provides a schematic illustration of an example barcoded oligonucleotide structure.
  • FIGS. 19A and 19B show example operations for performing RNA analysis.
  • FIG. 19A discloses SEQ ID NOS 167, 167 and 167, respectively, in order of appearance.
  • FIG. 19B discloses SEQ ID NOS 168-169, 168-169, 169, 169 and 169, respectively, in order of appearance.
  • FIG. 20 shows a schematic for enriching VDJ sequences from immune molecules such as TCRs, BCRs, and immunoglobulins.
  • FIG. 20 discloses SEQ ID NOS 167 and 167, respectively, in order of appearance.
  • FIGS. 21A-21C show enrichment of target sequences (A) after cDNA amplification
  • FIG. 22 shows cDNA yields from 12,000; 6,000; or 3,000 cells using gel-beads in an emulsion-reverse transcription reaction (GEM-RT).
  • FIG. 23 shows sequencing results from cDNA that has been enriched using constant region primers compared to unenriched cDNA.
  • FIG. 24 shows cDNA yields using differing concentrations of a template switch oligo (TSO) were tested.
  • FIGS. 25A and 25B show cDNA yields from TSO immobilized to gel beads (GB-TSO) using either 6,000 primary T cells (A) or 2,200 Jurkat cells (B).
  • FIGS. 26A and 26B show cDNA yields from an enrichment using an in solution RT reaction (A) or a GEM RT reaction (B) using nested enrichment primers.
  • FIGS. 27A-27C show enrichment of TCR cDNA using p7 primers only (A), variable region primers with TCR beta chain constant region primers (B), and variable region primers with TCR alpha chain constant region primers (C).
  • FIGS. 28A-28D show a comparison of enriched product generated with either 8 ⁇ M or 200 ⁇ M TSO gel beads using P7 primers with TCR alpha chain constant region primers (A), variable region primers with TCR beta chain constant region primers (B), variable region primers with TCR alpha chain constant region primers (C), and variable region primers with TCR beta chain constant region primers (D).
  • A TCR alpha chain constant region primers
  • B variable region primers with TCR beta chain constant region primers
  • C variable region primers with TCR alpha chain constant region primers
  • D variable region primers with TCR beta chain constant region primers
  • FIGS. 29A and 29B show variations of a schematic for generating labeled polynucleotides.
  • FIG. 29A discloses SEQ ID NOS 170, 170, 170, 170 and 170, respectively, in order of appearance.
  • barcode generally refers to a label, or identifier, that can be part of an analyte to convey information about the analyte.
  • a barcode can be a tag attached to an analyte (e.g., nucleic acid molecule) or a combination of the tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)).
  • the barcode may be unique. Barcodes can have a variety of different formats, for example, barcodes can include: polynucleotide barcodes; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences.
  • a barcode can be attached to an analyte in a reversible or irreversible manner.
  • the barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and/or after sequencing of the sample.
  • Barcodes can allow for identification and/or quantification of individual sequencing-reads in real time.
  • the barcode is generated in a combinatorial manner. Barcodes that may be used with methods, devices and systems of the present disclosure, including methods for forming such barcodes, are described in, for example, U.S. Patent Pub. No. 2014/0378350, which is entirely incorporated herein by reference.
  • subject generally refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species, or other organism, such as a plant.
  • the subject can be a vertebrate, a mammal, a mouse, a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets.
  • a subject can be a healthy individual, an individual that has or is suspected of having a disease or a pre-disposition to the disease, or an individual that is in need of therapy or suspected of needing therapy.
  • a subject can be a patient.
  • a genome generally refers to an entirety of a subject's hereditary information.
  • a genome can be encoded either in DNA or in RNA.
  • a genome can comprise coding regions that code for proteins as well as non-coding regions.
  • a genome can include the sequence of all chromosomes together in an organism. For example, the human genome has a total of 46 chromosomes. The sequence of all of these together may constitute a human genome.
  • adaptor(s) can be used synonymously.
  • An adaptor or tag can be coupled to a polynucleotide sequence to be “tagged” by any approach including ligation, hybridization, or other approaches.
  • sequence of nucleotide bases in one or more polynucleotides generally refers to methods and technologies for determining the sequence of nucleotide bases in one or more polynucleotides.
  • the polynucleotides can be, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA).
  • Sequencing can be performed by various systems currently available, such as, with limitation, a sequencing system by Illumina, Pacific Biosciences, Oxford Nanopore, or Life Technologies (Ion Torrent).
  • Such devices may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human), as generated by the device from a sample provided by the subject. In some situations, systems and methods provided herein may be used with proteomic information.
  • variant generally refers to a genetic variant, such as a nucleic acid molecule comprising a polymorphism.
  • a variant can be a structural variant or copy number variant, which can be genomic variants that are larger than single nucleotide variants or short indels.
  • a variant can be an alteration or polymorphism in a nucleic acid sample or genome of a subject.
  • Single nucleotide polymorphisms SNPs
  • Polymorphisms can include single nucleotide variations (SNVs), insertions, deletions, repeats, small insertions, small deletions, small repeats, structural variant junctions, variable length tandem repeats, and/or flanking sequences.
  • Copy number variants (CNVs) transversions and other rearrangements are also forms of genetic variation.
  • a genomic alternation may be a base change, insertion, deletion, repeat, copy number variation, or transversion.
  • the term “bead,” as used herein, generally refers to a particle.
  • the bead may be a solid or semi-solid particle.
  • the bead may be a gel.
  • the bead may be formed of a polymeric material.
  • the bead may be magnetic or non-magnetic.
  • sample generally refers to a biological sample of a subject.
  • the sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate.
  • the sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample.
  • the sample may be a skin sample.
  • the sample may be a cheek swap.
  • the sample may be a plasma or serum sample.
  • the sample may be a cell-free or cell free sample.
  • a cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from a group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.
  • primer generally refers to a strand of RNA or DNA that serves as a starting point for nucleic acid (e.g., DNA) synthesis.
  • a primer may be used in a primer extension reaction, which may be a nucleic acid amplification reaction, such as, for example, polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR).
  • the primer may have a sequence that is capable of coupling to a nucleic acid molecule.
  • sequence may be complementary to the nucleic acid molecule, such as a poly-T sequence or a predetermined sequence, or a sequence that is otherwise capable of coupling (e.g., hybridizing) to the nucleic acid molecule, such as a universal primer.
  • Nucleic acid sequencing technologies have yielded substantial results in sequencing biological materials, including providing substantial sequence information on individual organisms, and relatively pure biological samples. However, these systems have traditionally not been effective at being able to identify and characterize cells at the single cell level.
  • nucleic acid sequencing technologies derive the nucleic acids that they sequence from collections of cells obtained from tissue or other samples, such as biological fluids (e.g., blood, plasma, etc).
  • the cells can be processed (e.g., all together) to extract the genetic material that represents an average of the population of cells, which can then be processed into sequencing ready DNA libraries that are configured for a given sequencing technology.
  • the nucleic acids derived from the cells may include DNA, or RNA, including, e.g., mRNA, total RNA, or the like, that may be processed to produce complementary DNA (cDNA) for sequencing.
  • cDNA complementary DNA
  • ensemble sample preparation methods can be, from the outset, predisposed to primarily identifying and characterizing the majority constituents in the sample of cells, and may not be designed to pick out the minority constituents, e.g., genetic material contributed by one cell, a few cells, or a small percentage of total cells in the sample.
  • an ensemble approach can be predisposed to presenting potentially inaccurate data from cell populations that are non-homogeneous in terms of expression levels. In some cases, where expression is high in a small minority of the cells in an analyzed population, and absent in the majority of the cells of the population, an ensemble method may indicate low level expression for the entire population.
  • next generation sequencing technologies e.g., massively parallel sequencing
  • amplification can be biased toward amplification of majority constituents in a sample, and may not preserve the starting ratios of such minority and majority components. While some of these difficulties may be addressed by utilizing different sequencing systems, such as single molecule systems that do not require amplification, the single molecule systems, as well as the ensemble sequencing methods of other next generation sequencing systems, can also have large input DNA requirements.
  • Some single molecule sequencing systems can have sample input DNA requirements of from 500 nanograms (ng) to upwards of 10 micrograms ( ⁇ g), which may not be obtainable from individual cells or even small subpopulations of cells.
  • other NGS systems can be optimized for starting amounts of sample DNA in the sample of from approximately 50 ng to about 1 ⁇ g.
  • the methods described herein may compartmentalize the analysis of individual cells or small populations of cells, including e.g., nucleic acids from individual cells or small groups of cells, and then allow that analysis to be attributed back to the individual cell or small group of cells from which the nucleic acids were derived. This can be accomplished regardless of whether the cell population represents a 50/50 mix of cell types, a 90/10 mix of cell types, or virtually any ratio of cell types, as well as a complete heterogeneous mix of different cell types, or any mixture between these.
  • Differing cell types may include cells from different tissue types of an individual or the same tissue type from different individuals, or biological organisms such as microorganisms from differing genera, species, strains, variants, or any combination of any or all of the foregoing.
  • differing cell types may include normal and tumor tissue from an individual, various cell types obtained from a human subject such as a variety of immune cells (e.g., B cells, T cells, and the like), multiple different bacterial species, strains and/or variants from environmental, forensic, microbiome or other samples, or any of a variety of other mixtures of cell types.
  • Methods and systems described herein may provide for the compartmentalization, depositing or partitioning of the nucleic acid contents of individual cells from a sample material containing cells, into discrete compartments or partitions (referred to interchangeably herein as partitions), where each partition maintains separation of its own contents from the contents of other partitions.
  • a partition is a droplet or well.
  • Unique identifiers e.g., barcodes
  • Barcodes may be delivered, for example in an oligonucleotide to a partition via any suitable mechanism, such as using beads (e.g., gel beads).
  • beads e.g., gel beads
  • cellular derivatives such as cells or constituents of such cells in matrix (e.g., gel or polymeric matrix), are compartmentalized or partitioned in partitions (e.g., droplets or wells).
  • barcoded oligonucleotides are delivered to a partition via a microcapsule.
  • barcoded oligonucleotides are initially associated with the microcapsule and then released from the microcapsule upon application of a stimulus which allows the oligonucleotides to dissociate or to be released from the microcapsule.
  • a microcapsule in some embodiments, comprises a bead.
  • a bead may be porous, non-porous, solid, semi-solid, semi-fluidic, or fluidic.
  • a bead may be dissolvable, disruptable, or degradable. In some cases, a bead may not be degradable.
  • the bead may be a gel bead.
  • a gel bead may be a hydrogel bead.
  • a gel bead may be formed from molecular precursors, such as a polymeric or monomeric species.
  • a semi-solid bead may be a liposomal bead.
  • Solid beads may comprise metals including iron oxide, gold, and silver. In some cases, the beads are silica beads. In some cases, the beads are rigid. In some cases, the beads may be flexible and/or compressible.
  • the bead may contain molecular precursors (e.g., monomers or polymers), which may form a polymer network via polymerization of the precursors.
  • a precursor may be an already polymerized species capable of undergoing further polymerization via, for example, a chemical cross-linkage.
  • a precursor comprises one or more of an acrylamide or a methacrylamide monomer, oligomer, or polymer.
  • the bead may comprise prepolymers, which are oligomers capable of further polymerization.
  • polyurethane beads may be prepared using prepolymers.
  • the bead may contain individual polymers that may be further polymerized together.
  • beads may be generated via polymerization of different precursors, such that they comprise mixed polymers, co-polymers, and/or block co-polymers.
  • a bead may comprise natural and/or synthetic materials.
  • a polymer can be a natural polymer or a synthetic polymer.
  • a bead comprises both natural and synthetic polymers.
  • natural polymers include proteins and sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum, Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate, or natural polymers thereof.
  • proteins and sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silk
  • Examples of synthetic polymers include acrylics, nylons, silicones, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and combinations (e.g., co-polymers) thereof. Bea
  • a chemical cross-linker may be a precursor used to cross-link monomers during polymerization of the monomers and/or may be used to attach oligonucleotides (e.g., barcoded oligonucleotides) to the bead.
  • polymers may be further polymerized with a cross-linker species or other type of monomer to generate a further polymeric network.
  • Non-limiting examples of chemical cross-linkers include cystamine, gluteraldehyde, dimethyl suberimidate, N-Hydroxysuccinimide crosslinker BS3, formaldehyde, carbodiimide (EDC), SMCC, Sulfo-SMCC, vinylsilane, N,N′diallyltartardiamide (DATD), N,N′-Bis(acryloyl)cystamine (BAC), or homologs thereof.
  • the crosslinker used in the present disclosure contains cystamine.
  • Crosslinking may be permanent or reversible, depending upon the particular crosslinker used. Reversible crosslinking may allow for the polymer to linearize or dissociate under appropriate conditions. In some cases, reversible cross-linking may also allow for reversible attachment of a material bound to the surface of a bead. In some cases, a cross-linker may form disulfide linkages. In some cases, the chemical cross-linker forming disulfide linkages may be cystamine or a modified cystamine.
  • disulfide linkages can be formed between molecular precursor units (e.g., monomers, oligomers, or linear polymers) or precursors incorporated into a bead and oligonucleotides.
  • Cystamine (including modified cystamines), for example, is an organic agent comprising a disulfide bond that may be used as a crosslinker agent between individual monomeric or polymeric precursors of a bead.
  • Polyacrylamide may be polymerized in the presence of cystamine or a species comprising cystamine (e.g., a modified cystamine) to generate polyacrylamide gel beads comprising disulfide linkages (e.g., chemically degradable beads comprising chemically-reducible cross-linkers).
  • the disulfide linkages may permit the bead to be degraded (or dissolved) upon exposure of the bead to a reducing agent.
  • chitosan a linear polysaccharide polymer
  • glutaraldehyde via hydrophilic chains to form a bead.
  • Crosslinking of chitosan polymers may be achieved by chemical reactions that are initiated by heat, pressure, change in pH, and/or radiation.
  • the bead may comprise covalent or ionic bonds between polymeric precursors (e.g., monomers, oligomers, linear polymers), oligonucleotides, primers, and other entities.
  • polymeric precursors e.g., monomers, oligomers, linear polymers
  • oligonucleotides e.g., primers, and other entities.
  • the covalent bonds comprise carbon-carbon bonds or thioether bonds.
  • a bead may comprise an acrydite moiety, which in certain aspects may be used to attach one or more oligonucleotides (e.g., barcode sequence, barcoded oligonucleotide, primer, or other oligonucleotide) to the bead.
  • an acrydite moiety can refer to an acrydite analogue generated from the reaction of acrydite with one or more species, such as, the reaction of acrydite with other monomers and cross-linkers during a polymerization reaction.
  • Acrydite moieties may be modified to form chemical bonds with a species to be attached, such as an oligonucleotide (e.g., barcode sequence, barcoded oligonucleotide, primer, or other oligonucleotide).
  • Acrydite moieties may be modified with thiol groups capable of forming a disulfide bond or may be modified with groups already comprising a disulfide bond.
  • the thiol or disulfide via disulfide exchange) may be used as an anchor point for a species to be attached or another part of the acrydite moiety may be used for attachment.
  • attachment is reversible, such that when the disulfide bond is broken (e.g., in the presence of a reducing agent), the attached species is released from the bead.
  • an acrydite moiety comprises a reactive hydroxyl group that may be used for attachment.
  • Functionalization of beads for attachment of oligonucleotides may be achieved through a wide range of different approaches, including activation of chemical groups within a polymer, incorporation of active or activatable functional groups in the polymer structure, or attachment at the pre-polymer or monomer stage in bead production.
  • precursors e.g., monomers, cross-linkers
  • precursors that are polymerized to form a bead may comprise acrydite moieties, such that when a bead is generated, the bead also comprises acrydite moieties.
  • the acrydite moieties can be attached to an oligonucleotide, such as a primer (e.g., a primer for amplifying target nucleic acids, barcoded oligonucleotide, etc) that is desired to be incorporated into the bead.
  • the primer comprises a P5 sequence for attachment to a sequencing flow cell for Illumina sequencing.
  • the primer comprises a P7 sequence for attachment to a sequencing flow cell for Illumina sequencing.
  • the primer comprises a barcode sequence. In some cases, the primer further comprises a unique molecular identifier (UMI). In some cases, the primer comprises an R1 primer sequence for Illumina sequencing. In some cases, the primer comprises an R2 primer sequence for Illumina sequencing.
  • UMI unique molecular identifier
  • precursors comprising a functional group that is reactive or capable of being activated such that it becomes reactive can be polymerized with other precursors to generate gel beads comprising the activated or activatable functional group.
  • the functional group may then be used to attach additional species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads.
  • additional species e.g., disulfide linkers, primers, other oligonucleotides, etc.
  • some precursors comprising a carboxylic acid (COOH) group can co-polymerize with other precursors to form a gel bead that also comprises a COOH functional group.
  • acrylic acid a species comprising free COOH groups
  • acrylamide acrylamide
  • bis(acryloyl)cystamine can be co-polymerized together to generate a gel bead comprising free COOH groups.
  • the COOH groups of the gel bead can be activated (e.g., via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) or 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)) such that they are reactive (e.g., reactive to amine functional groups where EDC/NHS or DMTMM are used for activation).
  • EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • NHS N-Hydroxysuccinimide
  • DTMM 4-(4,6-Dimethoxy
  • the activated COOH groups can then react with an appropriate species (e.g., a species comprising an amine functional group where the carboxylic acid groups are activated to be reactive with an amine functional group) comprising a moiety to be linked to the bead.
  • an appropriate species e.g., a species comprising an amine functional group where the carboxylic acid groups are activated to be reactive with an amine functional group
  • Beads comprising disulfide linkages in their polymeric network may be functionalized with additional species via reduction of some of the disulfide linkages to free thiols.
  • the disulfide linkages may be reduced via, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.) to generate free thiol groups, without dissolution of the bead.
  • Free thiols of the beads can then react with free thiols of a species or a species comprising another disulfide bond (e.g., via thiol-disulfide exchange) such that the species can be linked to the beads (e.g., via a generated disulfide bond).
  • free thiols of the beads may react with any other suitable group.
  • free thiols of the beads may react with species comprising an acrydite moiety.
  • the free thiol groups of the beads can react with the acrydite via Michael addition chemistry, such that the species comprising the acrydite is linked to the bead.
  • uncontrolled reactions can be prevented by inclusion of a thiol capping agent such as N-ethylmalieamide or iodoacetate.
  • Activation of disulfide linkages within a bead can be controlled such that only a small number of disulfide linkages are activated. Control may be exerted, for example, by controlling the concentration of a reducing agent used to generate free thiol groups and/or concentration of reagents used to form disulfide bonds in bead polymerization. In some cases, a low concentration (e.g., molecules of reducing agent:gel bead ratios of less than about 10,000; 100,000; 1,000,000; 10,000,000; 100,000,000; 1,000,000,000; 10,000,000,000; or 100,000,000,000) of reducing agent may be used for reduction. Controlling the number of disulfide linkages that are reduced to free thiols may be useful in ensuring bead structural integrity during functionalization. In some cases, optically-active agents, such as fluorescent dyes may be may be coupled to beads via free thiol groups of the beads and used to quantify the number of free thiols present in a bead and/or track a bead.
  • addition of moieties to a gel bead after gel bead formation may be advantageous.
  • addition of an oligonucleotide (e.g., barcoded oligonucleotide) after gel bead formation may avoid loss of the species during chain transfer termination that can occur during polymerization.
  • smaller precursors e.g., monomers or cross linkers that do not comprise side chain groups and linked moieties
  • functionalization after gel bead synthesis can minimize exposure of species (e.g., oligonucleotides) to be loaded with potentially damaging agents (e.g., free radicals) and/or chemical environments.
  • the generated gel may possess an upper critical solution temperature (UCST) that can permit temperature driven swelling and collapse of a bead.
  • UCT upper critical solution temperature
  • Such functionality may aid in oligonucleotide (e.g., a primer) infiltration into the bead during subsequent functionalization of the bead with the oligonucleotide.
  • Post-production functionalization may also be useful in controlling loading ratios of species in beads, such that, for example, the variability in loading ratio is minimized.
  • Species loading may also be performed in a batch process such that a plurality of beads can be functionalized with the species in a single batch.
  • an acrydite moiety linked to precursor, another species linked to a precursor, or a precursor itself comprises a labile bond, such as chemically, thermally, or photo-sensitive bonds e.g., disulfide bonds, UV sensitive bonds, or the like.
  • acrydite moieties or other moieties comprising a labile bond are incorporated into a bead, the bead may also comprise the labile bond.
  • the labile bond may be, for example, useful in reversibly linking (e.g., covalently linking) species (e.g., barcodes, primers, etc.) to a bead.
  • a thermally labile bond may include a nucleic acid hybridization based attachment, e.g., where an oligonucleotide is hybridized to a complementary sequence that is attached to the bead, such that thermal melting of the hybrid releases the oligonucleotide, e.g., a barcode containing sequence, from the bead or microcapsule.
  • a nucleic acid hybridization based attachment e.g., where an oligonucleotide is hybridized to a complementary sequence that is attached to the bead, such that thermal melting of the hybrid releases the oligonucleotide, e.g., a barcode containing sequence, from the bead or microcapsule.
  • labile bonds may result in the generation of a bead capable of responding to varied stimuli.
  • Each type of labile bond may be sensitive to an associated stimulus (e.g., chemical stimulus, light, temperature, etc.) such that release of species attached to a bead via each labile bond may be controlled by the application of the appropriate stimulus.
  • Such functionality may be useful in controlled release of species from a gel bead.
  • another species comprising a labile bond may be linked to a gel bead after gel bead formation via, for example, an activated functional group of the gel bead as described above.
  • barcodes that are releasably, cleavably or reversibly attached to the beads described herein include barcodes that are released or releasable through cleavage of a linkage between the barcode molecule and the bead, or that are released through degradation of the underlying bead itself, allowing the barcodes to be accessed or accessible by other reagents, or both.
  • the barcodes that are releasable as described herein may sometimes be referred to as being activatable, in that they are available for reaction once released.
  • an activatable barcode may be activated by releasing the barcode from a bead (or other suitable type of partition described herein).
  • Other activatable configurations are also envisioned in the context of the described methods and systems.
  • labile bonds that may be coupled to a precursor or bead include an ester linkage (e.g., cleavable with an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), a sulfone linkage (e.g., cleavable via a base), a silyl ether linkage (e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable via an amylase), a peptide linkage (e.g., cleavable via a protease), or a phosphodiester linkage (e.g., cleavable via a nuclease (
  • Species that do not participate in polymerization may also be encapsulated in beads during bead generation (e.g., during polymerization of precursors). Such species may be entered into polymerization reaction mixtures such that generated beads comprise the species upon bead formation. In some cases, such species may be added to the gel beads after formation.
  • Such species may include, for example, oligonucleotides, reagents for a nucleic acid amplification reaction (e.g., primers, polymerases, dNTPs, co-factors (e.g., ionic co-factors)) including those described herein, reagents for enzymatic reactions (e.g., enzymes, co-factors, substrates), or reagents for a nucleic acid modification reactions such as polymerization, ligation, or digestion. Trapping of such species may be controlled by the polymer network density generated during polymerization of precursors, control of ionic charge within the gel bead (e.g., via ionic species linked to polymerized species), or by the release of other species. Encapsulated species may be released from a bead upon bead degradation and/or by application of a stimulus capable of releasing the species from the bead.
