WO2025166174A1

WO2025166174A1 - Methods and compositions for bead deconvolution

Info

Publication number: WO2025166174A1
Application number: PCT/US2025/014051
Authority: WO
Inventors: Eric K. POMARANSKI; Ronald Lebofsky; Jeffrey W. Perry; Adam M. Mccoy
Original assignee: BIO-RAD; Bio Rad Laboratories Inc
Current assignee: BIO-RAD; Bio Rad Laboratories Inc
Priority date: 2024-02-01
Filing date: 2025-01-31
Publication date: 2025-08-07
Anticipated expiration: 2026-08-01

Abstract

Methods and compositions are provided for detecting multiple barcodes in a partition for sequencing applications. Bead-specific barcoding oligonucleotides and/or detection oligonucleotides are used to introduce a bead-specific barcode sequence and a universal adapter sequence to target sequences from a single cell or nucleus. Also provided are beads comprising one or more bead-specific barcoding nucleic acids. In addition, the disclosure provides a plurality of partitions that include a single cell or nucleus or nucleic acids from a single cell or nucleus, beads comprising one or more bead-specific barcoding oligonucleotides, and a DNA polymerase.

Description

METHODSAND COMPOSITIONS FOR BEAD DECONVOLUTION

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present application claims benefit of priority to U.S. Provisional Patent Application No. 63/548,742. filed February 1. 2024, which is incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

[0002] Beads conjugated to oligonucleotides are used in high throughput sequencing applications having many different partitions. The oligonucleotides can be delivered to the partitions using beads as the delivery vehicle. The beads may deliver many copies of an oligonucleotide to a partition, and the oligonucleotide may have barcode sequences that are unique for the bead to which the oligonucleotide is linked. Tagging nucleic acids with a barcode sequence in partitions can provide important information through the sequencing and analysis of the barcode sequences along with the tagged nucleic acids. However, to ensure that partitions have only one bead and thus are uniquely labeled by a single barcode, bead concentrations are typically quite low, resulting in low utilization of the partitions and an increased amount of the sample and reagents required. Increasing bead concentrations results in higher partition occupancy and greater utilization of partitions, decreasing the required amount of sample and reagents needed. However, higher bead concentrations lead to a greater number of partitions having more than one bead, and therefore, some partitioned samples may be labeled by more than one barcode sequence. As a result, the presence of more than one bead-specific barcoded oligonucleotide in a partition can interfere with sequence analysis and quantification, resulting in a loss of sensitivity unless this issue can be identified. The present disclosure provides solutions to the potential loss of sensitivity, enabling a determination that multiple bead barcodes are from the same partition and accounting for that in the sequencing analysis. BRIEF SUMMARY OF THE INVENTION

[0003] In one aspect, methods of detecting multiple barcodes in a partition are provided. In some embodiments, the method comprises: providing a plurality of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality of copies of a capture oligonucleotide, a first deconvolution oligonucleotide, and a second deconvolution oligonucleotide, wherein the capture oligonucleotide comprises 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence; wherein the first deconvolution oligonucleotide comprises 5 ’-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a first bead capture sequence; wherein the second deconvolution oligonucleotide comprises 5’-3’: a third PCR priming sequence, the bead-specific barcode sequence, and a second bead capture sequence that is reverse complementary to the first bead capture sequence; and wherein the bead comprises more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides;

(ii) a DNA polymerase; and

(iii) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a capture oligonucleotide and first and second deconvolution oligonucleotides that comprise a first bead-specific barcode sequence, and wherein the second bead is linked to a capture oligonucleotide and first and second deconvolution oligonucleotides that comprise a second bead-specific barcode sequence; in the partitions, performing strand synthesis, wherein the performing comprises annealing sequences of cellular nucleic acids from the single cell or nucleus to some copies of the capture oligonucleotide and extending the capture oligonucleotides using the cellular nucleic acids as a template, wherein the strands synthesized comprise the first PCR priming sequence, the bead-specific barcode sequence, the capture sequence, and a target gene sequence; in the partitions, annealing some copies of the first bead capture sequence of the first deconvolution oligonucleotide to some copies of the second bead capture sequence of the second deconvolution oligonucleotide, extending the first deconvolution oligonucleotide with the DNA polymerase using the second deconvolution oligonucleotide as a template, and extending the second deconvolution oligonucleotide with the DNA polymerase using the first deconvolution oligonucleotide as a template, thereby generating double-stranded nucleic acids comprising the second PCR priming sequence, the bead-specific barcode sequence, the first bead capture sequence, the bead-specific barcode reverse complement sequence, and the third PCR priming reverse complement sequence; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the double-stranded nucleic acids, wherein if two different bead-specific barcode sequences or reverse complement sequences are linked on some of the double-stranded nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences or reverse complement sequences are from the same partition.

[0004] In some embodiments, the target capture sequence includes a poly T sequence. In other embodiments, the target capture sequence is a portion of a target gene sequence.

[0005] In some embodiments, performing strand synthesis comprises annealing sequences of cellular mRNAs from the single cell or nucleus to some copies of the capture oligonucleotide and forming first strand gene-specific cDNAs by extending the capture oligonucleotides with a reverse transcriptase using the cellular mRNAs as a template; and in the partitions, performing second strand synthesis using the DNA polymerase to form second strand cDNAs using first strand gene-specific cDNAs as templates and the cellular mRNAs as primers, thereby forming double-stranded gene-specific cDNAs from a plurality of different RNAs.

[0006] In some embodiments, the providing a plurality of partitions comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions. In other embodiments, the providing a plurality of partitions comprises providing partitions comprising fixed cells.

[0007] In some embodiments, the methods further comprise releasing the barcoding oligonucleotides from the beads.

[0008] In some embodiments, the methods further comprising, after performing strand synthesis, inactivating the polymerase and reverse transcriptase, if present, in the partitions. In some embodiments, inactivating includes applying heat to the partitions. In certain embodiments, inactivating comprises incubating the partitions at 75-90 degrees Celsius.

[0009] In some embodiments, the partitions are droplets in an emulsion or microwells. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

[0010] Also provided are a plurality⁷ of partitions. In some embodiments, the different partitions include different beads, each bead linked to a plurality of copies of a capture oligonucleotide, a first deconvolution oligonucleotide, and a second deconvolution oligonucleotide, wherein the capture oligonucleotide comprises 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence; wherein the first deconvolution oligonucleotide comprises 5 ’-3’: a second PCR priming sequence, the beadspecific barcode sequence and a first bead capture sequence; wherein the second deconvolution oligonucleotide comprises 5 ’-3’: a third PCR priming sequence, the beadspecific barcode sequence, and a second bead capture sequence that is reverse complementary⁷ to the first bead capture sequence; and wherein the bead comprises more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides. The partitions also include a DNA polymerase; a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; and optionally, a reverse transcriptase; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a capture oligonucleotide and first and second deconvolution oligonucleotides comprising a first bead-specific barcode sequence, and wherein the second bead is linked to a capture and first and second deconvolution oligonucleotides comprising a second bead-specific barcode sequence. In some embodiments, the target capture sequence is a poly T sequence. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

[0011] Also provided are beads. In some embodiments, the beads include a plurality of copies of a capture oligonucleotide, wherein the capture oligonucleotide comprises 5’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence; a plurality⁷ of copies of a first deconvolution oligonucleotide, wherein the first deconvolution oligonucleotide comprises 5’-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a first bead capture sequence; and a plurality of copies of a second deconvolution oligonucleotide, wherein the second deconvolution oligonucleotide comprises 5 ’-3’: a third PCR priming sequence, the bead-specific barcode sequence and a second bead capture sequence that is reverse complementary to the first bead capture sequence; wherein the bead comprises more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides.

[0012] In other aspects, additional methods for detecting barcodes in a partition are provided. providing a plurality of partitions, wherein different partitions comprise

(i) different beads, each bead linked to a plurality of copies of a first barcoding oligonucleotide and a second barcoding oligonucleotide, wherein the first barcoding oligonucleotide comprises 5’-3’: a first PCR priming sequence, a beadspecific barcode sequence, and a first target capture sequence: wherein the second barcoding oligonucleotide comprises 5^?-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a second target capture sequence; and wherein the bead comprises more copies of the first barcoding oligonucleotide than the second barcoding oligonucleotide;

(ii) a reverse transcriptase;