  • reagents for a nucleic acid amplification reaction e.g.,
  • Beads may be of uniform size or heterogeneous size.
  • the diameter of a bead may be about 1 ⁇ m, 5 ⁇ m, 10 ⁇ m, 20 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ m, 60 ⁇ m, 70 ⁇ m, 80 ⁇ m, 90 ⁇ m, 100 ⁇ m, 250 ⁇ m, 500 ⁇ m, or 1 mm.
  • a bead may have a diameter of at least about 1 ⁇ m, 5 ⁇ m, 10 ⁇ m, 20 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ m, 60 ⁇ m, 70 ⁇ m, 80 ⁇ m, 90 ⁇ m, 100 ⁇ m, 250 ⁇ m, 500 ⁇ m, 1 mm, or more. In some cases, a bead may have a diameter of less than about 1 ⁇ m, 5 ⁇ m, 10 ⁇ m, 20 ⁇ m, 30 ⁇ m, 40 ⁇ m, 50 ⁇ m, 60 ⁇ m, 70 ⁇ m, 80 ⁇ m, 90 ⁇ m, 100 ⁇ m, 250 ⁇ m, 500 ⁇ m, or 1 mm.
  • a bead may have a diameter in the range of about 40-75 ⁇ m, 30-75 ⁇ m, 20-75 ⁇ m, 40-85 ⁇ m, 40-95 ⁇ m, 20-100 ⁇ m, 10-100 ⁇ m, 1-100 ⁇ m, 20-250 ⁇ m, or 20-500 ⁇ m.
  • beads are provided as a population or plurality of beads having a relatively monodisperse size distribution. Where it may be desirable to provide relatively consistent amounts of reagents within partitions, maintaining relatively consistent bead characteristics, such as size, can contribute to the overall consistency.
  • the beads described herein may have size distributions that have a coefficient of variation in their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, or even less than 5%.
  • Beads may be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.
  • the beads may be degradable, disruptable, or dissolvable spontaneously or upon exposure to one or more stimuli (e.g., temperature changes, pH changes, exposure to particular chemical species or phase, exposure to light, reducing agent, etc.).
  • a bead may be dissolvable, such that material components of the beads are solubilized when exposed to a particular chemical species or an environmental change, such as a change temperature or a change in pH.
  • a gel bead is degraded or dissolved at elevated temperature and/or in basic conditions.
  • a bead may be thermally degradable such that when the bead is exposed to an appropriate change in temperature (e.g., heat), the bead degrades.
  • Degradation or dissolution of a bead bound to a species e.g., an oligonucleotide, e.g., barcoded oligonucleotide
  • a species e.g., an oligonucleotide, e.g., barcoded oligonucleotide
  • a degradable bead may comprise one or more species with a labile bond such that, when the bead/species is exposed to the appropriate stimuli, the bond is broken and the bead degrades.
  • the labile bond may be a chemical bond (e.g., covalent bond, ionic bond) or may be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.).
  • a crosslinker used to generate a bead may comprise a labile bond.
  • the labile bond can be broken and the bead degraded. For example, upon exposure of a polyacrylamide gel bead comprising cystamine crosslinkers to a reducing agent, the disulfide bonds of the cystamine can be broken and the bead degraded.
  • a degradable bead may be useful in more quickly releasing an attached species (e.g., an oligonucleotide, a barcode sequence, a primer, etc) from the bead when the appropriate stimulus is applied to the bead as compared to a bead that does not degrade.
  • an attached species e.g., an oligonucleotide, a barcode sequence, a primer, etc
  • the species may have greater mobility and accessibility to other species in solution upon degradation of the bead.
  • a species may also be attached to a degradable bead via a degradable linker (e.g., disulfide linker).
  • the degradable linker may respond to the same stimuli as the degradable bead or the two degradable species may respond to different stimuli.
  • a barcode sequence may be attached, via a disulfide bond, to a polyacrylamide bead comprising cystamine.
  • the bead Upon exposure of the barcoded-bead to a reducing agent, the bead degrades and the barcode sequence is released upon breakage of both the disulfide linkage between the barcode sequence and the bead and the disulfide linkages of the cystamine in the bead.
  • a degradable bead may be introduced into a partition, such as a droplet of an emulsion or a well, such that the bead degrades within the partition and any associated species (e.g., oligonucleotides) are released within the droplet when the appropriate stimulus is applied.
  • the free species e.g., oligonucleotides
  • a polyacrylamide bead comprising cystamine and linked, via a disulfide bond, to a barcode sequence, may be combined with a reducing agent within a droplet of a water-in-oil emulsion.
  • the reducing agent breaks the various disulfide bonds resulting in bead degradation and release of the barcode sequence into the aqueous, inner environment of the droplet.
  • heating of a droplet comprising a bead-bound barcode sequence in basic solution may also result in bead degradation and release of the attached barcode sequence into the aqueous, inner environment of the droplet.
  • degradation may refer to the disassociation of a bound or entrained species from a bead, both with and without structurally degrading the physical bead itself.
  • entrained species may be released from beads through osmotic pressure differences due to, for example, changing chemical environments.
  • alteration of bead pore sizes due to osmotic pressure differences can generally occur without structural degradation of the bead itself.
  • an increase in pore size due to osmotic swelling of a bead can permit the release of entrained species within the bead.
  • osmotic shrinking of a bead may cause a bead to better retain an entrained species due to pore size contraction.
  • degradable beads it may be desirable to avoid exposing such beads to the stimulus or stimuli that cause such degradation prior to the desired time, in order to avoid premature bead degradation and issues that arise from such degradation, including for example poor flow characteristics and aggregation.
  • beads comprise reducible cross-linking groups, such as disulfide groups
  • reducing agents e.g., DTT or other disulfide cleaving reagents.
  • treatment to the beads described herein will, in some cases be provided free of reducing agents, such as DTT.
  • reducing agent free (or DTT free) enzyme preparations in treating the beads described herein.
  • enzymes include, e.g., polymerase enzyme preparations, reverse transcriptase enzyme preparations, ligase enzyme preparations, as well as many other enzyme preparations that may be used to treat the beads described herein.
  • the terms “reducing agent free” or “DTT free” preparations can refer to a preparation having less than 1/10th, less than 1/50th, and even less than 1/100th of the lower ranges for such materials used in degrading the beads.
  • the reducing agent free preparation will typically have less than 0.01 mM, 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even less than 0.0001 mM DTT. In many cases, the amount of DTT will be undetectable.
  • a stimulus may be used to trigger degradation of the bead, which may result in the release of contents from the bead.
  • a stimulus may cause degradation of the bead structure, such as degradation of the covalent bonds or other types of physical interaction.
  • These stimuli may be useful in inducing a bead to degrade and/or to release its contents. Examples of stimuli that may be used include chemical stimuli, thermal stimuli, optical stimuli (e.g., light) and any combination thereof, as described more fully below.
  • Numerous chemical triggers may be used to trigger the degradation of beads. Examples of these chemical changes may include, but are not limited to pH-mediated changes to the integrity of a component within the bead, degradation of a component of a bead via cleavage of cross-linked bonds, and depolymerization of a component of a bead.
  • a bead may be formed from materials that comprise degradable chemical crosslinkers, such as BAC or cystamine. Degradation of such degradable crosslinkers may be accomplished through a number of mechanisms.
  • a bead may be contacted with a chemical degrading agent that may induce oxidation, reduction or other chemical changes.
  • a chemical degrading agent may be a reducing agent, such as dithiothreitol (DTT).
  • reducing agents may include ⁇ -mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.
  • a reducing agent may degrade the disulfide bonds formed between gel precursors forming the bead, and thus, degrade the bead.
  • a change in pH of a solution such as an increase in pH, may trigger degradation of a bead.
  • exposure to an aqueous solution, such as water may trigger hydrolytic degradation, and thus degradation of the bead.
  • Beads may also be induced to release their contents upon the application of a thermal stimulus.
  • a change in temperature can cause a variety of changes to a bead. For example, heat can cause a solid bead to liquefy. A change in heat may cause melting of a bead such that a portion of the bead degrades. In other cases, heat may increase the internal pressure of the bead components such that the bead ruptures or explodes. Heat may also act upon heat-sensitive polymers used as materials to construct beads.
  • a chemical degrading agent may be a reducing agent, such as DTT, wherein DTT may degrade the disulfide bonds formed between a crosslinker and gel precursors, thus degrading the bead.
  • a reducing agent may be added to degrade the bead, which may or may not cause the bead to release its contents.
  • reducing agents may include dithiothreitol (DTT), ⁇ -mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.
  • the reducing agent may be present at a concentration of about 0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10 mM.
  • the reducing agent may be present at a concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, or greater.
  • the reducing agent may be present at concentration of at most about 0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10 mM.
  • nucleic acid molecules e.g., primer, e.g., barcoded oligonucleotide
  • primer e.g., barcoded oligonucleotide
  • the pre-defined concentration of the primer is limited by the process of producing oligonucleotide bearing beads.
  • the partitions refer to containers or vessels (such as wells, microwells, tubes, vials, through ports in nanoarray substrates, e.g., BioTrove nanoarrays, or other containers).
  • the compartments or partitions comprise partitions that are flowable within fluid streams. These partitions may comprise, e.g., micro-vesicles that have an outer barrier surrounding an inner fluid center or core, or, in some cases, they may comprise a porous matrix that is capable of entraining and/or retaining materials within its matrix.
  • partitions comprise droplets of aqueous fluid within a non-aqueous continuous phase, e.g., an oil phase.
  • a variety of different vessels are described in, for example, U.S.
  • Patent Application Publication No. 20140155295 the full disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in detail in, e.g., U.S. Patent Application Publication No. 20100105112, the full disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • allocating individual cells to discrete partitions may generally be accomplished by introducing a flowing stream of cells in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams.
  • the occupancy of the resulting partitions e.g., number of cells per partition
  • the relative flow rates of the fluids can be selected such that, on average, the partitions contain less than one cell per partition, in order to ensure that those partitions that are occupied, are primarily singly occupied.
  • the relative flow rates of the fluids can be selected such that a majority of partitions are occupied, e.g., allowing for only a small percentage of unoccupied partitions.
  • the flows and channel architectures are controlled as to ensure a desired number of singly occupied partitions, less than a certain level of unoccupied partitions and less than a certain level of multiply occupied partitions.
  • the systems and methods described herein can be operated such that a majority of occupied partitions include no more than one cell per occupied partition.
  • the partitioning process is conducted such that fewer than 25% of the occupied partitions contain more than one cell, and in many cases, fewer than 20% of the occupied partitions have more than one cell. In some cases, fewer than 10% or even fewer than 5% of the occupied partitions include more than one cell per partition.
  • the flow of one or more of the cells, or other fluids directed into the partitioning zone are conducted such that, in many cases, no more than 50% of the generated partitions, no more than 25% of the generated partitions, or no more than 10% of the generated partitions are unoccupied.
  • these flows are controlled so as to present non-Poissonian distribution of single occupied partitions while providing lower levels of unoccupied partitions.
  • the above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above.
  • the use of the systems and methods described herein creates resulting partitions that have multiple occupancy rates of less than 25%, less than 20%, less than 15%, less than 10%, and in many cases, less than 5%, while having unoccupied partitions of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, and in some cases, less than 5%.
  • a substantial percentage of the overall occupied partitions can include both a microcapsule (e.g., bead) comprising barcoded oligonucleotides and a cell.
  • multiply occupied partitions e.g., containing two, three, four or more cells and/or microcapsules (e.g., beads) comprising barcoded oligonucleotides within a single partition.
  • the flow characteristics of the cell and/or bead containing fluids and partitioning fluids may be controlled to provide for such multiply occupied partitions.
  • the flow parameters may be controlled to provide a desired occupancy rate at greater than 50% of the partitions, greater than 75%, and in some cases greater than 80%, 90%, 95%, or higher.
  • additional microcapsules are used to deliver additional reagents to a partition.
  • the flow and frequency of the different beads into the channel or junction may be controlled to provide for the desired ratio of microcapsules from each source, while ensuring the desired pairing or combination of such beads into a partition with the desired number of cells.
  • the partitions described herein may comprise small volumes, e.g., less than 10 ⁇ L, less than 54, less than 14, less than 900 picoliters (pL), less than 800 pL, less than 700 pL, less than 600 pL, less than 500 pL, less than 400 pL, less than 300 pL, less than 200 pL, less than 100 pL, less than 50 pL, less than 20 pL, less than 10 pL, less than 1 pL, less than 500 nanoliters (nL), or even less than 100 nL, 50 nL, or even less.
  • small volumes e.g., less than 10 ⁇ L, less than 54, less than 14, less than 900 picoliters (pL), less than 800 pL, less than 700 pL, less than 600 pL, less than 500 pL, less than 400 pL, less than 300 pL, less than 200 pL, less than 100 pL, less than 50 pL,
  • the droplets may have overall volumes that are less than 1000 pL, less than 900 pL, less than 800 pL, less than 700 pL, less than 600 pL, less than 500 pL, less than 400 pL, less than 300 pL, less than 200 pL, less than 100 pL, less than 50 pL, less than 20 pL, less than 10 pL, or even less than 1 pL.
  • sample fluid volume e.g., including co-partitioned cells
  • the sample fluid volume within the partitions may be less than 90% of the above described volumes, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or even less than 10% the above described volumes.
  • partitioning species may generate a population or plurality of partitions.
  • any suitable number of partitions can be generated to generate the plurality of partitions.
  • a plurality of partitions may be generated that comprises at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions or at least about 1,000,000,000 partitions.
  • the plurality of partitions may comprise both unoccupied partitions (e.g., empty partitions) and occupied partitions
  • Microfluidic channel networks can be utilized to generate partitions as described herein.
  • Alternative mechanisms may also be employed in the partitioning of individual cells, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.
  • FIG. 1 An example of a simplified microfluidic channel structure for partitioning individual cells is illustrated in FIG. 1 .
  • the majority of occupied partitions include no more than one cell per occupied partition and, in some cases, some of the generated partitions are unoccupied.
  • some of the occupied partitions may include more than one cell.
  • the partitioning process may be controlled such that fewer than 25% of the occupied partitions contain more than one cell, and in many cases, fewer than 20% of the occupied partitions have more than one cell, while in some cases, fewer than 10% or even fewer than 5% of the occupied partitions include more than one cell per partition.
  • the channel structure can include channel segments 102 , 104 , 106 and 108 communicating at a channel junction 110 .
  • a first aqueous fluid 112 that includes suspended cells 114 may be transported along channel segment 102 into junction 110 , while a second fluid 116 that is immiscible with the aqueous fluid 112 is delivered to the junction 110 from channel segments 104 and 106 to create discrete droplets 118 of the aqueous fluid including individual cells 114 , flowing into channel segment 108 .
  • this second fluid 116 comprises an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, e.g., inhibiting subsequent coalescence of the resulting droplets.
  • a fluorinated oil such as a fluorinated oil
  • fluorosurfactant for stabilizing the resulting droplets, e.g., inhibiting subsequent coalescence of the resulting droplets.
  • cells may be encapsulated within a microcapsule that comprises an outer shell or layer or porous matrix in which is entrained one or more individual cells or small groups of cells, and may include other reagents. Encapsulation of cells may be carried out by a variety of processes. Such processes combine an aqueous fluid containing the cells to be analyzed with a polymeric precursor material that may be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor.
  • Such stimuli include, e.g., thermal stimuli (either heating or cooling), photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g., through crosslinking, polymerization initiation of the precursor (e.g., through added initiators), or the like.
  • microcapsules comprising cells may be carried out by a variety of methods.
  • air knife droplet or aerosol generators may be used to dispense droplets of precursor fluids into gelling solutions in order to form microcapsules that include individual cells or small groups of cells.
  • membrane based encapsulation systems may be used to generate microcapsules comprising encapsulated cells as described herein.
  • microfluidic systems like that shown in FIG. 1 may be readily used in encapsulating cells as described herein.
  • FIG. 1 may be readily used in encapsulating cells as described herein.
  • non-aqueous fluid 116 may also include an initiator to cause polymerization and/or crosslinking of the polymer precursor to form the microcapsule that includes the entrained cells.
  • initiator to cause polymerization and/or crosslinking of the polymer precursor to form the microcapsule that includes the entrained cells.
  • the activation agent may comprise a cross-linking agent, or a chemical that activates a cross-linking agent within the formed droplets.
  • the activation agent may comprise a polymerization initiator.
  • the polymer precursor comprises a mixture of acrylamide monomer with a N,N′-bis-(acryloyl)cystamine (BAC) comonomer
  • an agent such as tetraethylmethylenediamine (TEMED) may be provided within the second fluid streams in channel segments 104 and 106 , which initiates the copolymerization of the acrylamide and BAC into a cross-linked polymer network or, hydrogel.
  • TEMED tetraethylmethylenediamine
  • the TEMED may diffuse from the second fluid 116 into the aqueous first fluid 112 comprising the linear polyacrylamide, which will activate the crosslinking of the polyacrylamide within the droplets, resulting in the formation of the gel, e.g., hydrogel, microcapsules 118 , as solid or semi-solid beads or particles entraining the cells 114 .
  • the gel e.g., hydrogel, microcapsules 118
  • solid or semi-solid beads or particles entraining the cells 114 e.g., hydrogel, microcapsules 118
  • other ‘activatable’ encapsulation compositions may also be employed in the context of the methods and compositions described herein.
  • encapsulated cells can be selectively releasable from the microcapsule, e.g., through passage of time, or upon application of a particular stimulus, that degrades the microcapsule sufficiently to allow the cell, or its contents to be released from the microcapsule, e.g., into a partition, such as a droplet.
  • degradation of the microcapsule may be accomplished through the introduction of an appropriate reducing agent, such as DTT or the like, to cleave disulfide bonds that cross link the polymer matrix (See, e.g., U.S. Patent Application Publication No. 20140378345, the full disclosures of which are hereby incorporated herein by reference in their entirety for all purposes.
  • an appropriate reducing agent such as DTT or the like
  • Encapsulated cells or cell populations provide certain potential advantages of being storable, and more portable than droplet based partitioned cells. Furthermore, in some cases, it may be desirable to allow cells to be analyzed to incubate for a select period of time, in order to characterize changes in such cells over time, either in the presence or absence of different stimuli.
  • encapsulation of individual cells may allow for longer incubation than partitioning in emulsion droplets, although in some cases, droplet partitioned cells may also be incubated for different periods of time, e.g., at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 10 hours or more.
  • the encapsulation of cells may constitute the partitioning of the cells into which other reagents are co-partitioned.
  • encapsulated cells may be readily deposited into other partitions, e.g., droplets, as described above.
  • the cells may be partitioned along with lysis reagents in order to release the contents of the cells within the partition.
  • the lysis agents can be contacted with the cell suspension concurrently with, or immediately prior to the introduction of the cells into the partitioning junction/droplet generation zone, e.g., through an additional channel or channels upstream of channel junction 110 .
  • lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc. (St Louis, Mo.), as well as other commercially available lysis enzymes.
  • Other lysis agents may additionally or alternatively be co-partitioned with the cells to cause the release of the cell's contents into the partitions.
  • surfactant based lysis solutions may be used to lyse cells, although these may be less desirable for emulsion based systems where the surfactants can interfere with stable emulsions.
  • lysis solutions may include non-ionic surfactants such as, for example, TritonX-100 and Tween 20.
  • lysis solutions may include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS).
  • Electroporation, thermal, acoustic or mechanical cellular disruption may also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of cells that may be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a desired size, following cellular disruption.
  • non-emulsion based partitioning such as encapsulation of cells that may be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a desired size, following cellular disruption.
  • reagents can also be co-partitioned with the cells, including, for example, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids.
  • DNase and RNase inactivating agents or inhibitors such as proteinase K
  • chelating agents such as EDTA
  • the cells may be exposed to an appropriate stimulus to release the cells or their contents from a co-partitioned microcapsule.
  • a chemical stimulus may be co-partitioned along with an encapsulated cell to allow for the degradation of the microcapsule and release of the cell or its contents into the larger partition.
  • this stimulus may be the same as the stimulus described elsewhere herein for release of oligonucleotides from their respective microcapsule (e.g., bead).
  • this may be a different and non-overlapping stimulus, in order to allow an encapsulated cell to be released into a partition at a different time from the release of oligonucleotides into the same partition.
  • Additional reagents may also be co-partitioned with the cells, such as endonucleases to fragment the cell's DNA, DNA polymerase enzymes and dNTPs used to amplify the cell's nucleic acid fragments and to attach the barcode oligonucleotides to the amplified fragments.
  • Additional reagents may also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA.
  • template switching can be used to append a predefined nucleic acid sequence to the cDNA.
  • cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA in a template independent manner.
  • Switch oligos can include sequences complementary to the additional nucleotides, e.g., polyG.
  • the additional nucleotides (e.g., polyC) on the cDNA can hybridize to the additional nucleotides (e.g., polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA.
  • Template switching oligonucleotides may comprise a hybridization region and a template region.
  • the hybridization region can comprise any sequence capable of hybridizing to the target.
  • the hybridization region comprises a series of G bases to complement the overhanging C bases at the 3′ end of a cDNA molecule.
  • the series of G bases may comprise 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or more than 5 G bases.
  • the template sequence can comprise any sequence to be incorporated into the cDNA.
  • the template region comprises at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/or functional sequences.
  • Switch oligos may comprise deoxyribonucleic acids; ribonucleic acids; modified nucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC, 2′-deoxylnosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro
  • the length of a switch oligo may be 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, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,
  • the length of a switch oligo may be at least 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, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114
  • the length of a switch oligo may be at most 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, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114
  • the nucleic acids contained therein may be further processed within the partitions.
  • the nucleic acid contents of individual cells can be provided with unique identifiers such that, upon characterization of those nucleic acids they may be attributed as having been derived from the same cell or cells.
  • the ability to attribute characteristics to individual cells or groups of cells is provided by the assignment of unique identifiers specifically to an individual cell or groups of cells.
  • Unique identifiers e.g., in the form of nucleic acid barcodes can be assigned or associated with individual cells or populations of cells, in order to tag or label the cell's components (and as a result, its characteristics) with the unique identifiers.
  • unique identifiers can then be used to attribute the cell's components and characteristics to an individual cell or group of cells. In some aspects, this is carried out by co-partitioning the individual cells or groups of cells with the unique identifiers.
  • the unique identifiers are provided in the form of oligonucleotides that comprise nucleic acid barcode sequences that may be attached to or otherwise associated with the nucleic acid contents of individual cells, or to other components of the cells, and particularly to fragments of those nucleic acids.
  • the oligonucleotides are partitioned such that as between oligonucleotides in a given partition, the nucleic acid barcode sequences contained therein are the same, but as between different partitions, the oligonucleotides can, and do have differing barcode sequences, or at least represent a large number of different barcode sequences across all of the partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, although in some cases, two or more different barcode sequences may be present.