(iii) a DNA polymerase; and

(iv) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to first and second barcoding oligonucleotides comprising a first bead-specific barcode sequence, and wherein the second bead is linked to first and second barcoding oligonucleotides comprising a second bead-specific barcode sequence; in the partitions, performing reverse transcription, wherein the performing comprises annealing sequences of cellular RNAs from the single cell or nucleus to some copies of the first barcoding oligonucleotide and forming first strand gene-specific cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template, thereby producing a DNA:RNA heteroduplex sequence, wherein the first strand gene-specific cDNAs comprise the first PCR priming sequence, the bead-specific barcode sequence, the first target capture sequence, and the target gene sequence; in the partitions, degrading the RNA in the DNA:RNA heteroduplex; in the partitions, annealing some copies of the second target capture sequence of the second barcoding oligonucleotide to the first strand gene-specific cDNAs, extending the second barcoding oligonucleotide with the DNA polymerase using the first strand gene-specific cDNA as a template, thereby generating nucleic acids comprising 5’-3’: the second PCR priming sequence, the bead-specific barcode sequence, a portion of the target gene sequence, the reverse complement of the first target capture sequence, the reverse complement of the bead-specific barcode sequence, and the reverse complement of the first PCR priming sequence; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the nucleic acids, wherein if two different beadspecific barcode sequences or reverse complement of the bead-specific barcode sequences are linked to the portion of the target gene sequence in the nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences are from the same partition.

[0013] In some embodiments, the first capture sequence comprises a poly T sequence. In other embodiments, the first capture sequence comprises a portion of a target gene sequence. In some embodiments, the second target capture sequence binds to a sequence that is 50-600 nucleotides away from the first target capture sequence. In some embodiments, the second target capture sequence binds to a cDNA of a target gene that is highly expressed in the cell.

[0014] In some embodiments, the methods further comprise releasing the barcoding oligonucleotides from the beads.

[0015] In some embodiments, the providing a plurality of partitions comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions. In other embodiments, the providing a plurality of partitions comprises providing partitions comprising fixed cells.

[0016] In some embodiments, the methods further include, after performing the last strand synthesis, inactivating the polymerase and reverse transcriptase in the partition. In some embodiments, the inactivating comprises applying heat to the partitions. In certain embodiments, the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

[0017] In some embodiments, the partitions are droplets in emulsions or microwells. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead. [0018] Also provided are a plurality of partitions. In some embodiments, the partitions comprise different beads, each bead linked to a plurality’ of copies of a first barcoding oligonucleotide and a second barcoding oligonucleotide, wherein the first barcoding oligonucleotide comprises 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target capture sequence; wherein the second barcoding oligonucleotide comprises 5'-3’: a second PCR priming sequence, the bead-specific barcode sequence, a second target capture sequence; and wherein the bead comprises more copies of the first barcoding oligonucleotide than copies of the second barcoding oligonucleotide; a reverse transcriptase; and a single fixed, lysed, or permeabilized cell or nucleus or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to first and second barcoding oligonucleotides comprising a first bead-specific barcode sequence, and wherein the second bead is linked to first and second barcoding oligonucleotides comprising a second bead-specific barcode sequence. In some embodiments, the first target capture sequence comprises a poly T sequence. In some embodiments, the second target capture sequence binds to a sequence that is 50-600 nucleotides away from the first target gene capture sequence. In some embodiments, the second target capture sequence binds to a cDNA of a gene that was highly expressed in the cell. In some embodiments, the partitions are droplets in an emulsion or microwells. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

[0019] Also provided are beads that comprise a plurality of copies of a first barcoding oligonucleotide, wherein the first barcoding oligonucleotide comprises 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target capture sequence; and a plurality of copies of a second barcoding oligonucleotide; wherein the second barcoding oligonucleotide comprises 5^?-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a second target capture sequence; wherein the bead comprises more copies of the first barcoding oligonucleotide than copies of the second barcoding oligonucleotide. In some embodiments, the first target capture sequence comprises a poly T sequence. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

[0020] In other aspects, additional methods for detecting multiple barcodes in a partition are provided. In some embodiments, the methods comprise: providing a plurality of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality of copies of a barcoding oligonucleotide, comprising 5’-3’: a first PCR priming sequence, a beadspecific barcode sequence, and a first target gene capture sequence;

(ii) a detection oligonucleotide comprising 5’-3’: a second PCR priming sequence, a unique molecular identifier (UMI), and a second target gene capture sequence;

(iii) a reverse transcriptase;