  • the nucleic acid barcode sequences can include from 6 to about 20 or more nucleotides within the sequence of the oligonucleotides.
  • the length of a barcode sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.
  • the length of a barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.
  • the length of a barcode sequence may be at most 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.
  • nucleotides may be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they may be separated into two or more separate subsequences that are separated by 1 or more nucleotides.
  • separated barcode subsequences can be from about 4 to about 16 nucleotides in length.
  • the barcode subsequence may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer.
  • the barcode subsequence may be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer.
  • the barcode subsequence may be at most 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.
  • the co-partitioned oligonucleotides can also comprise other functional sequences useful in the processing of the nucleic acids from the co-partitioned cells. These sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual cells within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences.
  • sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual cells within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences.
  • oligonucleotides may also be employed, including, e.g., coalescence of two or more droplets, where one droplet contains oligonucleotides, or microdispensing of oligonucleotides into partitions, e.g., droplets within microfluidic systems.
  • microcapsules such as beads
  • each include large numbers of the above described barcoded oligonucleotides releasably attached to the beads, where all of the oligonucleotides attached to a particular bead will include the same nucleic acid barcode sequence, but where a large number of diverse barcode sequences are represented across the population of beads used.
  • hydrogel beads e.g., comprising polyacrylamide polymer matrices
  • hydrogel beads are used as a solid support and delivery vehicle for the oligonucleotides into the partitions, as they are capable of carrying large numbers of oligonucleotide molecules, and may be configured to release those oligonucleotides upon exposure to a particular stimulus, as described elsewhere herein.
  • the population of beads will provide a diverse barcode sequence library that includes at least 1,000 different barcode sequences, at least 5,000 different barcode sequences, at least 10,000 different barcode sequences, at least at least 50,000 different barcode sequences, at least 100,000 different barcode sequences, at least 1,000,000 different barcode sequences, at least 5,000,000 different barcode sequences, or at least 10,000,000 different barcode sequences. Additionally, each bead can be provided with large numbers of oligonucleotide molecules attached.
  • the number of molecules of oligonucleotides including the barcode sequence on an individual bead can be at least 1,000 oligonucleotide molecules, at least 5,000 oligonucleotide molecules, at least 10,000 oligonucleotide molecules, at least 50,000 oligonucleotide molecules, at least 100,000 oligonucleotide molecules, at least 500,000 oligonucleotides, at least 1,000,000 oligonucleotide molecules, at least 5,000,000 oligonucleotide molecules, at least 10,000,000 oligonucleotide molecules, at least 50,000,000 oligonucleotide molecules, at least 100,000,000 oligonucleotide molecules, and in some cases at least 1 billion oligonucleotide molecules.
  • the resulting population of partitions can also include a diverse barcode library that includes at least 1,000 different barcode sequences, at least 5,000 different barcode sequences, at least 10,000 different barcode sequences, at least at least 50,000 different barcode sequences, at least 100,000 different barcode sequences, at least 1,000,000 different barcode sequences, at least 5,000,000 different barcode sequences, or at least 10,000,000 different barcode sequences.
  • each partition of the population can include at least 1,000 oligonucleotide molecules, at least 5,000 oligonucleotide molecules, at least 10,000 oligonucleotide molecules, at least 50,000 oligonucleotide molecules, at least 100,000 oligonucleotide molecules, at least 500,000 oligonucleotides, at least 1,000,000 oligonucleotide molecules, at least 5,000,000 oligonucleotide molecules, at least 10,000,000 oligonucleotide molecules, at least 50,000,000 oligonucleotide molecules, at least 100,000,000 oligonucleotide molecules, and in some cases at least 1 billion oligonucleotide molecules.
  • a mixed, but known barcode sequences set may provide greater assurance of identification in the subsequent processing, e.g., by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.
  • the oligonucleotides are releasable from the beads upon the application of a particular stimulus to the beads.
  • the stimulus may be a photo-stimulus, e.g., through cleavage of a photo-labile linkage that releases the oligonucleotides.
  • a thermal stimulus may be used, where elevation of the temperature of the beads environment will result in cleavage of a linkage or other release of the oligonucleotides form the beads.
  • a chemical stimulus is used that cleaves a linkage of the oligonucleotides to the beads, or otherwise results in release of the oligonucleotides from the beads.
  • such compositions include the polyacrylamide matrices described above for encapsulation of cells, and may be degraded for release of the attached oligonucleotides through exposure to a reducing agent, such as DTT.
  • the beads including the attached oligonucleotides are co-partitioned with the individual cells, such that a single bead and a single cell are contained within an individual partition.
  • single cell/single bead occupancy is the most desired state, it will be appreciated that multiply occupied partitions (either in terms of cells, beads or both), or unoccupied partitions (either in terms of cells, beads or both) will often be present.
  • An example of a microfluidic channel structure for co-partitioning cells and beads comprising barcode oligonucleotides is schematically illustrated in FIG. 2 .
  • a substantial percentage of the overall occupied partitions will include both a bead and a cell and, in some cases, some of the partitions that are generated will be unoccupied. In some cases, some of the partitions may have beads and cells that are not partitioned 1:1. In some cases, it may be desirable to provide multiply occupied partitions, e.g., containing two, three, four or more cells and/or beads within a single partition.
  • channel segments 202 , 204 , 206 , 208 and 210 are provided in fluid communication at channel junction 212 .
  • An aqueous stream comprising the individual cells 214 , is flowed through channel segment 202 toward channel junction 212 . As described above, these cells may be suspended within an aqueous fluid, or may have been pre-encapsulated, prior to the partitioning process.
  • an aqueous stream comprising the barcode carrying beads 216 , is flowed through channel segment 204 toward channel junction 212 .
  • a non-aqueous partitioning fluid 216 is introduced into channel junction 212 from each of side channels 206 and 208 , and the combined streams are flowed into outlet channel 210 .
  • the two combined aqueous streams from channel segments 202 and 204 are combined, and partitioned into droplets 218 , that include co-partitioned cells 214 and beads 216 .
  • partitioning can be optimized to achieve a desired occupancy level of beads, cells or both, within the partitions 218 that are generated.
  • lysis agents e.g., cell lysis enzymes
  • the bead stream e.g., flowing through channel segment 204
  • Additional reagents may also be added to the partition in this configuration, such as endonucleases to fragment the cell's DNA, DNA polymerase enzyme and dNTPs used to amplify the cell's nucleic acid fragments and to attach the barcode oligonucleotides to the amplified fragments.
  • a chemical stimulus such as DTT, may be used to release the barcodes from their respective beads into the partition.
  • the chemical stimulus along with the cell-containing stream in channel segment 202 , such that release of the barcodes only occurs after the two streams have been combined, e.g., within the partitions 218 .
  • introduction of a common chemical stimulus e.g., that both releases the oligonucleotides form their beads, and releases cells from their microcapsules may generally be provided from a separate additional side channel (not shown) upstream of or connected to channel junction 212 .
  • a number of other reagents may be co-partitioned along with the cells, beads, lysis agents and chemical stimuli, including, for example, protective reagents, like proteinase K, chelators, nucleic acid extension, replication, transcription or amplification reagents such as polymerases, reverse transcriptases, transposases which can be used for transposon based methods (e.g., Nextera), nucleoside triphosphates or NTP analogues, primer sequences and additional cofactors such as divalent metal ions used in such reactions, ligation reaction reagents, such as ligase enzymes and ligation sequences, dyes, labels, or other tagging reagents.
  • protective reagents like proteinase K, chelators, nucleic acid extension, replication, transcription or amplification reagents such as polymerases, reverse transcriptases, transposases which can be used for transposon based methods (e.g., Nextera), nucle
  • the channel networks can be fluidly coupled to appropriate fluidic components.
  • the inlet channel segments e.g., channel segments 202 , 204 , 206 and 208 are fluidly coupled to appropriate sources of the materials they are to deliver to channel junction 212 .
  • channel segment 202 will be fluidly coupled to a source of an aqueous suspension of cells 214 to be analyzed, while channel segment 204 may be fluidly coupled to a source of an aqueous suspension of beads 216 .
  • Channel segments 206 and 208 may then be fluidly connected to one or more sources of the non-aqueous fluid.
  • outlets may include any of a variety of different fluidic components, from simple reservoirs defined in or connected to a body structure of a microfluidic device, to fluid conduits that deliver fluids from off-device sources, manifolds, or the like.
  • the outlet channel segment 210 may be fluidly coupled to a receiving vessel or conduit for the partitioned cells. Again, this may be a reservoir defined in the body of a microfluidic device, or it may be a fluidic conduit for delivering the partitioned cells to a subsequent process operation, instrument or component.
  • FIG. 8 shows images of individual Jurkat cells co-partitioned along with barcode oligonucleotide containing beads in aqueous droplets in an aqueous in oil emulsion.
  • individual cells may be readily co-partitioned with individual beads.
  • optimization of individual cell loading may be carried out by a number of methods, including by providing dilutions of cell populations into the microfluidic system in order to achieve the desired cell loading per partition as described elsewhere herein.
  • fragmentation may be accomplished through the co-partitioning of shearing enzymes, such as endonucleases, in order to fragment the nucleic acids into smaller fragments.
  • shearing enzymes such as endonucleases
  • endonucleases may include restriction endonucleases, including type II and type IIs restriction endonucleases as well as other nucleic acid cleaving enzymes, such as nicking endonucleases, and the like.
  • fragmentation may not be desired, and full length nucleic acids may be retained within the partitions, or in the case of encapsulated cells or cell contents, fragmentation may be carried out prior to partitioning, e.g., through enzymatic methods, e.g., those described herein, or through mechanical methods, e.g., mechanical, acoustic or other shearing.
  • the oligonucleotides disposed upon the bead may be used to barcode and amplify fragments of those nucleic acids.
  • the oligonucleotides present on the beads that are co-partitioned with the cells are released from their beads into the partition with the cell's nucleic acids.
  • the oligonucleotides can include, along with the barcode sequence, a primer sequence at its 5′ end. This primer sequence may be a random oligonucleotide sequence intended to randomly prime numerous different regions on the cell's nucleic acids, or it may be a specific primer sequence targeted to prime upstream of a specific targeted region of the cell's genome.
  • the primer portion of the oligonucleotide can anneal to a complementary region of the cell's nucleic acid.
  • Extension reaction reagents e.g., DNA polymerase, nucleoside triphosphates, co-factors (e.g., Mg2+ or Mn2+), that are also co-partitioned with the cells and beads, then extend the primer sequence using the cell's nucleic acid as a template, to produce a complementary fragment to the strand of the cell's nucleic acid to which the primer annealed, which complementary fragment includes the oligonucleotide and its associated barcode sequence.
  • Annealing and extension of multiple primers to different portions of the cell's nucleic acids will result in a large pool of overlapping complementary fragments of the nucleic acid, each possessing its own barcode sequence indicative of the partition in which it was created.
  • these complementary fragments may themselves be used as a template primed by the oligonucleotides present in the partition to produce a complement of the complement that again, includes the barcode sequence.
  • this replication process is configured such that when the first complement is duplicated, it produces two complementary sequences at or near its termini, to allow formation of a hairpin structure or partial hairpin structure, the reduces the ability of the molecule to be the basis for producing further iterative copies.
  • the cell's nucleic acids may include any desired nucleic acids within the cell including, for example, the cell's DNA, e.g., genomic DNA, RNA, e.g., messenger RNA, and the like.
  • the methods and systems described herein are used in characterizing expressed mRNA, including, e.g., the presence and quantification of such mRNA, and may include RNA sequencing processes as the characterization process.
  • the reagents partitioned along with the cells may include reagents for the conversion of mRNA into cDNA, e.g., reverse transcriptase enzymes and reagents, to facilitate sequencing processes where DNA sequencing is employed.
  • the nucleic acids to be characterized comprise RNA, e.g., mRNA, schematic illustration of one example of this is shown in FIG. 3 .
  • oligonucleotides that include a barcode sequence are co-partitioned in, e.g., a droplet 302 in an emulsion, along with a sample nucleic acid 304 .
  • the oligonucleotides 308 may be provided on a bead 306 that is co-partitioned with the sample nucleic acid 304 , which oligonucleotides are releasable from the bead 306 , as shown in panel A.
  • the oligonucleotides 308 include a barcode sequence 312 , in addition to one or more functional sequences, e.g., sequences 310 , 314 and 316 .
  • oligonucleotide 308 is shown as comprising barcode sequence 312 , as well as sequence 310 that may function as an attachment or immobilization sequence for a given sequencing system, e.g., a P5 sequence used for attachment in flow cells of an Illumina Hiseq® or Miseq® system.
  • the oligonucleotides also include a primer sequence 316 , which may include a random or targeted N-mer for priming replication of portions of the sample nucleic acid 304 .
  • oligonucleotide 308 is also included within oligonucleotide 308 .
  • sequence 314 may provide a sequencing priming region, such as a “read1” or R1 priming region, that is used to prime polymerase mediated, template directed sequencing by synthesis reactions in sequencing systems.
  • the functional sequences may be selected to be compatible with a variety of different sequencing systems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and the requirements thereof.
  • the barcode sequence 312 , immobilization sequence 310 and R1 sequence 314 may be common to all of the oligonucleotides attached to a given bead.
  • the primer sequence 316 may vary for random N-mer primers, or may be common to the oligonucleotides on a given bead for certain targeted applications.
  • the functional sequences may include primer sequences useful for RNA-seq applications.
  • the oligonucleotides may include poly-T primers for priming reverse transcription of RNA for RNA-seq.
  • oligonucleotides in a given partition e.g., included on an individual bead, may include multiple types of primer sequences in addition to the common barcode sequences, such as both DNA-sequencing and RNA sequencing primers, e.g., poly-T primer sequences included within the oligonucleotides coupled to the bead.
  • a single partitioned cell may be both subjected to DNA and RNA sequencing processes.
  • the oligonucleotides can prime the sample nucleic acid as shown in panel B, which allows for extension of the oligonucleotides 308 and 308 a using polymerase enzymes and other extension reagents also co-partitioned with the bead 306 and sample nucleic acid 304 .
  • panel C following extension of the oligonucleotides that, for random N-mer primers, may anneal to multiple different regions of the sample nucleic acid 304 ; multiple overlapping complements or fragments of the nucleic acid are created, e.g., fragments 318 and 320 .
  • sequence portions that are complementary to portions of sample nucleic acid e.g., sequences 322 and 324
  • these constructs are generally referred to herein as comprising fragments of the sample nucleic acid 304 , having the attached barcode sequences.
  • the barcoded nucleic acid fragments may then be subjected to characterization, e.g., through sequence analysis, or they may be further amplified in the process, as shown in panel D.
  • additional oligonucleotides e.g., oligonucleotide 308 b , also released from bead 306 , may prime the fragments 318 and 320 . This shown for fragment 318 .
  • the oligonucleotide anneals with the fragment 318 , and is extended to create a complement 326 to at least a portion of fragment 318 which includes sequence 328 , that comprises a duplicate of a portion of the sample nucleic acid sequence. Extension of the oligonucleotide 308 b continues until it has replicated through the oligonucleotide portion 308 of fragment 318 .
  • the oligonucleotides may be configured to prompt a stop in the replication by the polymerase at a desired point, e.g., after replicating through sequences 316 and 314 of oligonucleotide 308 that is included within fragment 318 .
  • this may be accomplished by different methods, including, for example, the incorporation of different nucleotides and/or nucleotide analogues that are not capable of being processed by the polymerase enzyme used.
  • this may include the inclusion of uracil containing nucleotides within the sequence region 312 to prevent a non-uracil tolerant polymerase to cease replication of that region.
  • a fragment 326 is created that includes the full-length oligonucleotide 308 b at one end, including the barcode sequence 312 , the attachment sequence 310 , the R1 primer region 314 , and the random N-mer sequence 316 b .
  • the complement 316 ′ to the random N-mer of the first oligonucleotide 308 may be included the complement 316 ′ to the random N-mer of the first oligonucleotide 308 , as well as a complement to all or a portion of the R1 sequence, shown as sequence 314 ′.
  • the R1 sequence 314 and its complement 314 ′ are then able to hybridize together to form a partial hairpin structure 328 .
  • sequence 316 ′ which is the complement to random N-mer 316
  • sequence 316 b may not be expected to be complementary to random N-mer sequence 316 b . This may not be the case for other applications, e.g., targeted primers, where the N-mers may be common among oligonucleotides within a given partition.
  • partial hairpin structures By forming these partial hairpin structures, it allows for the removal of first level duplicates of the sample sequence from further replication, e.g., preventing iterative copying of copies.
  • the partial hairpin structure also provides a useful structure for subsequent processing of the created fragments, e.g., fragment 326 .
  • the amplification of the cell's nucleic acids is carried out until the barcoded overlapping fragments within the partition constitute at least 1 ⁇ coverage of the particular portion or all of the cell's genome, at least 2 ⁇ , at least 3 ⁇ , at least 4 ⁇ , at least 5 ⁇ , at least 10 ⁇ , at least 20 ⁇ , at least 40 ⁇ or more coverage of the genome or its relevant portion of interest.
  • barcoded fragments may be directly sequenced on an appropriate sequencing system, e.g., an Illumina Hiseq®, Miseq® or X10 system, or they may be subjected to additional processing, such as further amplification, attachment of other functional sequences, e.g., second sequencing primers, for reverse reads, sample index sequences, and the like.
  • an appropriate sequencing system e.g., an Illumina Hiseq®, Miseq® or X10 system
  • additional processing such as further amplification, attachment of other functional sequences, e.g., second sequencing primers, for reverse reads, sample index sequences, and the like.
  • All of the fragments from multiple different partitions may then be pooled for sequencing on high throughput sequencers as described herein, where the pooled fragments comprise a large number of fragments derived from the nucleic acids of different cells or small cell populations, but where the fragments from the nucleic acids of a given cell will share the same barcode sequence.
  • the sequence of that fragment may be attributed back to that cell or those cells based upon the presence of the barcode, which will also aid in applying the various sequence fragments from multiple partitions to assembly of individual genomes for different cells. This is schematically illustrated in FIG. 4 .
  • a first nucleic acid 404 from a first cell 400 , and a second nucleic acid 406 from a second cell 402 are each partitioned along with their own sets of barcode oligonucleotides as described above.
  • the nucleic acids may comprise a chromosome, entire genome or other large nucleic acid from the cells.
  • each cell's nucleic acids 404 and 406 is then processed to separately provide overlapping set of second fragments of the first fragment(s), e.g., second fragment sets 408 and 410 .
  • This processing also provides the second fragments with a barcode sequence that is the same for each of the second fragments derived from a particular first fragment.
  • the barcode sequence for second fragment set 408 is denoted by “1” while the barcode sequence for fragment set 410 is denoted by “2”.
  • a diverse library of barcodes may be used to differentially barcode large numbers of different fragment sets. However, it is not necessary for every second fragment set from a different first fragment to be barcoded with different barcode sequences. In fact, in many cases, multiple different first fragments may be processed concurrently to include the same barcode sequence. Diverse barcode libraries are described in detail elsewhere herein.
  • the barcoded fragments may then be pooled for sequencing using, for example, sequence by synthesis technologies available from Illumina or Ion Torrent division of Thermo-Fisher, Inc.
  • sequence reads 412 can be attributed to their respective fragment set, e.g., as shown in aggregated reads 414 and 416 , at least in part based upon the included barcodes, and in some cases, in part based upon the sequence of the fragment itself.
  • the attributed sequence reads for each fragment set are then assembled to provide the assembled sequence for each cell's nucleic acids, e.g., sequences 418 and 420 , which in turn, may be attributed to individual cells, e.g., cells 400 and 402 .
  • the methods and systems described herein may have much broader applicability, including the ability to characterize other aspects of individual cells or cell populations, by allowing for the allocation of reagents to individual cells, and providing for the attributable analysis or characterization of those cells in response to those reagents. These methods and systems are particularly valuable in being able to characterize cells for, e.g., research, diagnostic, pathogen identification, and many other purposes.
  • cell surface features e.g., cell surface proteins like cluster of differentiation or CD proteins, have significant diagnostic relevance in characterization of diseases like cancer.
  • the methods and systems described herein may be used to characterize cell features, such as cell surface features, e.g., proteins, receptors, etc.
  • the methods described herein may be used to attach reporter molecules to these cell features, that when partitioned as described above, may be barcoded and analyzed, e.g., using DNA sequencing technologies, to ascertain the presence, and in some cases, relative abundance or quantity of such cell features within an individual cell or population of cells.
  • a library of potential cell binding ligands e.g., antibodies, antibody fragments, cell surface receptor binding molecules, or the like, maybe provided associated with a first set of nucleic acid reporter molecules, e.g., where a different reporter oligonucleotide sequence is associated with a specific ligand, and therefore capable of binding to a specific cell surface feature.
  • different members of the library may be characterized by the presence of a different oligonucleotide sequence label, e.g., an antibody to a first type of cell surface protein or receptor may have associated with it a first known reporter oligonucleotide sequence, while an antibody to a second receptor protein may have a different known reporter oligonucleotide sequence associated with it.
  • the cells Prior to co-partitioning, the cells may be incubated with the library of ligands, that may represent antibodies to a broad panel of different cell surface features, e.g., receptors, proteins, etc., and which include their associated reporter oligonucleotides.
  • Unbound ligands are washed from the cells, and the cells are then co-partitioned along with the barcode oligonucleotides described above.
  • the partitions will include the cell or cells, as well as the bound ligands and their known, associated reporter oligonucleotides.
  • reporter oligonucleotides may then subject the reporter oligonucleotides to the barcoding operations described above for cellular nucleic acids, to produce barcoded, reporter oligonucleotides, where the presence of the reporter oligonucleotides can be indicative of the presence of the particular cell surface feature, and the barcode sequence will allow the attribution of the range of different cell surface features to a given individual cell or population of cells based upon the barcode sequence that was co-partitioned with that cell or population of cells. As a result, one may generate a cell-by-cell profile of the cell surface features within a broader population of cells. This aspect of the methods and systems described herein, is described in greater detail below.
  • FIG. 5 This example is schematically illustrated in FIG. 5 .
  • a population of cells represented by cells 502 and 504 are incubated with a library of cell surface associated reagents, e.g., antibodies, cell surface binding proteins, ligands or the like, where each different type of binding group includes an associated nucleic acid reporter molecule associated with it, shown as ligands and associated reporter molecules 506 , 508 , 510 and 512 (with the reporter molecules being indicated by the differently shaded circles).
  • ligands and associated reporter molecules can become associated or coupled with the cell surface.
  • Individual cells are then partitioned into separate partitions, e.g., droplets 514 and 516 , along with their associated ligand/reporter molecules, as well as an individual barcode oligonucleotide bead as described elsewhere herein, e.g., beads 522 and 524 , respectively.
  • the barcoded oligonucleotides are released from the beads and used to attach the barcode sequence the reporter molecules present within each partition with a barcode that is common to a given partition, but which varies widely among different partitions. For example, as shown in FIG.
  • the reporter molecules that associate with cell 502 in partition 514 are barcoded with barcode sequence 518
  • the reporter molecules associated with cell 504 in partition 516 are barcoded with barcode 520 .
  • a library of oligonucleotides that reflects the surface ligands of the cell, as reflected by the reporter molecule, but which is substantially attributable to an individual cell by virtue of a common barcode sequence, allowing a single cell level profiling of the surface characteristics of the cell.
  • this process is not limited to cell surface receptors but may be used to identify the presence of a wide variety of specific cell structures, chemistries or other characteristics.
  • Single cell processing and analysis methods and systems described herein can be utilized for a wide variety of applications, including analysis of specific individual cells, analysis of different cell types within populations of differing cell types, analysis and characterization of large populations of cells for environmental, human health, epidemiological forensic, or any of a wide variety of different applications.
  • a particularly valuable application of the single cell analysis processes described herein is in the sequencing and characterization of a diseased cell.
  • a diseased cell can have altered metabolic properties, gene expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer.
  • cancer cells Of particular interest are cancer cells.
  • conventional analytical techniques including the ensemble sequencing processes alluded to above, are not highly adept at picking small variations in genomic make-up of cancer cells, particularly where those exist in a sea of normal tissue cells.
  • wide variations can exist and can be masked by the ensemble approaches to sequencing (See, e.g., Patel, et al., Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma, Science DOI: 10.1126/science.1254257 (Published online Jun. 12, 2014).
  • Cancer cells may be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells, and subjected to the partitioning processes described above. Upon analysis, one can identify individual cell sequences as deriving from a single cell or small group of cells, and distinguish those over normal tissue cell sequences.
  • Non-limiting examples of cancer cells include cells of cancers such as Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell
  • primer sequences useful in any of the various operations for attaching barcode sequences and/or amplification reactions may comprise gene specific sequences which target genes or regions of genes associated with or suspected of being associated with cancer.
  • this can include genes or regions of genes where the presence of mutations (e.g., insertions, deletions, polymorphisms, copy number variations, and gene fusions) associated with a cancerous condition are suspected to be present in a cell population.
  • fetal health or abnormality through the analysis of fetal cells is a difficult task using conventional techniques.
  • relatively invasive procedures such as amniocentesis obtaining fetal cell samples can employ harvesting those cells from the maternal circulation.
  • circulating fetal cells make up an extremely small fraction of the overall cellular population of that circulation.
  • complex analyses are performed in order to characterize what of the obtained data is likely derived from fetal cells as opposed to maternal cells.
  • the single cell characterization methods and systems described herein one can attribute genetic make up to individual cells, and categorize those cells as maternal or fetal based upon their respective genetic make-up.
  • the genetic sequence of fetal cells may be used to identify any of a number of genetic disorders, including, e.g., aneuploidy such as Down syndrome, Edwards syndrome, and Patau syndrome.
  • immune cells Also of interest are immune cells. Methods and compositions disclosed herein can be utilized for sequence analysis of the immune repertoire. Analysis of sequence information underlying the immune repertoire can provide a significant improvement in understanding the status and function of the immune system.
  • Non-limiting examples of immune cells which can be analyzed utilizing the methods described herein include B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and T helper cells), natural killer cells, cytokine induced killer (CIK) cells; myeloid cells, such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes/hypersegmented neutrophils), monocytes/macrophages, mast cell, thrombocytes/megakaryocytes, and dendritic cells.
  • T cells e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and T helper cells
  • CIK cytokine induced killer
  • myeloid cells such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes/hypersegmented neutrophils), monocytes/macrophages,
  • Immune cells express various adaptive immunological receptors relating to immune function, such as T cell receptors and B cell receptors.
  • T cell receptors and B cells receptors play a part in the immune response by specifically recognizing and binding to antigens and aiding in their destruction.
  • the T cell receptor is a molecule found on the surface of T cells that is generally responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR is generally a heterodimer of two chains, each of which is a member of the immunoglobulin superfamily, possessing an N-terminal variable (V) domain, and a C terminal constant domain.
  • V N-terminal variable
  • V N-terminal variable
  • C terminal constant domain In humans, in 95% of T cells the TCR consists of an alpha ( ⁇ ) and beta ( ⁇ ) chain, whereas in 5% of T cells the TCR consists of gamma and delta ( ⁇ / ⁇ ) chains. This ratio can change during ontogeny and in diseased states as well as in different species.
  • Each of the two chains of a TCR contains multiple copies of gene segments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, and a joining T gene segment.
  • the TCR alpha chain is generated by recombination of V and J segments, while the beta chain is generated by recombination of V, D, and J segments.
  • generation of the TCR gamma chain involves recombination of V and J gene segments, while generation of the TCR delta chain occurs by recombination of V, D, and J gene segments.
  • the intersection of these specific regions corresponds to the CDR3 region that is important for antigen-MHC recognition.
  • Complementarity determining regions are sequences in the variable domains of antigen receptors (e.g., T cell receptor and immunoglobulin) that can complement an antigen.
  • antigen receptors e.g., T cell receptor and immunoglobulin
  • Most of the diversity of CDRs is found in CDR3, with the diversity being generated by somatic recombination events during the development of T lymphocytes.
  • a unique nucleotide sequence that arises during the gene arrangement process can be referred to as a clonotype.
  • the B cell receptor is a molecule found on the surface of B cells.
  • the antigen binding portion of a BCR is composed of a membrane-bound antibody that, like most antibodies (e.g., immunoglobulins), has a unique and randomly determined antigen-binding site.
  • the antigen binding portion of a BCR includes membrane-bound immunoglobulin molecule of one isotype (e.g., IgD, IgM, IgA, IgG, or IgE).
  • the BCR is composed of two genes IgH and IgK (or IgL) coding for antibody heavy and light chains. Immunoglobulins are formed by recombination among gene segments, sequence diversification at the junctions of these segments, and point mutations throughout the gene. Each heavy chain gene contains multiple copies of three different gene segments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, and a joining T gene segment. Each light chain gene contains multiple copies of two different gene segments for the variable region of the protein—a variable ‘V’ gene segment and a joining T gene segment. The recombination can generate a molecule with one of each of the V, D, and J segments.
  • N and P nucleotides may be deleted and others added (called N and P nucleotides) at each of the two junctions, thereby generating further diversity.
  • B cell activation a process of affinity maturation through somatic hypermutation occurs. In this process progeny cells of the activated B cells accumulate distinct somatic mutations throughout the gene with higher mutation concentration in the CDR regions leading to the generation of antibodies with higher affinity to the antigens.
  • activated B cells undergo the process of isotype switching.
  • Antibodies with the same variable segments can have different forms (isotypes) depending on the constant segment. Whereas all na ⁇ ve B cells express IgM (or IgD), activated B cells mostly express IgG but also IgM, IgA and IgE.
  • IgM and/or IgD
  • IgG IgG
  • IgA IgA
  • IgE IgE
  • a unique nucleotide sequence that arises during the gene arrangement process can similarly be referred to as a clonotype.
  • the methods, compositions and systems disclosed herein are utilized to analyze the various sequences of TCRs and BCRs from immune cells, for example various clonotypes.
  • methods, compositions and systems disclosed herein are used to analyze the sequence of a TCR alpha chain, a TCR beta chain, a TCR delta chain, a TCR gamma chain, or any fragment thereof (e.g., variable regions including VDJ or VJ regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations of fragments thereof).
  • methods, compositions and systems disclosed herein are used to analyze the sequence of a B cell receptor heavy chain, B cell receptor light chain, or any fragment thereof (e.g., variable regions including VDJ or VJ regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations of fragments thereof).
  • primer sequences useful in any of the various operations for attaching barcode sequences and/or amplification reactions may comprise gene specific sequences which target genes or regions of genes of immune cell proteins, for example immune receptors.
  • gene sequences include, but are not limited to, sequences of various T cell receptor alpha variable genes (TRAV genes), T cell receptor alpha joining genes (TRAJ genes), T cell receptor alpha constant genes (TRAC genes), T cell receptor beta variable genes (TRBV genes), T cell receptor beta diversity genes (TRBD genes), T cell receptor beta joining genes (TRBJ genes), T cell receptor beta constant genes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), T cell receptor gamma joining genes (TRGJ genes), T cell receptor gamma constant genes (TRGC genes), T cell receptor delta variable genes (TRDV genes), T cell receptor delta diversity genes (TRDD genes), T cell receptor delta joining genes (TRDJ genes), and T cell receptor delta constant genes (TRDC genes).
  • TRAV genes T cell receptor alpha variable genes
  • TRAJ genes
  • samples may, by their nature, be made up of diverse populations of cells and other material that “contaminate” the sample, relative to the cells for which the sample is being tested, e.g., environmental indicator organisms, toxic organisms, and the like for, e.g., environmental and food safety testing, victim and/or perpetrator cells in forensic analysis for sexual assault, and other violent crimes, and the like.
  • the methods and compositions disclosed herein allow the determination of not only the immune repertoire and different clonotypes, but the functional characteristics (e.g., the transcriptome) of the cells associated with a clonotype or plurality of clonotypes that bind to the same or similar antigen.
  • These functional characteristics can comprise transcription of cytokine, chemokine, or cell-surface associated molecules, such as, costimulatory molecules, checkpoint inhibitors, cell surface maturation markers, or cell-adhesion molecules.
  • Such analysis allows a cell or cell population expressing a particular T cell receptor, B cell receptor, or immunoglobulin to be associated with certain functional characteristics.
  • any given antigen there will be multiple clonotypes of T cell receptor, B cell receptor, or immunoglobulin that specifically bind to that antigen.
  • Multiple clonotypes that bind to the same antigen are known as the idiotype.
  • neural cells can include long interspersed nuclear elements (LINEs), or ‘jumping’ genes that can move around the genome, which cause each neuron to differ from its neighbor cells.
  • LINEs long interspersed nuclear elements
  • Research has shown that the number of LINES in human brain exceeds that of other tissues, e.g., heart and liver tissue, with between 80 and 300 unique insertions (See, e.g., Coufal, N. G. et al. Nature 460, 1127-1131 (2009)). These differences have been postulated as being related to a person's susceptibility to neuro-logical disorders (see, e.g., Muotri, A. R. et al. Nature 468, 443-446 (2010)), or provide the brain with a diversity with which to respond to challenges.
  • the methods described herein may be used in the sequencing and characterization of individual neural cells.
  • the single cell analysis methods described herein are also useful in the analysis of gene expression, as noted above, both in terms of identification of RNA transcripts and their quantitation.
  • the barcode oligonucleotides may be configured to prime, replicate and consequently yield barcoded fragments of RNA from individual cells.
  • the barcode oligonucleotides may include mRNA specific priming sequences, e.g., poly-T primer segments that allow priming and replication of mRNA in a reverse transcription reaction or other targeted priming sequences.
  • random RNA priming may be carried out using random N-mer primer segments of the barcode oligonucleotides.
  • FIG. 6 provides a schematic of one example method for RNA expression analysis in individual cells using the methods described herein.
  • a cell containing sample is sorted for viable cells, which are quantified and diluted for subsequent partitioning.
  • the individual cells separately co-partitioned with gel beads bearing the barcoding oligonucleotides as described herein.
  • the cells are lysed and the barcoded oligonucleotides released into the partitions at operation 606 , where they interact with and hybridize to the mRNA at operation 608 , e.g., by virtue of a poly-T primer sequence, which is complementary to the poly-A tail of the mRNA.
  • a reverse transcription reaction is carried out at operation 610 to synthesize a cDNA transcript of the mRNA that includes the barcode sequence.
  • the barcoded cDNA transcripts are then subjected to additional amplification at operation 612 , e.g., using a polymerase chain reaction (PCR) process, purification at operation 614 , before they are placed on a nucleic acid sequencing system for determination of the cDNA sequence and its associated barcode sequence(s).
  • PCR polymerase chain reaction
  • operations 602 through 608 can occur while the reagents remain in their original droplet or partition, while operations 612 through 616 can occur in bulk (e.g., outside of the partition).
  • a partition is a droplet in an emulsion
  • the emulsion can be broken and the contents of the droplet pooled in order to complete operations 612 through 616 .
  • barcode oligonucleotides may be digested with exonucleases after the emulsion is broken. Exonuclease activity can be inhibited by ethylenediaminetetraacetic acid (EDTA) following primer digestion.
  • operation 610 may be performed either within the partitions based upon co-partitioning of the reverse transcription mixture, e.g., reverse transcriptase and associated reagents, or it may be performed in bulk.
  • the structure of the barcode oligonucleotides may include a number of sequence elements in addition to the oligonucleotide barcode sequence.
  • a barcode oligonucleotide for use in RNA analysis as described above is shown in FIG. 7 .
  • the overall oligonucleotide 702 is coupled to a bead 704 by a releasable linkage 706 , such as a disulfide linker.
  • the oligonucleotide may include functional sequences that are used in subsequent processing, such as functional sequence 708 , which may include one or more of a sequencer specific flow cell attachment sequence, e.g., a P5 sequence for Illumina sequencing systems, as well as sequencing primer sequences, e.g., a R1 primer for Illumina sequencing systems.
  • a barcode sequence 710 is included within the structure for use in barcoding the sample RNA.
  • An mRNA specific priming sequence, such as poly-T sequence 712 is also included in the oligonucleotide structure.
  • An anchoring sequence segment 714 may be included to ensure that the poly-T sequence hybridizes at the sequence end of the mRNA.
  • This anchoring sequence can include a random short sequence of nucleotides, e.g., 1-mer, 2-mer, 3-mer or longer sequence, which will ensure that the poly-T segment is more likely to hybridize at the sequence end of the poly-A tail of the mRNA.
  • An additional sequence segment 716 may be provided within the oligonucleotide sequence. In some cases, this additional sequence provides a unique molecular identifier (UMI) sequence segment, e.g., as a random sequence (e.g., such as a random N-mer sequence) that varies across individual oligonucleotides coupled to a single bead, whereas barcode sequence 710 can be constant among oligonucleotides tethered to an individual bead.
  • UMI unique molecular identifier
  • This unique sequence serves to provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA.
  • individual bead can include tens to hundreds of thousands or even millions of individual oligonucleotide molecules, where, as noted, the barcode segment can be constant or relatively constant for a given bead, but where the variable or unique sequence segment will vary across an individual bead.
  • This unique molecular identifier (UMI) sequence segment may include from 5 to about 8 or more nucleotides within the sequence of the oligonucleotides.
  • the unique molecular identifier (UMI) sequence segment can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length or longer. In some cases, the unique molecular identifier (UMI) sequence segment can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length or longer. In some cases, the unique molecular identifier (UMI) sequence segment can be at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length or shorter.
  • a cell is co-partitioned along with a barcode bearing bead and lysed while the barcoded oligonucleotides are released from the bead.
  • the poly-T portion of the released barcode oligonucleotide then hybridizes to the poly-A tail of the mRNA.
  • the poly-T segment then primes the reverse transcription of the mRNA to produce a cDNA transcript of the mRNA, but which includes each of the sequence segments 708 - 716 of the barcode oligonucleotide.
  • the oligonucleotide 702 includes an anchoring sequence 714 , it will more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA.
  • all of the cDNA transcripts of the individual mRNA molecules will include a common barcode sequence segment 710 .
  • the transcripts made from different mRNA molecules within a given partition will vary at this unique sequence. This provides a quantitation feature that can be identifiable even following any subsequent amplification of the contents of a given partition, e.g., the number of unique segments associated with a common barcode can be indicative of the quantity of mRNA originating from a single partition, and thus, a single cell.
  • the transcripts are then amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the unique sequence segment.
  • the primer sequence can be a gene specific primer sequence which targets specific genes for reverse transcription.
  • target genes comprise T cell receptor genes, B cell receptor genes or immunoglobulin receptor genes.
  • the gel bead bound oligonucleotides may be used to hybridize and capture the mRNA on the solid phase of the gel beads, in order to facilitate the separation of the RNA from other cell contents.
  • FIG. 9A An additional example of a barcode oligonucleotide for use in RNA analysis, including messenger RNA (mRNA, including mRNA obtained from a cell) analysis, is shown in FIG. 9A .
  • the overall oligonucleotide 902 can be coupled to a bead 904 by a releasable linkage 906 , such as a disulfide linker.
  • the oligonucleotide may include functional sequences that are used in subsequent processing, such as functional sequence 908 , which may include a sequencer specific flow cell attachment sequence, e.g., a P5 sequence for Illumina sequencing systems, as well as functional sequence 910 , which may include sequencing primer sequences, e.g., a R1 primer binding site for Illumina sequencing systems.
  • a barcode sequence 912 is included within the structure for use in barcoding the sample RNA.
  • An RNA specific (e.g., mRNA specific) priming sequence, such as poly-T sequence 914 is also included in the oligonucleotide structure.
  • An anchoring sequence segment (not shown) may be included to ensure that the poly-T sequence hybridizes at the sequence end of the mRNA.
  • An additional sequence segment 916 may be provided within the oligonucleotide sequence.
  • This additional sequence can provide a unique molecular identifier (UMI) sequence segment, e.g., as a random N-mer sequence that varies across individual oligonucleotides coupled to a single bead, whereas barcode sequence 912 can be constant among oligonucleotides tethered to an individual bead.
  • UMI unique molecular identifier
  • this unique sequence can serve to provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA, e.g., mRNA counting.
  • individual beads can include tens to hundreds of thousands or even millions of individual oligonucleotide molecules, where, as noted, the barcode segment can be constant or relatively constant for a given bead, but where the variable or unique sequence segment will vary across an individual bead.
  • a cell is co-partitioned along with a barcode bearing bead, switch oligo 924 , and other reagents such as reverse transcriptase, a reducing agent and dNTPs into a partition (e.g., a droplet in an emulsion).
  • a partition e.g., a droplet in an emulsion
  • the cell is lysed while the barcoded oligonucleotides 902 are released from the bead (e.g., via the action of the reducing agent) and the poly-T segment 914 of the released barcode oligonucleotide then hybridizes to the poly-A tail of mRNA 920 that is released from the cell.
  • the poly-T segment 914 is extended in a reverse transcription reaction using the mRNA as a template to produce a cDNA transcript 922 complementary to the mRNA and also includes each of the sequence segments 908 , 912 , 910 , 916 and 914 of the barcode oligonucleotide.
  • Terminal transferase activity of the reverse transcriptase can add additional bases to the cDNA transcript (e.g., polyC).
  • the switch oligo 924 may then hybridize with the additional bases added to the cDNA transcript and facilitate template switching.
  • a sequence complementary to the switch oligo sequence can then be incorporated into the cDNA transcript 922 via extension of the cDNA transcript 922 using the switch oligo 924 as a template.
  • all of the cDNA transcripts of the individual mRNA molecules will include a common barcode sequence segment 912 . However, by including the unique random N-mer sequence 916 , the transcripts made from different mRNA molecules within a given partition will vary at this unique sequence.
  • this provides a quantitation feature that can be identifiable even following any subsequent amplification of the contents of a given partition, e.g., the number of unique segments associated with a common barcode can be indicative of the quantity of mRNA originating from a single partition, and thus, a single cell.
  • the cDNA transcript 922 is then amplified with primers 926 (e.g., PCR primers) in operation 954 .
  • primers 926 e.g., PCR primers
  • the amplified product is then purified (e.g., via solid phase reversible immobilization (SPRI)) in operation 956 .
  • SPRI solid phase reversible immobilization
  • the amplified product is then sheared, ligated to additional functional sequences, and further amplified (e.g., via PCR).
  • the functional sequences may include a sequencer specific flow cell attachment sequence 930 , e.g., a P7 sequence for Illumina sequencing systems, as well as functional sequence 928 , which may include a sequencing primer binding site, e.g., for a R2 primer for Illumina sequencing systems, as well as functional sequence 932 , which may include a sample index, e.g., an i7 sample index sequence for Illumina sequencing systems.
  • operations 950 and 952 can occur in the partition, while operations 954 , 956 and 958 can occur in bulk solution (e.g., in a pooled mixture outside of the partition).
  • a partition is a droplet in an emulsion
  • the emulsion can be broken and the contents of the droplet pooled in order to complete operations 954 , 956 and 958 .
  • operation 954 may be completed in the partition.
  • barcode oligonucleotides may be digested with exonucleases after the emulsion is broken. Exonuclease activity can be inhibited by ethylenediaminetetraacetic acid (EDTA) following primer digestion.
  • EDTA ethylenediaminetetraacetic acid
  • RNA e.g., cellular RNA
  • functional sequence 908 may be a P7 sequence and functional sequence 910 may be a R2 primer binding site.
  • the functional sequence 930 may be a P5 sequence
  • functional sequence 928 may be a R1 primer binding site
  • functional sequence 932 may be an i5 sample index sequence for Illumina sequencing systems.
  • the configuration of the constructs generated by such a barcode oligonucleotide can help minimize (or avoid) sequencing of the poly-T sequence during sequencing.
  • FIG. 9B Shown in FIG. 9B is another example method for RNA analysis, including cellular mRNA analysis.
  • the switch oligo 924 is co-partitioned with the individual cell and barcoded bead along with reagents such as reverse transcriptase, a reducing agent and dNTPs into a partition (e.g., a droplet in an emulsion).
  • the switch oligo 924 may be labeled with an additional tag 934 , e.g., biotin.
  • the cell is lysed while the barcoded oligonucleotides 902 (e.g., as shown in FIG.
  • sequence 908 is a P7 sequence and sequence 910 is a R2 primer binding site. In other cases, sequence 908 is a P5 sequence and sequence 910 is a R1 primer binding site.
  • the poly-T segment 914 of the released barcode oligonucleotide hybridizes to the poly-A tail of mRNA 920 that is released from the cell.
  • the poly-T segment 914 is then extended in a reverse transcription reaction using the mRNA as a template to produce a cDNA transcript 922 complementary to the mRNA and also includes each of the sequence segments 908 , 912 , 910 , 916 and 914 of the barcode oligonucleotide. Terminal transferase activity of the reverse transcriptase can add additional bases to the cDNA transcript (e.g., polyC).
  • the switch oligo 924 may then hybridize with the cDNA transcript and facilitate template switching. A sequence complementary to the switch oligo sequence can then be incorporated into the cDNA transcript 922 via extension of the cDNA transcript 922 using the switch oligo 924 as a template.
  • an isolation operation 960 can be used to isolate the cDNA transcript 922 from the reagents and oligonucleotides in the partition.
  • the additional tag 934 e.g., biotin
  • an interacting tag 936 e.g., streptavidin, which may be attached to a magnetic bead 938 .
  • the cDNA can be isolated with a pull-down operation (e.g., via magnetic separation, centrifugation) before amplification (e.g., via PCR) in operation 955 , followed by purification (e.g., via solid phase reversible immobilization (SPRI)) in operation 957 and further processing (shearing, ligation of sequences 928 , 932 and 930 and subsequent amplification (e.g., via PCR)) in operation 959 .
  • sequence 908 is a P7 sequence and sequence 910 is a R2 primer binding site
  • sequence 930 is a P5 sequence
  • sequence 928 is a R1 primer binding site
  • sequence 932 is an i5 sample index sequence.
  • sequence 908 is a P5 sequence and sequence 910 is a R1 primer binding site
  • sequence 930 is a P7 sequence and sequence 928 is a R2 primer binding site
  • sequence 932 is an i7 sample index sequence.