(iv) a DNA polymerase; and

(v) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a first bead-specific barcode sequence, and wherein the second bead is linked to a second bead-specific barcode sequence; in the partitions, performing reverse transcription, wherein the performing comprises annealing sequences of cellular RNAs from the single cell or nucleus to some copies of the barcoding oligonucleotide and forming first strand gene-specific cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template, thereby producing an RNA:DNA heteroduplex, wherein the first strand genespecific cDNAs comprise the first PCR priming sequence, the bead-specific barcode sequence, the first target gene capture sequence, and target gene sequence; in the partitions, degrading the RNA in the heteroduplex; in the partitions, performing second strand synthesis to form second strand cDNAs using first strand cDNAs as templates and the detection oligonucleotide as a primer, wherein the second strand cDNA comprises 5'-3’: the second PCR priming sequence, the UMI, the second target capture sequence, a portion of the target gene sequence, the bead-specific barcode reverse complement sequence, and the first PCR priming reverse complement sequence; in the partitions, annealing some copies of the barcoding oligonucleotide to some copies of the second strand cDNA, extending the barcoding oligonucleotide using the second strand cDNA as a template, thereby generating double-stranded nucleic acids, the first strand comprising the first PCR priming sequence, the bead-specific barcode sequence, the portion of the target gene sequence, the UMI reverse complement sequence, and the second PCR primer reverse complement sequence; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the nucleic acids, wherein if two different beadspecific barcode sequences or bead-specific barcode reverse complement sequences are linked to the UMI in the nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences or bead-specific barcode reverse complement sequences are from the same partition.

[0021] In some embodiments, the methods further comprise releasing the barcoding oligonucleotides from the beads.

[0022] In some embodiments, the first target gene capture sequence comprises a poly T sequence.

[0023] In some embodiments, the providing a plurality of partitions comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions. In other embodiments, the providing a plurality of partitions comprises providing partitions comprising fixed cells.

[0024] In some embodiments, the methods further comprise, after performing the last strand synthesis, inactivating the polymerase and reverse transcriptase in the partitions. In some embodiments, the inactivating comprises applying heat to the partitions. In certain embodiments, the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

[0025] In some embodiments, the partitions are droplets in an emulsion or microwells. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

[0026] Also provided are a plurality of different partitions. In some embodiments, the different partitions comprise different beads, each bead linked to a plurality of copies of a barcoding oligonucleotide, comprising ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence; a detection oligonucleotide comprising 5 ’-3’: a second PCR priming sequence, a unique molecular identifier (UMI), and a second target gene capture sequence; a reverse transcriptase; a DNA polymerase; and a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a first bead-specific barcode sequence, and wherein the second bead is linked to a second bead-specific barcode sequence. In some embodiments, the first target gene capture sequence comprises a poly T sequence. In some embodiments, the partitions are droplets in an emulsion or microwells. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

[0027] In other aspects, additional methods for detecting barcodes in a partition are provided. In some embodiments, the methods comprise: providing a plurality of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality of copies of a barcoding oligonucleotide, comprising 5'-3’: a palindromic sequence, a PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence;

(ii) optionally, a detection oligonucleotide comprising a second target gene capture sequence;

(iii) a reverse transcriptase;

(iv) a DNA polymerase; and

(v) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a first bead-specific barcode sequence, and wherein the second bead is linked to a second bead-specific barcode sequence; in the partitions, performing reverse transcription, wherein the performing comprises annealing sequences of some cellular RNAs from the single cell or nucleus to some copies of the barcoding oligonucleotide and forming first strand cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template, thereby producing an RNA:DNA heteroduplex, wherein the first strand cDNAs comprise 5 '-3': the palindromic sequence, the first PCR priming sequence, the bead-specific barcode sequence, the first target gene capture sequence, and the target gene sequence; in the partitions, performing second strand synthesis using the first strand cDNAs as a template and extending from the cellular RNAs. thereby producing double stranded nucleic acids having an RNA:DNA heteroduplex, wherein the second strand comprises the palindromic sequence at its 3’ end; in the partitions, separating the first strand cDNAs from the second strand nucleic acids; in the partitions, performing additional strand synthesis, wherein the palindromic sequences of some of the second strand nucleic acids anneal to the palindromic sequences of different second strand nucleic acids, and wherein strand synthesis uses the second strand nucleic acids as template and extends from the palindromic sequence, thereby producing double-stranded nucleic acids comprising 5’-3‘: the target gene sequence, the bead-specific barcode reverse complement sequence, the PCR priming reverse complement sequence, the palindromic sequence, the PCR priming sequence, the bead-specific barcode sequence, and target gene reverse complement sequence; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the double-stranded nucleic acids, wherein if two different bead-specific barcode or bead-specific barcode reverse complement sequences are linked to the palindromic sequence in some of the double-stranded nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences or beadspecific barcode reverse complement sequences are from the same partition.