  • operations 951 and 953 can occur in the partition
  • operations 960 , 955 , 957 and 959 can occur in bulk solution (e.g., in a pooled mixture outside of the partition).
  • a partition is a droplet in an emulsion
  • the emulsion can be broken and the contents of the droplet pooled in order to complete operation 960 .
  • the operations 955 , 957 , and 959 can then be carried out following operation 960 after the transcripts are pooled for processing.
  • FIG. 9C Shown in FIG. 9C is another example method for RNA analysis, including cellular mRNA analysis.
  • the switch oligo 924 is co-partitioned with the individual cell and barcoded bead along with reagents such as reverse transcriptase, a reducing agent and dNTPs in a partition (e.g., a droplet in an emulsion).
  • a partition e.g., a droplet in an emulsion
  • the cell is lysed while the barcoded oligonucleotides 902 (e.g., as shown in FIG. 9A ) are released from the bead (e.g., via the action of the reducing agent).
  • sequence 908 is a P7 sequence and sequence 910 is a R2 primer binding site.
  • sequence 908 is a P5 sequence and sequence 910 is a R1 primer binding site.
  • the poly-T segment 914 of the released barcode oligonucleotide then hybridizes to the poly-A tail of mRNA 920 that is released from the cell.
  • the poly-T segment 914 is then extended in a reverse transcription reaction using the mRNA as a template to produce a cDNA transcript 922 complementary to the mRNA and also includes each of the sequence segments 908 , 912 , 910 , 916 and 914 of the barcode oligonucleotide. Terminal transferase activity of the reverse transcriptase can add additional bases to the cDNA transcript (e.g., polyC).
  • the switch oligo 924 may then hybridize with the cDNA transcript and facilitate template switching.
  • a sequence complementary to the switch oligo sequence can then be incorporated into the cDNA transcript 922 via extension of the cDNA transcript 922 using the switch oligo 924 as a template.
  • mRNA 920 and cDNA transcript 922 are denatured in operation 962 .
  • a second strand is extended from a primer 940 having an additional tag 942 , e.g., biotin, and hybridized to the cDNA transcript 922 .
  • the biotin labeled second strand can be contacted with an interacting tag 936 , e.g., streptavidin, which may be attached to a magnetic bead 938 .
  • the cDNA can be isolated with a pull-down operation (e.g., via magnetic separation, centrifugation) before amplification (e.g., via polymerase chain reaction (PCR)) in operation 965 , followed by purification (e.g., via solid phase reversible immobilization (SPRI)) in operation 967 and further processing (shearing, ligation of sequences 928 , 932 and 930 and subsequent amplification (e.g., via PCR)) in operation 969 .
  • PCR polymerase chain reaction
  • SPRI solid phase reversible immobilization
  • sequence 930 is a P5 sequence and sequence 928 is a R1 primer binding site and sequence 932 is an i5 sample index sequence.
  • sequence 930 is a P7 sequence and sequence 928 is a R2 primer binding site and sequence 932 is an i7 sample index sequence.
  • operations 961 and 963 can occur in the partition, while operations 962 , 964 , 965 , 967 , and 969 can occur in bulk (e.g., outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled in order to complete operations 962 , 964 , 965 , 967 and 969 .
  • FIG. 9D Shown in FIG. 9D is another example method for RNA analysis, including cellular mRNA analysis.
  • the switch oligo 924 is co-partitioned with the individual cell and barcoded bead along with reagents such as reverse transcriptase, a reducing agent and dNTPs.
  • the cell is lysed while the barcoded oligonucleotides 902 (e.g., as shown in FIG. 9A ) are released from the bead (e.g., via the action of the reducing agent).
  • sequence 908 is a P7 sequence and sequence 910 is a R2 primer binding site.
  • sequence 908 is a P5 sequence and sequence 910 is a R1 primer binding site.
  • the poly-T segment 914 of the released barcode oligonucleotide then hybridizes to the poly-A tail of mRNA 920 that is released from the cell.
  • the poly-T segment 914 is then extended in a reverse transcription reaction using the mRNA as a template to produce a cDNA transcript 922 complementary to the mRNA and also includes each of the sequence segments 908 , 912 , 910 , 916 and 914 of the barcode oligonucleotide. Terminal transferase activity of the reverse transcriptase can add additional bases to the cDNA transcript (e.g., polyC).
  • the switch oligo 924 may then hybridize with the cDNA transcript and facilitate template switching.
  • a sequence complementary to the switch oligo sequence can then be incorporated into the cDNA transcript 922 via extension of the cDNA transcript 922 using the switch oligo 924 as a template.
  • the mRNA 920 , cDNA transcript 922 and switch oligo 924 can be denatured, and the cDNA transcript 922 can be hybridized with a capture oligonucleotide 944 labeled with an additional tag 946 , e.g., biotin.
  • biotin-labeled capture oligonucleotide 944 which is hybridized to the cDNA transcript, can be contacted with an interacting tag 936 , e.g., streptavidin, which may be attached to a magnetic bead 938 .
  • an interacting tag 936 e.g., streptavidin
  • the cDNA transcript can be amplified (e.g., via PCR) with primers 926 at operation 975 , followed by purification (e.g., via solid phase reversible immobilization (SPRI)) in operation 977 and further processing (shearing, ligation of sequences 928 , 932 and 930 and subsequent amplification (e.g., via PCR)) in operation 979 .
  • SPRI solid phase reversible immobilization
  • sequence 930 is a P5 sequence and sequence 928 is a R1 primer binding site and sequence 932 is an i5 sample index sequence.
  • sequence 930 is a P7 sequence and sequence 928 is a R2 primer binding site and sequence 932 is an i7 sample index sequence.
  • operations 971 and 973 can occur in the partition, while operations 966 , 975 , 977 (purification), and 979 can occur in bulk (e.g., outside the partition).
  • the emulsion can be broken and the contents of the droplet pooled in order to complete operations 966 , 975 , 977 and 979 .
  • FIG. 9E Shown in FIG. 9E is another example method for RNA analysis, including cellular RNA analysis.
  • an individual cell is co-partitioned along with a barcode bearing bead, a switch oligo 990 , and other reagents such as reverse transcriptase, a reducing agent and dNTPs into a partition (e.g., a droplet in an emulsion).
  • a partition e.g., a droplet in an emulsion.
  • the cell is lysed while the barcoded oligonucleotides (e.g., 902 as shown in FIG. 9A ) are released from the bead (e.g., via the action of the reducing agent).
  • the barcoded oligonucleotides e.g., 902 as shown in FIG. 9A
  • sequence 908 is a P7 sequence and sequence 910 is a R2 primer binding site. In other cases, sequence 908 is a P5 sequence and sequence 910 is a R1 primer binding site.
  • the poly-T segment of the released barcode oligonucleotide then hybridizes to the poly-A tail of mRNA 920 released from the cell.
  • the poly-T segment is then extended in a reverse transcription reaction to produce a cDNA transcript 922 complementary to the mRNA and also includes each of the sequence segments 908 , 912 , 910 , 916 and 914 of the barcode oligonucleotide.
  • Terminal transferase activity of the reverse transcriptase can add additional bases to the cDNA transcript (e.g., polyC).
  • the switch oligo 990 may then hybridize with the cDNA transcript and facilitate template switching.
  • a sequence complementary to the switch oligo sequence and including a T7 promoter sequence, can be incorporated into the cDNA transcript 922 .
  • a second strand is synthesized and at operation 970 the T7 promoter sequence can be used by T7 polymerase to produce RNA transcripts in in vitro transcription.
  • the RNA transcripts can be purified (e.g., via solid phase reversible immobilization (SPRI)), reverse transcribed to form DNA transcripts, and a second strand can be synthesized for each of the DNA transcripts.
  • the RNA transcripts can be contacted with a DNase (e.g., DNAase I) to break down residual DNA.
  • a DNase e.g., DNAase I
  • the DNA transcripts are then fragmented and ligated to additional functional sequences, such as sequences 928 , 932 and 930 and, in some cases, further amplified (e.g., via PCR).
  • sequence 930 is a P5 sequence and sequence 928 is a R1 primer binding site and sequence 932 is an i5 sample index sequence.
  • sequence 930 is a P7 sequence and sequence 928 is a R2 primer binding site and sequence 932 is an i7 sample index sequence.
  • the DNA transcripts can be contacted with an RNase to break down residual RNA.
  • operations 981 and 983 can occur in the partition, while operations 968 , 970 , 985 and 987 can occur in bulk (e.g., outside the partition).
  • a partition is a droplet in an emulsion
  • the emulsion can be broken and the contents of the droplet pooled in order to complete operations 968 , 970 , 985 and 987 .
  • oligonucleotides coupled to beads may include primers with sequences that are targeted for such target regions (e.g., constant regions).
  • polyT primer regions can instead be gene specific primer sequences.
  • RNA analysis including messenger RNA (mRNA, including mRNA obtained from a cell) analysis
  • mRNA messenger RNA
  • FIG. 10 Another example of a barcode oligonucleotide for use in RNA analysis, including messenger RNA (mRNA, including mRNA obtained from a cell) analysis is shown in FIG. 10 .
  • the overall oligonucleotide 1002 is coupled to a bead 1004 by a releasable linkage 1006 , such as a disulfide linker.
  • the oligonucleotide may include functional sequences that are used in subsequent processing, such as functional sequence 1008 , which may include a sequencer specific flow cell attachment sequence, e.g., a P7 sequence, as well as functional sequence 1010 , which may include sequencing primer sequences, e.g., a R2 primer binding site.
  • functional sequence 1008 which may include a sequencer specific flow cell attachment sequence, e.g., a P7 sequence
  • a barcode sequence 1012 is included within the structure for use in barcoding the sample RNA.
  • An RNA specific (e.g., mRNA specific) priming sequence such as poly-T sequence 1014 may be included in the oligonucleotide structure.
  • An anchoring sequence segment (not shown) may be included to ensure that the poly-T sequence hybridizes at the sequence end of the mRNA.
  • An additional sequence segment 1016 may be provided within the oligonucleotide sequence. This additional sequence can provide a unique molecular identifier (UMI) sequence segment, as described elsewhere herein.
  • An additional functional sequence 1020 may be included for in vitro transcription, e.g., a T7 RNA polymerase promoter sequence.
  • individual beads can include tens to hundreds of thousands or even millions of individual oligonucleotide molecules, where, as noted, the barcode segment can be constant or relatively constant for a given bead, but where the variable or unique sequence segment will vary across an individual bead.
  • a cell is co-partitioned along with a barcode bearing bead, and other reagents such as reverse transcriptase, reducing agent and dNTPs into a partition (e.g., a droplet in an emulsion).
  • a partition e.g., a droplet in an emulsion.
  • the cell is lysed while the barcoded oligonucleotides 1002 are released (e.g., via the action of the reducing agent) from the bead, and the poly-T segment 1014 of the released barcode oligonucleotide then hybridizes to the poly-A tail of mRNA 1020 .
  • the poly-T segment is then extended in a reverse transcription reaction using the mRNA as template to produce a cDNA transcript 1022 of the mRNA and also includes each of the sequence segments 1020 , 1008 , 1012 , 1010 , 1016 , and 1014 of the barcode oligonucleotide.
  • all of the cDNA transcripts of the individual mRNA molecules will include a common barcode sequence segment 1012 .
  • the transcripts made from different mRNA molecules within a given partition will vary at this unique sequence.
  • this provides a quantitation feature that can be identifiable even following any subsequent amplification of the contents of a given partition, e.g., the number of unique segments associated with a common barcode can be indicative of the quantity of mRNA originating from a single partition, and thus, a single cell.
  • a second strand is synthesized and at operation 1056 the T7 promoter sequence can be used by T7 polymerase to produce RNA transcripts in in vitro transcription.
  • the transcripts are fragmented (e.g., sheared), ligated to additional functional sequences, and reverse transcribed.
  • the functional sequences may include a sequencer specific flow cell attachment sequence 1030 , e.g., a P5 sequence, as well as functional sequence 1028 , which may include sequencing primers, e.g., a R1 primer binding sequence, as well as functional sequence 1032 , which may include a sample index, e.g., an i5 sample index sequence.
  • the RNA transcripts can be reverse transcribed to DNA, the DNA amplified (e.g., via PCR), and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the unique sequence segment.
  • operations 1050 and 1052 can occur in the partition, while operations 1054 , 1056 , 1058 and 1060 can occur in bulk (e.g., outside the partition).
  • operations 1054 , 1056 , 1058 and 1060 can occur in bulk (e.g., outside the partition).
  • the emulsion can be broken and the contents of the droplet pooled in order to complete operations 1054 , 1056 , 1058 and 1060 .
  • functional sequence 1008 may be a P5 sequence and functional sequence 1010 may be a R1 primer binding site.
  • functional sequence 1030 may be a P7 sequence
  • functional sequence 1028 may be a R2 primer binding site
  • functional sequence 1032 may be an i7 sample index sequence.
  • RNA analysis including messenger RNA (mRNA, including mRNA obtained from a cell) analysis is shown in FIG. 11 .
  • the overall oligonucleotide 1102 is coupled to a bead 1104 by a releasable linkage 1106 , such as a disulfide linker.
  • the oligonucleotide may include functional sequences that are used in subsequent processing, such as functional sequence 1108 , which may include a sequencer specific flow cell attachment sequence, e.g., a P5 sequence, as well as functional sequence 1110 , which may include sequencing primer sequences, e.g., a R1 primer binding site.
  • sequence 1108 is a P7 sequence and sequence 1110 is a R2 primer binding site.
  • a barcode sequence 1112 is included within the structure for use in barcoding the sample RNA.
  • An additional sequence segment 1116 may be provided within the oligonucleotide sequence. In some cases, this additional sequence can provide a unique molecular identifier (UMI) sequence segment, as described elsewhere herein.
  • UMI unique molecular identifier
  • An additional sequence 1114 may be included to facilitate template switching, e.g., polyG.
  • individual beads can include tens to hundreds of thousands or even millions of individual oligonucleotide molecules, where, as noted, the barcode segment can be constant or relatively constant for a given bead, but where the variable or unique sequence segment will vary across an individual bead.
  • a cell is co-partitioned along with a microcapsule (e.g., bead bearing a barcoded oligonucleotide), polyT sequence, and other reagents such as a DNA polymerase, a reverse transcriptase, oligonucleotide primers, dNTPs, and reducing agent into a partition (e.g., a droplet in an emulsion).
  • a partition e.g., a droplet in an emulsion.
  • the partition can serve as a reaction volume.
  • the partition serving as the reaction volume can comprise a container or vessel such as a well, a microwell, vial, a tube, through ports in nanoarray substrates, or micro-vesicles having an outer barrier surrounding an inner fluid center or core, emulsion, or a droplet.
  • the partition comprises a droplet of aqueous fluid within a non-aqueous continuous phase, e.g., an oil phase.
  • the cell can be lysed and the barcoded oligonucleotides can be released from the bead (e.g., via the action of the reducing agent or other stimulus).
  • Cell lysis and release of the barcoded oligonucleotides from the microcapsule may occur simultaneously in the partition (e.g., a droplet in an emulsion) or the reaction volume.
  • cell lysis precedes release of the barcoded oligonucleotides from the microcapsule.
  • release of the barcoded oligonucleotides from the microcapsule precedes cell lysis.
  • the reaction volume can be subjected to an amplification reaction to generate an amplification product.
  • the polyT sequence hybridizes to the polyA tail of mRNA 1120 released from the cell as illustrated in operation 1150 .
  • the polyT sequence is then extended in a reverse transcription reaction using the mRNA as a template to produce a cDNA transcript 1122 complementary to the mRNA. Terminal transferase activity of the reverse transcriptase can add additional bases to the cDNA transcript (e.g., polyC) in a template independent manner.
  • the additional bases added to the cDNA transcript e.g., polyC
  • the barcoded oligonucleotide does not hybridize to the template polynucleotide.
  • the barcoded oligonucleotide upon release from the microcapsule, can be present in the reaction volume at any suitable concentration.
  • the barcoded oligonucleotide is present in the reaction volume at a concentration of about 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 50 ⁇ M, 100 ⁇ M, 150 ⁇ M, 200 ⁇ M, 250 ⁇ M, 300 ⁇ M, 400 ⁇ M, or 500 ⁇ M.
  • the barcoded oligonucleotide is present in the reaction volume at a concentration of at least about 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 50 ⁇ M, 100 ⁇ M, 150 ⁇ M, 200 ⁇ M, 250 ⁇ M, 300 ⁇ M, 400 ⁇ M, 500 ⁇ M or greater.
  • the barcoded oligonucleotide is present in the reaction volume at a concentration of at most about 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 35 ⁇ M, 40 ⁇ M, 50 ⁇ M, 100 ⁇ M, 150 ⁇ M, 200 ⁇ M, 250 ⁇ M, 300 ⁇ M, 400 ⁇ M, or 500 ⁇ M.
  • the transcripts can be further processed (e.g., amplified, portions removed, additional sequences added, etc.) and characterized as described elsewhere herein.
  • the transcripts are sequenced directly.
  • the transcripts are further processed (e.g., portions removed, additional sequences added, etc) and then sequenced.
  • the reaction volume is subjected to a second amplification reaction to generate an additional amplification product.
  • the transcripts or first amplification products can be used as the template for the second amplification reaction.
  • primers for the second amplification reaction comprise the barcoded oligonucleotide and polyT sequence.
  • primers for the second amplification reaction comprise additional primers co-partitioned with the cell.
  • these additional amplification products are sequenced directly.
  • these additional amplification products are further processed (e.g., portions removed, additional sequences added, etc) and then sequenced.
  • the configuration of the amplification products (e.g., first amplification products and second amplification products) generated by such a method can help minimize (or avoid) sequencing of the poly-T sequence during sequencing.
  • FIG. 12A An additional example of a barcode oligonucleotide for use in RNA analysis, including cellular RNA analysis is shown in FIG. 12A .
  • the overall oligonucleotide 1202 is coupled to a bead 1204 by a releasable linkage 1206 , such as a disulfide linker.
  • the oligonucleotide may include functional sequences that are used in subsequent processing, such as functional sequence 1208 , which may include a sequencer specific flow cell attachment sequence, e.g., a P5 sequence, as well as functional sequence 1210 , which may include sequencing primer sequences, e.g., a R1 primer binding site.
  • sequence 1208 is a P7 sequence and sequence 1210 is a R2 primer binding site.
  • a barcode sequence 1212 is included within the structure for use in barcoding the sample RNA.
  • An additional sequence segment 1216 may be provided within the oligonucleotide sequence. In some cases, this additional sequence can provide a unique molecular identifier (UMI) sequence segment, as described elsewhere herein.
  • UMI unique molecular identifier
  • individual beads can include tens to hundreds of thousands or even millions of individual oligonucleotide molecules, where, as noted, the barcode segment can be constant or relatively constant for a given bead, but where the variable or unique sequence segment will vary across an individual bead.
  • a cell is co-partitioned along with a barcode bearing bead and other reagents such as RNA ligase and a reducing agent into a partition (e.g., a droplet in an emulsion).
  • the cell is lysed while the barcoded oligonucleotides are released (e.g., via the action of the reducing agent) from the bead.
  • the barcoded oligonucleotides can then be ligated to the 5′ end of mRNA transcripts while in the partitions by RNA ligase.
  • Subsequent operations may include purification (e.g., via solid phase reversible immobilization (SPRI)) and further processing (shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)), and these operations may occur in bulk (e.g., outside the partition).
  • SPRI solid phase reversible immobilization
  • further processing shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)
  • these operations may occur in bulk (e.g., outside the partition).
  • a partition is a droplet in an emulsion
  • the emulsion can
  • FIG. 12B An additional example of a barcode oligonucleotide for use in RNA analysis, including cellular RNA analysis is shown in FIG. 12B .
  • the overall oligonucleotide 1222 is coupled to a bead 1224 by a releasable linkage 1226 , such as a disulfide linker.
  • the oligonucleotide may include functional sequences that are used in subsequent processing, such as functional sequence 1228 , which may include a sequencer specific flow cell attachment sequence, e.g., a P5 sequence, as well as functional sequence 1230 , which may include sequencing primer sequences, e.g., a R1 primer binding site.
  • sequence 1228 is a P7 sequence and sequence 1230 is a R2 primer binding site.
  • a barcode sequence 1232 is included within the structure for use in barcoding the sample RNA.
  • a priming sequence 1234 (e.g., a random priming sequence) can also be included in the oligonucleotide structure, e.g., a random hexamer.
  • An additional sequence segment 1236 may be provided within the oligonucleotide sequence. In some cases, this additional sequence provides a unique molecular identifier (UMI) sequence segment, as described elsewhere herein.
  • UMI unique molecular identifier
  • individual beads can include tens to hundreds of thousands or even millions of individual oligonucleotide molecules, where, as noted, the barcode segment can be constant or relatively constant for a given bead, but where the variable or unique sequence segment will vary across an individual bead.
  • the barcode segment can be constant or relatively constant for a given bead, but where the variable or unique sequence segment will vary across an individual bead.
  • a cell is co-partitioned along with a barcode bearing bead and additional reagents such as reverse transcriptase, a reducing agent and dNTPs into a partition (e.g., a droplet in an emulsion).
  • the cell is lysed while the barcoded oligonucleotides are released from the bead (e.g., via the action of the reducing agent).
  • sequence 1228 is a P7 sequence and sequence 1230 is a R2 primer binding site.
  • sequence 1228 is a P5 sequence and sequence 1230 is a R1 primer binding site.
  • the priming sequence 1234 of random hexamers can randomly hybridize cellular mRNA.
  • the random hexamer sequence can then be extended in a reverse transcription reaction using mRNA from the cell as a template to produce a cDNA transcript complementary to the mRNA and also includes each of the sequence segments 1228 , 1232 , 1230 , 1236 , and 1234 of the barcode oligonucleotide.
  • Subsequent operations may include purification (e.g., via solid phase reversible immobilization (SPRI)), further processing (shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)), and these operations may occur in bulk (e.g., outside the partition).
  • SPRI solid phase reversible immobilization
  • a partition is a droplet in an emulsion
  • the emulsion can be broken and the contents of the droplet pooled for additional operations.
  • Additional reagents that may be co-partitioned along with the barcode bearing bead may include oligonucleotides to block ribosomal RNA (rRNA) and nucleases to digest genomic DNA and cDNA from cells.
  • rRNA removal agents may be applied during additional processing operations.
  • the configuration of the constructs generated by such a method can help minimize (or avoid) sequencing of the poly-T sequence during sequencing.
  • the priming sequence 1234 may be a random N-mer.
  • sequence 1228 is a P7 sequence and sequence 1230 is a R2 primer binding site.
  • sequence 1228 is a P5 sequence and sequence 1230 is a R1 primer binding site.
  • the individual cell is co-partitioned along with a barcode bearing bead, poly-T sequence, and other reagents such as reverse transcriptase, polymerase, a reducing agent and dNTPs into a partition (e.g., droplet in an emulsion).
  • the cell is lysed while the barcoded oligonucleotides are released from the bead (e.g., via the action of the reducing agent) and the poly-T sequence hybridizes to the poly-A tail of cellular mRNA.
  • a reverse transcription reaction using the mRNA as template, cDNA transcripts of cellular mRNA can be produced.
  • the RNA can then be degraded with an RNase.
  • the priming sequence 1234 in the barcoded oligonucleotide can then randomly hybridize to the cDNA transcripts.
  • the oligonucleotides can be extended using polymerase enzymes and other extension reagents co-partitioned with the bead and cell similar to as shown in FIG.
  • amplification products e.g., barcoded fragments
  • the barcoded nucleic acid fragments may, in some cases subjected to further processing (e.g., amplification, addition of additional sequences, clean up processes, etc. as described elsewhere herein) characterized, e.g., through sequence analysis.
  • sequencing signals can come from full length RNA.
  • the barcode sequence can be appended to the 3′ end of the template polynucleotide sequence (e.g., mRNA). Such configuration may be desired, for example, if the sequence at the 3′ end of the template polynucleotide is desired to be analyzed.
  • the template polynucleotide sequence e.g., mRNA
  • the barcode sequence can be appended to the 5′ end of a template polynucleotide sequence (e.g., mRNA).
  • a template polynucleotide sequence e.g., mRNA
  • Such configuration may be desired, for example, if the sequence at the 5′ end of the template polynucleotide is desired to be analyzed.
  • a barcode sequence can be appended to the 3′ end of a first subset of the template polynucleotides, and a barcode sequence can be appended to the 5′ end of a second subset of the template polynucleotides.
  • the first subset of template polynucleotides and the second subset of template polynucleotides are appended to barcode sequences in the same partition.
  • the barcodes appended to the 3′ ends of template polynucleotides are different from the barcodes appended to the 5′ ends of template polynucleotides.
  • the barcodes appended to the 3′ ends may have a different barcode sequence compared to the barcodes appended to the 5′ end.
  • the barcodes appended to the 3′ ends of template polynucleotides have the same barcode sequence as the barcodes appended to the 5′ ends of template polynucleotides.
  • beads are used to deliver the barcode oligonucleotides to partitions.
  • the different barcodes can be attached to the same or different bead.
  • a barcode sequence can be appended to the 5′ end of a template polynucleotide sequence by any suitable method.
  • the template polynucleotide is a messenger RNA, mRNA, molecule.
  • the barcode sequence can be appended to the 5′ end of a template polynucleotide sequence by use of a primer comprising the barcode sequence in a primer extension reaction.
  • the barcode may be present in a primer used for a primer extension reaction in which the template polynucleotide or a derivative thereof, for example an amplification product, is used as the template for primer extension.
  • the barcode may be present on a template switching oligonucleotide participating in a primer extension reaction.
  • the barcode sequence can be appended to the 5′ end of a template polynucleotide by ligating an oligonucleotide comprising the barcode sequence directly to the template polynucleotide.
  • the present disclosure provides a method of appending a barcode sequence to the 5′ end of a template polynucleotide sequence by a primer extension reaction using a primer comprising a barcode sequence and the template polynucleotide or a derivative thereof as the template for primer extension.
  • the primer extension reaction may occur in a partition.
  • a cell, or a nucleic acid derivative thereof is co-partitioned with a primer capable of primer extension and a template switching oligo comprising a barcode sequence.
  • the primer capable of primer extension may hybridize to a nucleic acid of the cell or to a nucleic acid derivative.
  • the template switching oligo comprising the barcode sequence is releasably attached to a bead, e.g., a gel bead.
  • a cell, or a nucleic acid derivative thereof is co-partitioned with a primer having a sequence towards a 3′ end that hybridizes to the template polynucleotide, a template switching oligonucleotide having a first predefined sequence towards a 5′ end, and a microcapsule, such as a bead, having barcoded oligonucleotides releasably coupled thereto.
  • the oligonucleotides coupled to the bead include barcode sequences that are identical (e.g., all oligonucleotides sharing the same barcode sequence).
  • the oligonucleotides coupled to the beads additionally include unique molecular identifier (UMI) sequence segments (e.g., all oligonucleotides having different unique molecular identifier sequences).
  • UMI unique molecular identifier
  • FIG. 18 shows a barcoded oligonucleotide coupled to a bead.
  • the overall oligonucleotide 1802 is coupled to a bead 1804 by a releasable linkage 1806 , such as a disulfide linker.
  • the oligonucleotide may include functional sequences that are useful for subsequent processing, such as functional sequence 1808 , which may include a sequencer specific flow cell attachment sequence, e.g., a P5 sequence, as well as functional sequence 1810 , which may include sequencing primer sequences, e.g., a R1 primer binding site.
  • sequence 1808 is a P7 sequence
  • sequence 1810 is a R2 primer binding site.
  • a barcode sequence 1812 can be included within the structure for use in barcoding the template polynucleotide.
  • the functional sequences may be selected for compatibility with a variety of different sequencing systems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and the requirements thereof.
  • the barcode sequence 1812 , functional sequences 1808 (e.g., flow cell attachment sequence) and 1810 e.g., sequencing primer sequences
  • the barcoded oligonucleotide can also comprise a sequence 1816 to facilitate template switching (e.g., a polyG sequence).
  • the additional sequence provides a unique molecular identifier (UMI) sequence segment, as described elsewhere herein.
  • UMI unique molecular identifier
  • individual beads can include tens to hundreds of thousands or even millions of individual oligonucleotide molecules, where, as previously noted herein, the barcode sequence can be constant or relatively constant for a given bead.
  • a cell or a nucleic acid derived therefrom is co-partitioned with a bead bearing a barcoded oligonucleotide and reagents such as reverse transcriptase, poly-T primers, dNTPs, and a chemical stimulus (e.g., reducing agent) into a partition.
  • the barcoded oligonucleotide attached to the bead can comprise a sequence to facilitate template switching (e.g., polyG or riboG).
  • the partition can be a droplet in an emulsion. In cases where a cell is provided in the partition, the partition can further comprise a lysis reagent to lyse the cell.
  • the barcoded oligonucleotide can be released from the bead when contacted with the chemical stimulus (e.g., reducing agent). Following release from the bead, the barcoded oligonucleotide can be present in the partition at any suitable concentration. In some embodiments, the barcoded oligonucleotide is present in the partition at a concentration that is suitable for generating a sufficient yield of amplification products for downstream processing and analysis, including, but not limited to, sequencing adaptor attachment and sequencing analysis.
  • the oligonucleotide is extended yielding an anti-sense strand 1922 A which is appended by multiple cytidines on the 3′ end.
  • the template switching sequence 1990 A (e.g., polyG or riboG) of the barcoded oligonucleotide pairs with the cytidines of the anti-sense strand 1922 A and the anti-sense strand is extended using the barcoded oligonucleotide as template.
  • the barcoded oligonucleotide can comprise additional functional sequences 1908 A, 1912 A, and 1910 A.
  • the barcoded oligonucleotide comprises a unique molecular identifier (UMI, for example 1908 A), a barcode sequence (for example 1912 A), and a Read 1 sequence (R1, for example 1910 A).
  • Operations 1901 A, 1902 A, and 1903 A may be performed in the partition (e.g., droplet or well).
  • the extension in 1902 A and 1903 A can be facilitated by an enzyme comprising polymerase activity.
  • the extension can be facilitated by a DNA-dependent polymerase or a reverse-transcriptase (e.g., RNA dependent).
  • the extension comprises polymerase chain reaction.
  • the extension comprises reverse transcription.
  • the enzyme can add nucleotides in a template independent manner. In some cases, at least three cytidines are appended to the 3′ end of the cDNA transcript in a template independent manner.
  • the nucleic acid product (e.g., cDNA product) may be released from the partition and subject to further processing reactions such as additional amplification.
  • the nucleic acid product is pooled with products from other partitions for subsequent processing in bulk.
  • a portion of the amplified product can be subjected to enrichment to obtain a subset of nucleic acids corresponding to genes of interest.
  • enrichment to obtain a subset of nucleic acids corresponding to genes of interest comprises one or more amplification reactions.
  • One or more gene specific primers can be used for primer extension using the cDNA molecule as a template. Any of a variety of polymerases can be used in embodiments herein for primer extension, non-limiting examples of which include exonuclease minus DNA Polymerase I large (Klenow) Fragment, Phi29 DNA polymerase, Taq DNA Polymerase, T4 DNA polymerase, T7 DNA polymerase, and the like.
  • thermostable polymerases including but not limited to, Thermus thermophilus HB8; Thermus oshimai; Thermus scotoductus; Thermus thermophilus 1B21; Thermus thermophilus GK24; Thermus aquaticus polymerase AmpliTaq® FS or Taq (G46D; F667Y), Taq (G46D; F667Y; E6811), and Taq (G46D; F667Y; T664N; R660G); Pyrococcus furiosus polymerase; Thermococcus gorgonarius polymerase; Pyrococcus species GB-D polymerase; Thermococcus sp.
  • thermostable polymerases including but not limited to, Thermus thermophilus HB8; Thermus oshimai; Thermus scotoductus; Thermus thermophilus 1B21; Thermus thermophilus GK24; Thermus aquaticus polymerase AmpliTaq®
  • a hot start polymerase is used.
  • a hot start polymerase is a modified form of a DNA polymerase that can be activated by incubation at elevated temperatures.
  • Additional functional sequences can be added to the nucleic acid product or an amplification product thereof.
  • the additional functional sequences may allow for amplification or sample identification. This may occur in the partition or, alternatively, in bulk.
  • the amplification products can be sheared, ligated to adapters and amplified to add additional functional sequences.
  • both the enriched and unenriched amplification products are subject to analysis.
  • a cell is co-partitioned along with a bead bearing a barcoded oligonucleotide and additional reagents such as reverse transcriptase, primers, oligonucleotides (e.g., template switching oligonucleotides), dNTPs, and reducing agent into a partition (e.g., a droplet in an emulsion).
  • a partition e.g., a droplet in an emulsion.
  • the cell can be lysed to yield a plurality of template polynucleotides (e.g., DNA such as genomic DNA, RNA such as mRNA, etc).
  • the cell is lysed using a lysis reagent that is co-partitioned with the cell.
  • the barcoded oligonucleotide can be released from the bead following the application of stimulus as previously described herein. Following release from the bead, the barcoded oligonucleotide can be present in the partition at any suitable concentration. In some embodiments, the barcoded oligonucleotide is present in the partition at a concentration that is suitable for generating a sufficient yield of amplification products for downstream processing and analysis, including, but not limited to, sequencing adaptor attachment and sequencing analysis. In some embodiments, the concentration of the barcoded oligonucleotide is limited by the loading capacity of the barcode bearing bead, or the amount of oligonucleotides deliverable by the bead.
  • the template switching oligonucleotide which can be co-partitioned with the cell, bead bearing barcoded oligonucleotides, etc, can be present in the partition at any suitable concentration.
  • the template switching oligonucleotide is present in the partition at a concentration that is suitable for efficient template switching during an amplification reaction.
  • the concentration of the template switching oligonucleotide can be dependent on the reagents used for droplet generation.
  • the template switching oligonucleotide is among a plurality of template switching oligonucleotides.
  • the barcoded oligonucleotide and template switching oligonucleotide are present in the partition at similar concentrations. In some embodiments, the barcoded oligonucleotide and template switching oligonucleotides may be present in proportions reflective of the desired amount of amplification products to be generated using each oligonucleotide. In some embodiments, the template switching oligonucleotide is present in the partition at a greater concentration than the barcoded oligonucleotide. This difference in concentration can be due to limitations on the capacity of the barcode bearing bead.
  • the concentration of the template switching oligonucleotide in the reaction volume is at least 2, 5, 10, 20, 50, 100, 200 or more times that of the concentration of the barcoded oligonucleotide in the same reaction volume when the barcoded oligonucleotide is free in the partition (e.g., not attached to the bead).
  • a reaction mixture comprising a template polynucleotide from a cell 1920 B and (i) the primer 1924 B having a sequence towards a 3′ end that hybridizes to the template polynucleotide (e.g., polyT) and an additional sequence element 1900 B and (ii) a template switching oligonucleotide 1926 B that comprises a first predefined sequence 1810 towards a 5′ end can be subjected to an amplification reaction to yield a first amplification product.
  • the template polynucleotide e.g., polyT
  • the template polynucleotide is an mRNA with a polyA tail and the primer that hybridizes to the template polynucleotide comprises a polyT sequence towards a 3′ end, which is complementary to the polyA segment.
  • the first predefined sequence can comprise at least one of an adaptor sequence, a barcode sequence, a unique molecular identifier (UMI) sequence, a primer binding site, and a sequencing primer binding site or any combination thereof.
  • the first predefined sequence 1810 is a sequence that can be common to all partitions of a plurality of partitions.
  • the first predefined sequence may comprise a flow cell attachment sequence, an amplification primer binding site, or a sequencing primer binding site and the first amplification reaction facilitates the attachment the predefined sequence to the template polynucleotide from the cell.
  • the first predefined sequence comprises a primer binding site.
  • the first predefined sequence comprises a sequencing primer binding site.
  • the first predefined sequence comprises a barcode sequence. As illustrated in operation 1950 B, the sequence towards a 3′ end (e.g., polyT) of the primer 1924 B hybridizes to the template polynucleotide 1920 B.
  • extension reaction reagents e.g., reverse transcriptase, nucleoside triphosphates, co-factors (e.g., Mg2+ or Mn2+), that are also co-partitioned
  • reverse transcriptase can extend the primer 1924 B sequence using the cell's nucleic acid as a template, to produce a transcript, e.g., cDNA transcript, 1922 B having a fragment complementary to the nucleic acid to which the primer annealed.
  • the reverse transcriptase has terminal transferase activity and the reverse transcriptase adds additional nucleotides, e.g., polyC, to the cDNA transcript in a template independent manner.
  • the template switching oligonucleotide 1926 B for example a template switching oligonucleotide which includes a polyG sequence, can hybridize to the cDNA transcript 1922 B and facilitate template switching in the first amplification reaction.
  • the transcript therefore, may comprise the sequence of the primer 1924 B, a sequence complementary to the template polynucleotide from the cell, and a sequence complementary to the template switching oligonucleotide.
  • the primer and template switching oligonucleotide may be universal to all partitions.
  • the partitions may individually contain more than one cell, one cell, no cells, or nucleic acids derived from a cell.
  • the primer may comprise at least a polyT segment capable of hybridizing and priming an extension reaction from the polyA segment of an mRNA.
  • the primer may comprise a random sequence capable of hybridizing to and priming extension reactions randomly on various polynucleotide templates.
  • a template switching oligonucleotide having a sequence capable of hybridizing to the appended bases can be used for template switching in manner that is independent of the sequence of the polynucleotide templates to be analyzed.
  • the template switching oligonucleotide can comprise a first predefined sequence towards a 5′ end that does not specifically hybridize to the template.
  • analysis of particular genes is desired.
  • the primer may comprise a gene specific sequence capable of hybridizing to and priming extension reactions from templates comprising specific genes.
  • multiple genes are to be analyzed and a primer is among a plurality of primers. Individual primers of the plurality may target different genes. Each of the plurality of primers may have a sequence for a particular gene.
  • the first amplification product or transcript can be subjected to a second amplification reaction to generate a second amplification product.
  • additional sequences e.g., functional sequences such as flow cell attachment sequence, sequencing primer binding sequences, barcode sequences, etc
  • the first and second amplification reactions can be performed in the same volume, such as for example in a droplet or well.
  • the first amplification product is subjected to a second amplification reaction in the presence of a barcoded oligonucleotide to generate a second amplification product having a barcode sequence.
  • the barcode sequence can be unique to a partition, that is, each partition has a unique barcode sequence.
  • the barcoded oligonucleotide may comprise a sequence of at least a segment of the template switching oligonucleotide and at least a second predefined sequence.
  • the segment of the template switching oligonucleotide on the barcoded oligonucleotide can facilitate hybridization of the barcoded oligonucleotide to the transcript, e.g., cDNA transcript, to facilitate the generation of a second amplification product.
  • the barcoded oligonucleotide may comprise a second defined sequence such as at least one of an adaptor sequence, a unique molecular identifier (UMI) sequence, a primer binding site, and a sequencing primer binding site or any combination thereof.
  • UMI unique molecular identifier
  • the second amplification reaction uses the first amplification product as a template and the barcoded oligonucleotide as a primer.
  • the segment of the template switching oligonucleotide on the barcoded oligonucleotide 1928 B can hybridize to the portion of the cDNA transcript or complementary fragment 1922 B having a sequence complementary to the template switching oligonucleotide or that which was copied from the template switching oligonucleotide.
  • extension reaction reagents e.g., polymerase, nucleoside triphosphates, co-factors (e.g., Mg2+ or Mn2+), that are also co-partitioned, can extend the primer sequence using the first amplification product as template as illustrated in operation 1956 B.
  • the second amplification product can comprise a second predefined sequence (e.g., 1808 , 1812 , and 1810 ), a sequence of a segment of the template polynucleotide (e.g., mRNA), and a sequence complementary to the primer (e.g., 1924 B).
  • amplification products derived therefrom can comprise the corresponding DNA sequence, for example thymine instead of uracil bases.
  • the second amplification product uses the barcoded oligonucleotide as a template and at least a portion of the first amplification product as a primer.
  • the segment of the first amplification product e.g., cDNA transcript
  • the segment of the first amplification product having a sequence complementary to the template switching oligonucleotide can hybridize to the segment of the barcoded oligonucleotide comprising a sequence of at least a segment of the template switching oligonucleotide.
  • extension reaction reagents e.g., polymerase, nucleoside triphosphates, co-factors (e.g., Mg2+ or Mn2+), that are also co-partitioned, can extend the primer sequence (e.g., first amplification product) using the barcoded oligonucleotide as template as illustrated in operation 1958 B.
  • the second amplification product may comprise the sequence of the primer (e.g., 1924 B), a sequence which is complementary to the sequence of the template polynucleotide (e.g., mRNA), and a sequence complementary to the second predefined sequence (e.g., 1808 , 1812 , and 1810 ).
  • the second amplification reaction is performed subsequent to the first amplification reaction in the presence of an intervening purification step.
  • An intervening purification step can be used, for example, to purify the template (e.g., first amplification product) from excess reagents, including excess primers such as template switching oligonucleotides.
  • the amplification reaction is performed in the absence of an intervening purification step.
  • an intervening purification step is not performed so that all sample preparation is performed in a same reaction volume.
  • the template switching oligonucleotide may compete with barcoded oligonucleotide in the second amplification reaction as the barcoded oligonucleotide comprises at least a segment of the template switching oligonucleotide. Competition between the template switching oligonucleotide and barcoded oligonucleotide in the second amplification reaction to generate additional amplification product may result in a second amplification product lacking a barcode sequence. Such amplification products lacking a barcode sequence may be undesirable as they lack a barcode sequence which can provide unique identifying information of the template.
  • the template switching oligonucleotide may out-compete the barcoded oligonucleotide in the second amplification reaction if the template switching oligonucleotide is present at a higher concentration in the reaction volume than the barcoded oligonucleotide.
  • Various approaches can be utilized to favor the use of the barcoded oligonucleotide in the second amplification reaction to generate amplification products having a barcode sequence in situations where the barcoded oligonucleotide is present at a lower concentration than the template switching oligonucleotide in the reaction volume.
  • the template switching oligonucleotide is not available for primer extension during the second amplification reaction. In some embodiments, the template switching oligonucleotide is degraded prior to the second amplification reaction. In some embodiments, the template switching oligonucleotide is degraded during the second amplification reaction.
  • the template switching oligonucleotide may comprise ribonucleic acids (RNA).
  • RNA ribonucleic acids
  • the template switching oligonucleotide comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% RNA. In some embodiments, the template switching oligonucleotide comprises 100% RNA. In some embodiments, a first reaction rate of the second amplification reaction using the barcoded oligonucleotide is greater than a second reaction rate of the second amplification using the template switching oligonucleotide.
  • the barcoded oligonucleotide can hybridize to the first amplification product at a higher annealing temperature as compared to the template switching oligonucleotide.
  • the first amplification product and the barcoded oligonucleotide can have a higher melting temperature as compared to a melting temperature of the first amplification product and the template switching oligonucleotide.
  • the second amplification reaction may be performed with an annealing temperature at which the barcoded oligonucleotide is able to hybridize to the first amplification product and initiation primer extension and at which the template switching oligonucleotide is unable to hybridize to the first amplification product and initiate primer extension.
  • the primer annealing temperature of the second amplification reaction is at least about 0.5° C., 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C. or greater than a primer annealing temperature of the first amplification reaction.
  • the difference in melting temperatures can result from the presence of modified nucleotides in the template switching oligonucleotide.
  • the template switching oligonucleotide comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% modified nucleotides.
  • the template switching oligonucleotide comprises 100% modified oligonucleotides.
  • the difference in melting temperature can be the result of the presence of modified nucleotides in the barcoded oligonucleotide.
  • the barcoded oligonucleotide comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% modified nucleotides. In some embodiments, the barcoded oligonucleotide comprises 100% modified oligonucleotides.
  • Modified nucleotides include, but are not limited to, 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC, 2′-deoxylnosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, and 2′ Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G).
  • 2-Aminopurine 2,6-Diaminopurine
  • 2-Amino-dA inverted dT
  • 5-Methyl dC 2′-deoxylnosine
  • Super T 5-hydroxybutynl-2′-deoxy
  • the first amplification reaction is facilitated using an enzyme comprising polymerase activity.
  • the first amplification reaction can be facilitated by a DNA-dependent polymerase or a reverse-transcriptase (e.g., RNA dependent).
  • the first amplification reaction comprises polymerase chain reaction.
  • the first amplification reaction comprises reverse transcription.
  • the second amplification reaction is facilitated using an enzyme comprising polymerase activity.
  • the second amplification reaction can be facilitated by a DNA-dependent polymerase.
  • the second amplification reaction comprises polymerase chain reaction.
  • a template polynucleotide comprising mRNA may first be reverse transcribed to cDNA (e.g., an amplification product of the template polynucleotide).
  • the mRNA molecule can be reverse transcribed to cDNA using a reverse transcriptase enzyme and a primer, such as a poly-T primer.
  • a reverse transcriptase enzyme and a primer, such as a poly-T primer.
  • Non-limiting examples of enzymes that can be used for reverse transcription in embodiments herein include HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and variants, modified products and derivatives thereof.
  • a gene specific primer having a barcode sequence can then be used for primer extension using the cDNA molecule (e.g., amplification product of the template polynucleotide) as a template.
  • a primer comprising a barcode can hybridize to the cDNA molecule via sequence complementarity. Extension of the primer using the cDNA molecule as template may result in a polynucleotide product comprising the template polynucleotide sequence and the barcode sequence located at the 5′ end of the template polynucleotide sequence.
  • any of a variety of polymerases can be used in embodiments herein for primer extension, non-limiting examples of which include exonuclease minus DNA Polymerase I large (Klenow) Fragment, Phi29 DNA polymerase, Taq DNA Polymerase, T4 DNA polymerase, T7 DNA polymerase, and the like.
  • thermostable polymerases including but not limited to, Thermus thermophilus HB8; Thermus oshimai; Thermus scotoductus; Thermus thermophilus 1B21; Thermus thermophilus GK24; Thermus aquaticus polymerase AmpliTaq® FS or Taq (G46D; F667Y), Taq (G46D; F667Y; E6811), and Taq (G46D; F667Y; T664N; R660G); Pyrococcus furiosus polymerase; Thermococcus gorgonarius polymerase; Pyrococcus species GB-D polymerase; Thermococcus sp.
  • thermostable polymerases including but not limited to, Thermus thermophilus HB8; Thermus oshimai; Thermus scotoductus; Thermus thermophilus 1B21; Thermus thermophilus GK24; Thermus aquaticus polymerase AmpliTaq®
  • a hot start polymerase is used.
  • a hot start polymerase is a modified form of a DNA polymerase that can be activated by incubation at elevated temperatures. Such a polymerase can be used, for example, to further increase sensitivity, specificity, and yield; and/or to further improve low copy target amplification.
  • a barcode sequence is appended to the 5′ end of a template polynucleotide sequence by ligating an oligonucleotide comprising a barcode sequence directly to the 5′ end of the template polynucleotide.
  • Ligating an oligonucleotide comprising a barcode sequence to a template polynucleotide can be implemented by various methods.
  • ligating an oligonucleotide comprising a barcode sequence to a template polynucleotide involves an enzyme, such as a ligase (e.g., an RNA ligase or a DNA ligase).
  • Non-limiting examples of enzymes that can be used for ligation in embodiments herein include ATP-dependent double-stranded polynucleotide ligases, NAD+ dependent DNA or RNA ligases, and single-strand polynucleotide ligases.
  • Non-limiting examples of ligases which can be used in embodiments herein include CircLigase I and CircLigase II (Epicentre; Madison, Wis.), Escherichia coli DNA ligase, Thermus filiformis DNA ligase, Tth DNA ligase, Thermus scotoductus DNA ligase (I and II), T3 DNA ligase, T4 DNA ligase, T4 RNA ligase, T7 DNA ligase, Taq ligase, Ampligase (Epicentre® Technologies Corp.), VanC-type ligase, 9° N DNA Ligase, Tsp DNA ligase, DNA ligase I, DNA ligase III, DNA ligase IV, Sso7-T3 DNA ligase, Sso7-T4 DNA ligase, Sso7-T7 DNA ligase, Sso7-Taq DNA ligase, Sso7-
  • the mRNA molecule can be treated to yield a 5′ monophosphate group prior to ligating. Any suitable reaction may be employed to yield a 5′ monophosphate group.
  • the mRNA molecule can be treated with an enzyme such as a pyrophosphohydrolase.
  • An example of a pyrophosphohydrolase that can be used in embodiments herein is RNA 5′ phyrophosphohydrolase (RppH).
  • RppH RNA 5′ phyrophosphohydrolase
  • two phosphate groups are removed from a triphosphate group to yield a monophosphate.
  • a single enzyme both removes the phosphate groups present on the mRNA molecule and adds the monophosphate group.
  • a first enzyme removes the phosphate groups present on the mRNA molecule and a second enzyme adds the monophosphate group.
  • the phosphate groups are removed from the 5′ end of the mRNA molecule and the 5′ end is adenylated.
  • An enzyme which can be used for 5′ adenylation in embodiments herein includes Mth RNA ligase.
  • the oligonucleotide comprising the barcode sequence is ligated to the template polynucleotide within a partition (e.g., droplet or well).
  • a partition in some cases, comprises a polynucleotide sample comprising the template polynucleotide, an oligonucleotide having the barcode sequence, a ligase enzyme, and any other suitable reagents for ligation.
  • the ligase can implement the attachment of the oligonucleotide comprising the barcode sequence to the template polynucleotide within the partition.
  • the template polynucleotide is an mRNA molecule and the oligonucleotide ligated to it is a DNA molecule.
  • the oligonucleotide comprising the barcode sequence is ligated to the template polynucleotide outside of a partition.
  • the barcoded template can be subjected to further amplification.
  • one or more further amplification reactions are performed within the partition.
  • one or more further amplification reactions are performed outside of a partition.
  • a plurality of barcoded mRNA polynucleotides for example from a plurality of partitions, is pooled and subjected to further processing in bulk.
  • the barcoded template polynucleotide is subjected to polymerase chain reaction.
  • the template polynucleotide comprises mRNA and the barcoded template polynucleotide is subjected to reverse transcription, yielding a cDNA transcript.
  • the partitions can comprise primers having a poly-T region capable of hybridizing to the poly-A region of the barcoded mRNA.
  • the primer having a poly-T region can hybridize to the barcoded template and initiate primer extension in reverse transcription.
  • Non-limiting examples of enzymes that can be used for reverse transcription in embodiments herein include HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and variants, modified products and derivatives thereof.
  • a partition can contain a reverse transcriptase enzyme capable of reverse transcribing a template polynucleotide that is attached at its 5′ end to a barcoded oligonucleotide.
  • a plurality of barcoded mRNA polynucleotides from a plurality of partitions can be pooled for bulk processing.
  • the reaction volume for performing reverse transcription can comprise primers having a poly-T region capable of hybridizing to the poly-A region of a barcoded mRNA.
  • the primers for reverse transcription further comprise additional elements, such as tags, which can be used, for example, for isolating cDNA transcripts.
  • cDNA transcripts comprising biotin tags can be isolated from components of the reaction volume (e.g., excess primers, reverse transcriptase enzyme, barcoded mRNA molecules) by performing a purification reaction with streptavidin or other molecule capable of binding biotin.
  • components of the reaction volume e.g., excess primers, reverse transcriptase enzyme, barcoded mRNA molecules
  • subsequent operations may be performed, including purification (e.g., via solid phase reversible immobilization (SPRI)) or further processing (e.g., shearing, addition of functional sequences, and subsequent amplification (e.g., via PCR)).
  • Functional sequences such as flow cell sequences, may be added by ligation. These operations may occur in bulk (e.g., outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations.
  • Additional reagents that may be co-partitioned along with the barcode bearing bead may include oligonucleotides to block ribosomal RNA (rRNA) and nucleases to digest genomic DNA from cells. Alternatively, rRNA removal agents may be applied during additional processing operations.
  • the configuration of the constructs generated by such a method can help minimize (or avoid) sequencing of the poly-T sequence during sequencing and/or sequence the 5′ end of a polynucleotide sequence.
  • the amplification products for example first amplification products and/or second amplification products, may be subject to sequencing for sequence analysis.
  • the processes and systems described herein may also be used to characterize individual cells as a way to provide an overall profile of a cellular, or other organismal population.
  • a variety of applications require the evaluation of the presence and quantification of different cell or organism types within a population of cells, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g., in tracing contamination or the like.
  • the analysis processes described above may be used to individually characterize, sequence and/or identify large numbers of individual cells within a population. This characterization may then be used to assemble an overall profile of the originating population, which can provide important prognostic and diagnostic information.
  • shifts in human microbiomes including, e.g., gut, buccal, epidermal microbiomes, etc.
  • single cell analysis methods and systems described herein one can again, characterize, sequence and identify individual cells in an overall population, and identify shifts within that population that may be indicative of diagnostic ally relevant factors.
  • sequencing of bacterial 16S ribosomal RNA genes has been used as a highly accurate method for taxonomic classification of bacteria.
  • Using the targeted amplification and sequencing processes described above can provide identification of individual cells within a population of cells.
  • identification and diagnosis of infection or potential infection may also benefit from the single cell analyses described herein, e.g., to identify microbial species present in large mixes of other cells or other biological material, cells and/or nucleic acids, including the environments described above, as well as any other diagnostically relevant environments, e.g., cerebrospinal fluid, blood, fecal or intestinal samples, or the like.
  • the foregoing analyses may also be particularly useful in the characterization of potential drug resistance of different cells or pathogens, e.g., cancer cells, bacterial pathogens, etc., through the analysis of distribution and profiling of different resistance markers/mutations across cell populations in a given sample. Additionally, characterization of shifts in these markers/mutations across populations of cells over time can provide valuable insight into the progression, alteration, prevention, and treatment of a variety of diseases characterized by such drug resistance issues.
  • pathogens e.g., cancer cells, bacterial pathogens, etc.
  • any of a variety of individual biological organisms, or components of organisms are encompassed within this description, including, for example, cells, viruses, organelles, cellular inclusions, vesicles, or the like. Additionally, where referring to cells, it will be appreciated that such reference includes any type of cell, including without limitation prokaryotic cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or other animal cell types, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from single cell or multicellular organisms.
  • analysis of different environmental samples to profile the microbial organisms, viruses, or other biological contaminants that are present within such samples can provide important information about disease epidemiology, and potentially aid in forecasting disease outbreaks, epidemics an pandemics.
  • a sample is provided that contains cells that are to be analyzed and characterized as to their cell surface proteins.
  • a library of antibodies, antibody fragments, or other molecules having a binding affinity to the cell surface proteins or antigens (or other cell features) for which the cell is to be characterized also referred to herein as cell surface feature binding groups.
  • binding groups can include a reporter molecule that is indicative of the cell surface feature to which the binding group binds.
  • a binding group type that is specific to one type of cell surface feature will comprise a first reporter molecule, while a binding group type that is specific to a different cell surface feature will have a different reporter molecule associated with it.
  • these reporter molecules will comprise oligonucleotide sequences. Oligonucleotide based reporter molecules provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g., using sequencing or array technologies.
  • the binding groups include oligonucleotides attached to them.
  • a first binding group type e.g., antibodies to a first type of cell surface feature
  • a reporter oligonucleotide that has a first nucleotide sequence.
  • Different binding group types e.g., antibodies having binding affinity for other, different cell surface features
  • reporter oligonucleotides that comprise different nucleotide sequences, e.g., having a partially or completely different nucleotide sequence.
  • the reporter oligonucleotide sequence may be known and readily identifiable as being associated with the known cell surface feature binding group.
  • oligonucleotides may be directly coupled to the binding group, or they may be attached to a bead, molecular lattice, e.g., a linear, globular, cross-linked, or other polymer, or other framework that is attached or otherwise associated with the binding group, which allows attachment of multiple reporter oligonucleotides to a single binding group.
  • molecular lattice e.g., a linear, globular, cross-linked, or other polymer, or other framework that is attached or otherwise associated with the binding group, which allows attachment of multiple reporter oligonucleotides to a single binding group.
  • reporter molecules can comprise the same sequence, or a particular binding group will include a known set of reporter oligonucleotide sequences.
  • the reporter molecules can be different and attributable to the particular binding group.
  • Attachment of the reporter groups to the binding groups may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments.
  • oligonucleotide reporter groups associated with antibody based binding groups such oligonucleotides may be covalently attached to a portion of an antibody or antibody fragment using chemical conjugation techniques (e.g., Lightning-Link® antibody labeling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker.
  • chemical conjugation techniques e.g., Lightning-Link® antibody labeling kits available from Innova Biosciences
  • other non-covalent attachment mechanisms e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated
  • Antibody and oligonucleotide biotinylation techniques are available (See, e.g., Fang, et al., Fluoride - Cleavable Biotinylation Phosphoramidite for 5′- end - Labeling and Affinity Purification of Synthetic Oligonucleotides , Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, DNA 3′ End Biotinylation Kit, available from Thermo Scientific, the full disclosures of which are incorporated herein by reference in their entirety for all purposes).
  • protein and peptide biotinylation techniques have been developed and are readily available (See, e.g., U.S. Pat. No. 6,265,552, the full disclosures of which are incorporated herein by reference in their entirety for all purposes).
  • the reporter oligonucleotides may be provided having any of a range of different lengths, depending upon the diversity of reporter molecules desired or a given analysis, the sequence detection scheme employed, and the like. In some cases, these reporter sequences can be greater than about 5 nucleotides in length, greater than about 10 nucleotides in length, greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or even 200 nucleotides in length. In some cases, these reporter nucleotides may be less than about 250 nucleotides in length, less than about 200, 180, 150, 120 100, 90, 80, 70, 60, 50, 40, or even 30 nucleotides in length.
  • the reporter oligonucleotides may be selected to provide barcoded products that are already sized, and otherwise configured to be analyzed on a sequencing system. For example, these sequences may be provided at a length that ideally creates sequenceable products of a desired length for particular sequencing systems. Likewise, these reporter oligonucleotides may include additional sequence elements, in addition to the reporter sequence, such as sequencer attachment sequences, sequencing primer sequences, amplification primer sequences, or the complements to any of these.
  • a cell-containing sample is incubated with the binding molecules and their associated reporter oligonucleotides, for any of the cell surface features desired to be analyzed.
  • the cells are washed to remove unbound binding groups.
  • the cells are partitioned into separate partitions, e.g., droplets, along with the barcode carrying beads described above, where each partition includes a limited number of cells, e.g., in some cases, a single cell.
  • the barcoded replicates of the reporter molecules may additionally include functional sequences, such as primer sequences, attachment sequences or the like.
  • the barcoded reporter oligonucleotides are then subjected to sequence analysis to identify which reporter oligonucleotides bound to the cells within the partitions. Further, by also sequencing the associated barcode sequence, one can identify that a given cell surface feature likely came from the same cell as other, different cell surface features, whose reporter sequences include the same barcode sequence, i.e., they were derived from the same partition.
  • a cell surface profile of individual cells Based upon the reporter molecules that emanate from an individual partition based upon the presence of the barcode sequence, one may then create a cell surface profile of individual cells from a population of cells. Profiles of individual cells or populations of cells may be compared to profiles from other cells, e.g., ‘normal’ cells, to identify variations in cell surface features, which may provide diagnostically relevant information. In particular, these profiles may be particularly useful in the diagnosis of a variety of disorders that are characterized by variations in cell surface receptors, such as cancer and other disorders.
  • the present disclosure also provides methods for reducing nonspecific priming in a single-cell 5′ gene expression assay.
  • a cell barcode sequence barcode
  • UMI unique molecular identifier sequence
  • TSO template switching oligonucleotide
  • This oligonucleotide may be attached to the first strand cDNA via a template switching reaction where the reverse transcription (RT) enzyme 1) reverse transcribes a messenger RNA (mRNA) sequence into first-strand complementary DNA (cDNA) from a primer targeting the 3′ end of the mRNA, 2) adds nontemplated cytidines to the 5′ end of the first-strand cDNA, 3) switches template to the TSO, which may contain 3′ guanidines or guanidine-derivatives that hybridize to the added cytidines.
  • RT reverse transcription
  • the TSO may co-exist in solution with the RT enzyme and the total RNA contents of a cell. If the TSO is a single stranded DNA (ssDNA) molecule, it can participate as an RT primer rather than as a template-switching substrate. Given, for example, that the over 90% of the total RNA contents of a cell include noncoding ribosomal RNA (rRNA), this may produce barcoded off products that do not contribute to the 5′ gene expression or V(D)J sequencing assay but do consume sequencing reads, increasing the cost required to achieve the same sequencing depth. In addition, if the UMI is implemented as a randomer, the presence of this randomer at the 3′ end of the TSO greatly increases its ability to serve as a primer on rRNA template.
  • ssDNA single stranded DNA
  • rRNA noncoding ribosomal RNA
  • a TSO that is less likely to serve as an RT primer via the introduction of a particular spacer sequence between the UMI and terminal riboGs may be used.
  • Another approach is to design and include a set of auxiliary blocking oligonucleotides that may hybridize to rRNA and prevent binding of the TSO.
  • the spacer sequence can be optimized by selecting a sequence that minimizes the predicted melting temperature of the (spacer-GGG):rRNA duplex against all human ribosomal RNA molecules.
  • the blocker sequences can be optimized by selecting sequences that maximize the predicted melting temperature of the (blocker):rRNA duplex against all human ribosomal RNA molecules.
  • TSO that are less likely to serve as an RT primer via the introduction of a particular spacer sequence between the UMI and terminal riboGs. Additionally, described herein are auxiliary blocking oligonucleotides that hybridize to rRNA and prevent binding of the TSO.
  • Table 1 provides examples of spacer sequences that are optimized by selecting a sequence that minimizes the predicted melting temperature of the (spacer-GGG):rRNA duplex against all human ribosomal RNA molecules.
  • Table 2 provides examples of blocker sequences that are optimized by selecting sequences that maximize the predicted melting temperature of the (blocker):rRNA duplex against all human ribosomal RNA molecules.
  • Table 3 provides examples of full construct barcodes.
  • N and W represent any of ⁇ A,C,G,T ⁇ and any of ⁇ A,T ⁇ respectively that make up the UMI sequence. UMIs may be different across different oligos on the same bead.
  • N1 is any one of ⁇ A,C,G,T ⁇ ; N1 positions with ratios of 25%, 25%, 25% and 25% for the four nucleotides.
  • N2 is any one of ⁇ A,C,G,T ⁇ ; N2 positions with ratios of 40%, 10%, 10% and 40% for the four nucleotides.
  • a cell barcode may be a 16 base sequence that is a random choice from about 737,000 sequences.
  • the length of the barcode (16) can be altered.
  • the diversity of potential barcode sequences (737 k) can be alterable.
  • the defined nature of the barcode can be altered, for example, it may also be completely random (16 Ns) or semi-random (16 bases that come from a biased distribution of nucleotides).
  • the canonical UMI sequence may be a 10 nucleotide randomer.
  • the length of the UMI can be altered.
  • the random nature of the UMI can be altered, for example, it may be semi-random (bases that come from a biased distribution of nucleotides.)
  • the distribution of UMI nucleotide(s) may be biased; for example, UMI sequences that do not contain Gs or Cs may be less likely to serve as primers.
  • the spacer may alterable within given or predetermined parameters. For example one method may give an optimal sequence of TTTCTTATAT (SEQ ID NO: 159), but using a slightly different optimization strategy results in a sequence that is likely just as or nearly as good.
  • the selected template switching region can comprise 3 consecutive riboGs or more.
  • the selected template switching region can comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive riboGs or more.
  • Alternative nucleotide may be used such as deoxyribo Gs, LNA G's, and potentially any combination thereof.
  • the present disclosure also provides methods of enriching cDNA sequences. Enrichment may be useful for TCR, BCR, and immunoglobulin gene analysis since these genes may possess similar yet polymorphic variable region sequences. These sequences can be responsible for antigen binding and peptide-MHC interactions. For example, due to gene recombination events in individual developing T cells, a single human or mouse will naturally express many thousands of different TCR genes. This T cell repertoire can exceed 100,000 or more different TCR rearrangements occurring during T cell development, yielding a total T cell population that is highly polymorphic with respect to its TCR gene sequences especially for the variable region. For immunoglobulin genes, the same may apply, except even greater diversity may be present. As previously noted, each distinct sequence may correspond to a clonotype.
  • enrichment increases accuracy and sensitivity of methods for sequencing TCR, BCR and immunoglobulin genes at a single cell level. In certain embodiments, enrichment increases the number of sequencing reads that map to a TCR, BCR, or immunoglobulin gene. In some embodiments, enrichment leads to greater than or equal to 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of total sequencing reads mapping to a TCR, BCR or immunoglobulin gene.
  • enrichment leads to greater than or equal to 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of total sequencing reads mapping to a variable region of a TCR, BCR or immunoglobulin gene.
  • an enrichment step can be employed. Enrichment may be useful for the sequencing and analysis of genes that may be related yet highly polymorphic.
  • an enriched gene comprises a TCR sequence, a BCR sequence, or an immunoglobulin sequence.
  • an enriched gene comprises a mitochondrial gene or a cytochrome family gene.
  • enrichment is employed after an initial round of reverse transcription (e.g., cDNA production). In some embodiments, enrichment is employed after an initial round of reverse transcription and cDNA amplification for at least 5, 10, 15, 20, 25, 30, 40 or more cycles. In some embodiments, enrichment is employed after a cDNA amplification.
  • the amplified cDNA can be subjected to a clean up step before the enrichment step using a column, gel extraction, or beads in order to remove unincorporated primers, unincorporated nucleotides, very short or very long nucleic acid fragments and enzymes.
  • enrichment is followed by a clean-up step before sequencing library preparation.
  • Enrichment of gene or cDNA sequences can be facilitated by a primer that anneals within a known sequence of the target gene.
  • a primer that anneals to a constant region of the gene or cDNA can be paired with a sequencing primer that anneals to a TSO functional sequence.
  • the enriched cDNA falls into a length range that approximately corresponds to that genes variable region.
  • greater than about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more cDNA or cDNA fragments fall within a range of about 300 base pairs to about 900 base pairs, of about 400 base pairs to about 800 base pairs, of about 500 base pairs to about 700 base pairs, or of about 500 base pairs to about 600 base pairs.
  • FIG. 20 shows an example enrichment scheme.
  • an oligonucleotide with a poly-T sequence 2014 and in some cases an additional sequence 2016 that binds to, for example, a sequencing or PCR primer, anneals to a target RNA 2020 .
  • the oligonucleotide is extended yielding an anti-sense strand 2022 which is appended by multiple cytidines on the 3′ end.
  • a template switching oligonucleotide attached to a gel bead 2038 is provided and a riboG of the TSO pairs with the cytidines of the sense strand and is extended to create a sense and an antisense strand.
  • the template switching oligonucleotide is released from the gel bead during extension. In some cases, the template switching oligonucleotide is released from the gel bead prior to extension. In some cases, the template switching oligonucleotide is released from the gel bead after extension.
  • the TSO comprises a barcode 2012 and one or two additional functional sequences 2008 and 2012 .
  • the additional functional sequences can comprise a P7 or R2 sequence for attachment to an Illumina sequencing flow cell, for example.
  • Operations 2001 and 2002 may be performed in a partition (e.g., droplet or well). Subsequent to operation 2002 , the nucleic acid product from operations 2001 and 2002 may be removed from the partition and in some cases pooled with other products from other partitions for subsequent processing.
  • amplification or sample identification can be added that allow for amplification or sample identification. This may occur in a partition or in bulk.
  • This reaction yields amplified cDNA molecules as in 2003 which are mixed templates comprising a barcode and sequencing primers. In some cases, not all of these cDNA molecules will comprise a target variable region sequence.
  • a primer 2018 that anneals to a sequence 3′ of a TCR, BCR or immunoglobulin variable region 2020 specifically amplifies the variable region comprising cDNAs yielding products as shown in operation 2005 .
  • Such enrichment may be performed for various approaches described herein, such as, e.g., the approaches described above in the context of FIGS. 19A and 19B .
  • primer 2018 anneals in a constant region of a TCR (e.g., TCR-alpha or TCR-beta), BCR or immunoglobulin gene. After amplification the products are sheared, adaptors ligated and amplified a second time to add additional functional sequences 2007 and 2011 and a sample index 2009 as shown in operation 2006 .
  • the additional functional sequences can functionally complement the first pair 2008 and 2010 and comprise for example a P5 or R1 sequence.
  • FIG. 21 shows example size distributions after cDNA amplification but before enrichment (A), after enrichment but before sequencing library prep (B), and after sequencing library preparation (C).
  • the initial poly-T primer, comprising sequences 2016 and 2014 can be attached to a gel bead as opposed to the TSO.
  • the poly-T comprising primer comprises functional sequences and barcode sequences 2008 , 2010 , 2012 , and the TSO comprises sequence 2016 .
  • Operations 2003 - 2006 may be performed in bulk.
  • clonotype information derived from next-generation sequencing data of cDNA prepped from cellular RNA is combined with other targeted on non targeted cDNA enrichment to illuminate functional and ontological aspects of B-cell and T cells that express a given TCR, BCR, or immunoglobulin.
  • clonotype information is combined with analysis of expression of an immunologically relevant cDNA.
  • the cDNA encodes a cell lineage marker, a cell surface functional marker, immunoglobulin isotype, a cytokine and/or chemokine, an intracellular signaling polypeptide, a cell metabolism polypeptide, a cell-cycle polypeptide, an apoptosis polypeptide, a transcriptional activator/inhibitor, an miRNA or lncRNA.
  • TCR T-cell Receptor
  • Tools for assembling T-cell Receptor (TCR) sequences may use known sequences of V and C regions to “anchor” assemblies. This may make such tools only applicable to organisms with well characterized references (human and mouse). However, most mammalian T cell receptors have similar amino acid motifs and similar structure. In the absence of a reference, a method can scan assembled transcripts for regions that are diverse or semi-diverse, find the junction region which should be highly diverse, then scan for known amino acid motifs.
  • the complementary CDRs such as the CDR1, CDR2, or CDR3, region be accurately delimited, only that a diverse sequence is found that can uniquely identify the clonotype.
  • One advantage of this method is that the software may not require a set of reference sequences and can operate fully de novo, thus this method can enable immune research in eukaryotes with poorly characterized genomes/transcriptomes.
  • the methods described herein allow simultaneously obtaining single-cell gene expression information with single-cell immune receptor sequences (TCRs/BCRs). This can be achieved using the methods described herein, such as by amplifying genes relevant to lymphocyte function and state (either in a targeted or unbiased way) while simultaneously amplifying the TCR/BCR sequences for clonotyping. This can allow such applications as 1) interrogating changes in lymphocyte activation/response to an antigen, at the single clonotype or single cell level; or 2) classifying lymphocytes into subtypes based on gene expression while simultaneously sequencing their TCR/BCRs. UMIs are typically ignored during TCR (or generally transcriptome) assembly.
  • base quality estimates for assembled contigs can inform clonotype inference. Errors can make cells with the same (real) clonotype have mismatching assembled sequences. Further, combining base-quality estimates and clonotype abundances to correct clonotype assignments. For example, if 10 cells have clonotype X and one cell has a clonotype that differs by X in only a few bases and these bases have low quality, then this cell may be assigned to clonotype X. In some embodiments, clonotypes that differ by a single amino acid or nucleic acid may be discriminated. In some embodiments, clonotypes that differ by less than 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids or nucleic acids may be discriminated. An example, non limiting, base error calculation scheme is shown below in Example VII.
  • microfluidic devices used for partitioning the cells as described above.
  • Such microfluidic devices can comprise channel networks for carrying out the partitioning process like those set forth in FIGS. 1 and 2 .
  • these microfluidic devices can comprise channel networks, such as those described herein, for partitioning cells into separate partitions, and co-partitioning such cells with oligonucleotide barcode library members, e.g., disposed on beads.
  • channel networks can be disposed within a solid body, e.g., a glass, semiconductor or polymer body structure in which the channels are defined, where those channels communicate at their termini with reservoirs for receiving the various input fluids, and for the ultimate deposition of the partitioned cells, etc., from the output of the channel networks.
  • a reservoir fluidly coupled to channel 202 may be provided with an aqueous suspension of cells 214
  • a reservoir coupled to channel 204 may be provided with an aqueous suspension of beads 216 carrying the oligonucleotides.
  • Channel segments 206 and 208 may be provided with a non-aqueous solution, e.g., an oil, into which the aqueous fluids are partitioned as droplets at the channel junction 212 .
  • a non-aqueous solution e.g., an oil
  • an outlet reservoir may be fluidly coupled to channel 210 into which the partitioned cells and beads can be delivered and from which they may be harvested.
  • the channel segments may be coupled to any of a variety of different fluid sources or receiving components, including tubing, manifolds, or fluidic components of other systems.
  • kits for analyzing individual cells or small populations of cells may include one, two, three, four, five or more, up to all of partitioning fluids, including both aqueous buffers and non-aqueous partitioning fluids or oils, nucleic acid barcode libraries that are releasably associated with beads, as described herein, microfluidic devices, reagents for disrupting cells amplifying nucleic acids, and providing additional functional sequences on fragments of cellular nucleic acids or replicates thereof, as well as instructions for using any of the foregoing in the methods described herein.
  • FIG. 17 shows a computer system 1701 that is programmed or otherwise configured to implement methods of the disclosure including nucleic acid sequencing methods, interpretation of nucleic acid sequencing data and analysis of cellular nucleic acids, such as RNA (e.g., mRNA), and characterization of cells from sequencing data.
  • the computer system 1701 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system 1701 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1705 , which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 1701 also includes memory or memory location 1710 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1715 (e.g., hard disk), communication interface 1720 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1725 , such as cache, other memory, data storage and/or electronic display adapters.
  • the memory 1710 , storage unit 1715 , interface 1720 and peripheral devices 1725 are in communication with the CPU 1705 through a communication bus (solid lines), such as a motherboard.
  • the storage unit 1715 can be a data storage unit (or data repository) for storing data.
  • the computer system 1701 can be operatively coupled to a computer network (“network”) 1730 with the aid of the communication interface 1720 .
  • the network 1730 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network 1730 in some cases is a telecommunication and/or data network.
  • the network 1730 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network 1730 in some cases with the aid of the computer system 1701 , can implement a peer-to-peer network, which may enable devices coupled to the computer system 1701 to behave as a client or a server.
  • the CPU 1705 can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 1710 .
  • the instructions can be directed to the CPU 1705 , which can subsequently program or otherwise configure the CPU 1705 to implement methods of the present disclosure. Examples of operations performed by the CPU 1705 can include fetch, decode, execute, and writeback.
  • the CPU 1705 can be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the system 1701 can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • the storage unit 1715 can store files, such as drivers, libraries and saved programs.
  • the storage unit 1715 can store user data, e.g., user preferences and user programs.
  • the computer system 1701 in some cases can include one or more additional data storage units that are external to the computer system 1701 , such as located on a remote server that is in communication with the computer system 1701 through an intranet or the Internet.
  • the computer system 1701 can communicate with one or more remote computer systems through the network 1730 .
  • the computer system 1701 can communicate with a remote computer system of a user.
  • remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants.
  • the user can access the computer system 1701 via the network 1730 .
  • Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1701 , such as, for example, on the memory 1710 or electronic storage unit 1715 .
  • the machine executable or machine readable code can be provided in the form of software.
  • the code can be executed by the processor 1705 .
  • the code can be retrieved from the storage unit 1715 and stored on the memory 1710 for ready access by the processor 1705 .
  • the electronic storage unit 1715 can be precluded, and machine-executable instructions are stored on memory 1710 .
  • the code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.
  • “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • a machine readable medium such as computer-executable code
  • a tangible storage medium such as computer-executable code
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory, such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
  • Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.
  • RF radio frequency
  • IR infrared
  • Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data.
  • Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the computer system 1701 can include or be in communication with an electronic display 1735 that comprises a user interface (UI) 1740 for providing, for example, results of nucleic acid sequencing, analysis of nucleic acid sequencing data, characterization of nucleic acid sequencing samples, cell characterizations, etc.
  • UI user interface
  • Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • Methods and systems of the present disclosure can be implemented by way of one or more algorithms.
  • An algorithm can be implemented by way of software upon execution by the central processing unit 1705 .
  • the algorithm can, for example, initiate nucleic acid sequencing, process nucleic acid sequencing data, interpret nucleic acid sequencing results, characterize nucleic acid samples, characterize cells, etc.
  • reverse transcription with template switching and cDNA amplification is performed in emulsion droplets with operations as shown in FIG. 9A .
  • the reaction mixture that is partitioned for reverse transcription and cDNA amplification includes 1,000 cells or 10,000 cells or 10 ng of RNA, beads bearing barcoded oligonucleotides/0.2% Tx-100/5 ⁇ Kapa buffer, 2 ⁇ Kapa HS HiFi Ready Mix, 4 ⁇ M switch oligo, and Smartscribe. Where cells are present, the mixture is partitioned such that a majority or all of the droplets comprise a single cell and single bead.
  • the cells are lysed while the barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of mRNA that is released from the cell as in operation 950 .
  • the poly-T segment is extended in a reverse transcription reaction as in operation 952 and the cDNA transcript is amplified as in operation 954 .
  • the thermal cycling conditions are 42° C. for 130 minutes; 98° C. for 2 min; and 35 cycles of the following 98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 6 min. Following thermal cycling, the emulsion is broken and the transcripts are purified with Dynabeads and 0.6 ⁇ SPRI as in operation 956 .
  • the yield from template switch reverse transcription and PCR in emulsions is shown for 1,000 cells in FIG. 13A and 10,000 cells in FIG. 13C and 10 ng of RNA in FIG. 13B (Smartscribe line).
  • the cDNA transcripts from RT and PCR performed in emulsions for 10 ng RNA is sheared and ligated to functional sequences, cleaned up with 0.8 ⁇ SPRI, and is further amplified by PCR as in operation 958 .
  • the amplification product is cleaned up with 0.8 ⁇ SPRI.
  • the yield from this processing is shown in FIG. 13B (SSII line).
  • reverse transcription with template switching and cDNA amplification is performed in emulsion droplets with operations as shown in FIG. 9A .
  • the reaction mixture that is partitioned for reverse transcription and cDNA amplification includes Jurkat cells, beads bearing barcoded oligonucleotides/0.2% TritonX-100/5 ⁇ Kapa buffer, 2 ⁇ Kapa HS HiFi Ready Mix, 4 ⁇ M switch oligo, and Smartscribe.
  • the mixture is partitioned such that a majority or all of the droplets comprise a single cell and single bead.
  • the cells are lysed while the barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of mRNA that is released from the cell as in operation 950 .
  • the poly-T segment is extended in a reverse transcription reaction as in operation 952 and the cDNA transcript is amplified as in operation 954 .
  • the thermal cycling conditions are 42° C. for 130 minutes; 98° C. for 2 min; and 35 cycles of the following 98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 6 min.
  • reverse transcription is performed in emulsion droplets and cDNA amplification is performed in bulk in a manner similar to that as shown in FIG. 9C .
  • the reaction mixture that is partitioned for reverse transcription includes beads bearing barcoded oligonucleotides, 10 ng Jurkat RNA (e.g., Jurkat mRNA), 5 ⁇ First-Strand buffer, and Smartscribe.
  • the barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of the RNA as in operation 961 .
  • the poly-T segment is extended in a reverse transcription reaction as in operation 963 .
  • the thermal cycling conditions for reverse transcription are one cycle at 42° C. for 2 hours and one cycle at 70° C. for 10 min. Following thermal cycling, the emulsion is broken and RNA and cDNA transcripts are denatured as in operation 962 . A second strand is then synthesized by primer extension with a primer having a biotin tag as in operation 964 .
  • the reaction conditions for this primer extension include cDNA transcript as the first strand and biotinylated extension primer ranging in concentration from 0.5-3.0 ⁇ M.
  • the thermal cycling conditions are one cycle at 98° C. for 3 min and one cycle of 98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 30 min.
  • the second strand is pulled down with Dynabeads MyOne Streptavidin C1 and T1, and cleaned-up with Agilent SureSelect XT buffers.
  • the second strand is pre-amplified via PCR as in operation 965 with the following cycling conditions—one cycle at 98° C. for 3 min and one cycle of 98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 30 min.
  • the yield for various concentrations of biotinylated primer (0.5 ⁇ M, 1.0 ⁇ M, 2.0 ⁇ M, and 3.0 ⁇ M) is shown in FIG. 15 .
  • the mixture that is partitioned for reverse transcription includes beads bearing barcoded oligonucleotides which also include a T7 RNA polymerase promoter sequence, 10 ng human RNA (e.g., human mRNA), 5 ⁇ First-Strand buffer, and Smartscribe.
  • the mixture is partitioned such that a majority or all of the droplets comprise a single bead.
  • the barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of the RNA as in operation 1050 .
  • the poly-T segment is extended in a reverse transcription reaction as in operation 1052 .
  • the thermal cycling conditions are one cycle at 42° C. for 2 hours and one cycle at 70° C. for 10 min. Following thermal cycling, the emulsion is broken and the remaining operations are performed in bulk.
  • a second strand is then synthesized by primer extension as in operation 1054 .
  • the reaction conditions for this primer extension include cDNA transcript as template and extension primer.
  • the thermal cycling conditions are one cycle at 98° C. for 3 min and one cycle of 98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 30 min. Following this primer extension, the second strand is purified with 0.6 ⁇ SPRI.
  • in vitro transcription is then performed to produce RNA transcripts. In vitro transcription is performed overnight, and the transcripts are purified with 0.6 ⁇ SPRI. The RNA yields from in vitro transcription are shown in FIG. 16 .
  • TCRs T-Cell Receptors
  • T-cells are co-partitioned with gel beads comprising barcoded template switching oligonucleotides.
  • T-cells Prior to partitioning, T-cells are optionally enriched from a cell sample, for example by fluorescence activated cell sorting (FACS) or other sorting technique.
  • FACS fluorescence activated cell sorting
  • Additional reagents for generating labeled polynucleotides including, but not limited to, reverse transcriptase enzyme, poly(dT) primer, and dNTPs, are delivered to partitions as part of a master mix.
  • a T-cell derived template polynucleotide comprising mRNA (e.g., 1920 ), poly(dT) primer (e.g., 1924 ), and a template switching oligonucleotide (e.g., 1926 ) are subjected to an amplification reaction to yield a first amplification product.
  • the poly(dT) primer hybridizes to the polyA tail of the mRNA template polynucleotide and acts as a primer for reverse transcription by the reverse transcriptase enzyme that is co-partitioned with the T-cell (e.g., 1950 ).
  • the reverse transcriptase enzyme has terminal transferase activity and adds additional nucleotides, e.g., polyC, to the cDNA transcript in a template independent manner.
  • the template switching oligonucleotide (e.g., 1926 ) hybridizes to the cDNA transcript and facilitates template switching in the first amplification reaction (e.g., 1952 ).
  • reverse transcription performed within partitions generates unbiased cDNA comprising a sequencing adapter, a cell barcode and a unique molecular identifier (UMI) on the 5′ end of the transcript.
  • UMI unique molecular identifier
  • the first amplification reaction product or cDNA transcript is subjected to a second amplification reaction to generate a second amplification product.
  • Polymerase chain reaction (PCR) is performed with one primer for the 5′ end of the transcript and one or more primers for the desired TCR/Ig constant region(s) (e.g., primers targeting TCR alpha ( ⁇ ) and/or beta ( ⁇ ) chain, and in some cases gamma and/or delta ( ⁇ / ⁇ ) chains).
  • PCR Polymerase chain reaction
  • the contents of multiple partitions can be combined such that the second amplification reaction is performed in bulk.
  • amplification products are subjected to enzymatic fragmentation and further processed to attach sequencing adaptors to generate a sequencing library.
  • Additional sequences include functional sequences such as flow cell attachment sequences and sequencing primer binding sequences.
  • the labeled polynucleotides are sequenced to yield sequencing reads, and sequencing reads are used to assemble full or partial TCR receptor gene sequences.
  • Additional analysis includes transcript counting for which an analysis pipeline may include, for example, (i) barcode processing, (ii) read filtering, (iii) cell-by-cell consensus assembly, (iv) V(D)J annotation, and (v) clonotype inference and clustering.
  • B-cell receptors BCRs
  • Ig receptors Other receptors (e.g., B-cell receptors (BCRs) and Ig receptors) can be similarly analyzed using the methods described herein by partitioning the appropriate immune cell type for generating labeled polynucleotides and using receptor specific primers to generate amplification products.
  • BCRs B-cell receptors
  • Ig receptors Ig receptors
  • cellular suspensions of 3,000; 6,000; or 12,000 primary human T cells were loaded on a GemCode Single Cell Instrument (10 ⁇ Genomics, Pleasanton, Calif.) to generate single cell-gel bead emulsions (SC-GEMs).
  • the gel beads were modified to carry a template switching oligonucleotide (TSO) as shown in FIG. 18 or FIG. 20 at 8 ⁇ M, yielding a final concentration of 0.32 ⁇ M in GEM.
  • TSO template switching oligonucleotide
  • reverse transcription was performed on the cells in emulsion using a poly-T primer and reverse transcriptase for 5 minutes at 55° C., followed by 1 hour and 55 minutes at 52° C.
  • FIG. 22 shows the cDNA yields from all three cellular suspensions. cDNA yields from 12,000 cells was greater than either 6,000 or 3,000 cells, which yielded similar amounts.
  • Indexed sequencing libraries were constructed using the GemCode Single Cell 3′ Library Kit, following these steps: 1) end repair and A-tailing; 2) adapter ligation; 3) post-ligation cleanup with SPRIselect; 4) sample index PCR and cleanup. These sequencing libraries were sequenced using an Illumina MiSeq sequencer. Sequencing performance of poly-T primed libraries was compared to libraries constructed from enriched cDNA libraries created by using an enriched priming method which substituted the poly-T primer with primers that bound the constant region of TCR ⁇ , TCR ⁇ , or both. FIG.
  • TSO TSO immobilized to gel beads
  • GB-TSTO TSO immobilized to gel beads
  • cDNA from 3,000 primary T cells was prepared using poly-T priming followed by enrichment using primers that anneal to TCR constant regions, yielding 38.5% VDJ mappable reads, quantitation of this enrichment is shown in FIG. 26A .
  • GEM-RT emulsion-reverse transcription reaction
  • This nested enrichment comprised PCR using outer primers annealing to the TCR alpha and beta paired with a P7 primer for 10 cycles using 60° C. extensions, followed by PCR using inner primers annealing to the TCR alpha and beta paired with a P7 primer for 10 cycles using 60° C. extensions. Results of this are shown in FIG. 26B with the largest amount of enrichment exhibited using a lower concentration of gel beads (8 ⁇ M).
  • FIGS. 27A-27C show that using V region primers in conjunction with constant region primers specifically enrich TCR alpha ( FIG. 27B ) and TCR beta sequences ( FIG. 27C ) compared to P7 primers paired with constant region primers.
  • FIG. 28 further shows that by using specific enrichment ( 28 C and D; V region+C region primers) compared to general enrichment ( 28 A and B; P7 primer+C region primer) yields of specifically enriched product were increased by increasing the amount of TSO-GB (from 8 ⁇ M to 200 ⁇ m).
  • individual cells are lysed in partitions comprising gel bead emulsions (GEMs).
  • GEMs for example, can be aqueous droplets comprising gel beads.
  • a template polynucleotide comprising an mRNA molecule can be reverse transcribed by a reverse transcriptase and a primer comprising a poly(dT) region.
  • a template switching oligo (TSO) present in the GEM can facilitate template switching so that a resulting polynucleotide product or cDNA transcript from reverse transcription comprises the primer sequence, a reverse complement of the mRNA molecule sequence, and a sequence complementary to the template switching oligo.
  • the template switching oligo can comprise additional sequence elements, such as a unique molecular identifier (UMI), a barcode sequence (BC), and a Read1 sequence. See FIG. 29A .
  • UMI unique molecular identifier
  • BC barcode sequence
  • Read1 sequence Read1 sequence. See FIG. 29A .
  • a plurality of mRNA molecules from the cell is reverse transcribed within the GEM, yielding a plurality of polynucleotide products having various nucleic acid sequences.
  • the polynucleotide product can be subjected to target enrichment in bulk. Prior to target enrichment, the polynucleotide product can be optionally subjected to additional reaction(s) to yield double-stranded polynucleotides.
  • the target may comprise VDJ sequences of a T cell and/or B cell receptor gene sequence. As shown at the top of the right panel of FIG.
  • the polynucleotide product (shown as a double-stranded molecule, but can optionally be a single-stranded transcript) can be subjected to a first target enrichment polymerase chain reaction (PCR) using a primer that hybridizes to the Read 1 region and a second primer that hybridizes to a first region of the constant region (C) of the receptor sequence (e.g., TCR or BCR).
  • PCR polymerase chain reaction
  • the product of the first target enrichment PCR can be subjected to a second, optional target enrichment PCR.
  • a second primer that hybridizes to a second region of the constant region (C) of the receptor can be used.
  • This second primer can, in some cases, hybridize to a region of the constant region that is closer to the VDJ region that the primer used in the first target enrichment PCR.
  • the resulting polynucleotide product can be further processed to add additional sequences useful for downstream analysis, for example sequencing.
  • the polynucleotide products can be subjected to fragmentation, end repair, A-tailing, adapter ligation, and one or more clean-up/purification operations.
  • a first subset of the polynucleotide products from cDNA amplification can be subjected to target enrichment ( FIG. 29B , right panel) and a second subset of the polynucleotide products from cDNA amplification is not subjected to target enrichment ( FIG. 29B , bottom left panel).
  • the second subset can be subjected to further processing without enrichment to yield an unenriched, sequencing ready population of polynucleotides.
  • the second subset can be subjected to fragmentation, end repair, A-tailing, adapter ligation, and one or more clean-up/purification operations.
  • the labeled polynucleotides can then be subjected to sequencing analysis. Sequencing reads of the enriched polynucleotides can yield sequence information about a particular population of the mRNA molecules in the cell whereas the enriched polynucleotides can yield sequence information about various mRNA molecules in the cell.
  • transcript and UMI will be used inter-changeably, assuming that there is a 1-1 relationship between transcripts and UMIs.
  • R be the real template base at the given position and T u be the (unobserved) base at the given position on transcipt/UMI u.
  • Rui and Rui be the real (pre-sequencing errors) and observed (post-sequencing errors) bases on the i th read of UMI u and Qui be the corresponding base quality.
  • Equation I can be derived by summing over the unobserved value c of transcript u at the given position and the (also unobserved) real value d of each read at this position.
  • Equation II Equation II
  • Devices, systems, compositions and methods of the present disclosure may be used for various applications, such as, for example, processing a single analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein) form a single cell.
  • a biological particle e.g., a cell or cell bead
  • a partition e.g., droplet
  • multiple analytes from the biological particle are processed for subsequent processing.
  • the multiple analytes may be from the single cell. This may enable, for example, simultaneous proteomic, transcriptomic and genomic analysis of the cell.
US15/825,740 2016-10-19 2017-11-29 Methods and systems for barcoding nucleic acid molecules from individual cells or cell populations Abandoned US20180105808A1 (en)

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US15/825,740 US20180105808A1 (en) 2016-10-19 2017-11-29 Methods and systems for barcoding nucleic acid molecules from individual cells or cell populations
US17/131,494 US20210238581A1 (en) 2016-10-19 2020-12-22 Methods and systems for processing polynucleotides
US17/692,792 US20220333191A1 (en) 2016-10-19 2022-03-11 Methods and systems for processing polynucleotides
US17/692,758 US20220389503A1 (en) 2016-10-19 2022-03-11 Methods and systems for processing polynucleotides
US18/301,565 US20240067953A1 (en) 2016-10-19 2023-04-17 Methods and systems for processing polynucleotides

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