[0028] In some embodiments, the separating comprises heating to separate the first strand cDNAs from the second strand nucleic acids. In other embodiments, the separating and additional strand synthesis comprise in the partitions, degrading the RNA from the heteroduplex; in the partitions, performing strand synthesis with a displacing DNA polymerase, using first strand cDNAs as templates and the detection oligonucleotide as a primer, wherein the second strand comprises 5’-3’: a portion of the target gene sequence, a bead-specific barcode reverse complement sequence, and the palindromic sequence, and wherein the displaced strand comprises 5’-3’: the bead-specific barcode reverse complement sequence, and the palindromic sequence; and in the partitions, performing additional strand synthesis with the second strand nucleic acids and the displaced strand nucleic acids as templates, wherein the palindromic sequences of some of the displaced strands anneal to the palindromic sequences of different displaced stands, and wherein strand synthesis uses the bead-specific barcodes and PCR priming sequences as template and extends from the palindromic sequence, thereby producing double-stranded nucleic acids comprising 5 ’-3’: the PCR priming reverse complement sequence, the bead-specific barcode reverse complement sequence, the palindromic sequence, the bead-specific barcode sequence, and the PCR priming sequence. [0029] In some embodiments, the first target gene capture sequence comprises a poly T sequence.

[0030] In some embodiments, the providing a plurality of partitions comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions. In other embodiments, the providing a plurality of partitions comprises providing partitions comprising fixed cells.

[0031] In some embodiments, the method further comprises releasing the barcoding oligonucleotides from the beads.

[0032] In some embodiments, the methods further comprise, after the last strand synthesis, inactivating the polymerase and reverse transcriptase in the partitions. In some embodiments, the inactivating comprises applying heat to the partitions; In certain embodiments, the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

[0033] In some embodiments, the partitions are droplets in an emulsion or microwells. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

[0034] Also provided are a plurality of different partitions. In some embodiments, the different partitions comprise different beads, each bead linked to a plurality of copies of a barcoding oligonucleotide, comprising ’-3': a palindromic sequence, a PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence; optionally, a detection oligonucleotide comprising a second target gene capture sequence; a reverse transcriptase; a DNA polymerase; and a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a first bead-specific barcode sequence, and wherein the second bead is linked to a second beadspecific barcode sequence. In some embodiments, the first target gene capture sequence comprises a poly T sequence. In some embodiments, the partitions are droplets in an emulsion or microwells. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

[0035] Also provided are beads comprising a plurality of copies of a barcoding oligonucleotide, comprising 5 ‘-3’: a palindromic sequence, a PCR priming sequence, a beadspecific barcode sequence, and a first target gene capture sequence. In some embodiments, the first target gene capture sequence comprises a poly T sequence. In some embodiments. the partitions are droplets in an emulsion or microwells. In some embodiments, the cell is a mammalian cell. In some embodiments, the bead is a hydrogel bead.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIGS. 1A-1D are schematic drawings of compositions and method steps for one embodiment of methods for detecting multiple barcodes in a partition. FIG. 1A depicts a head linked to a capture oligonucleotide and first and second deconvolution oligonucleotides, each including a universal adapter sequence (e.g., linker sequence shown as UUU. and a PCR priming or sequencing adapter sequence “PCR Rl” (e.g., “sequencing handle” to amplify and/or facilitate sequencing of the nucleic acids resulting from the steps depicted)), an optional replication identifier sequence (e.g., unique molecule identifier or UMI)), and a bead-specific barcode sequence (shown as CBC). The bead includes more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides. In addition to the universal adapter sequence, the optional UMI, and the bead-specific barcode sequence, the capture oligonucleotide also includes a target capture sequence. In the exemplified embodiment, the target capture sequence is depicted as a poly T sequence (SEQ ID NO: 1). In addition to the universal adapter sequence, the optional UMI, and the beadspecific barcode sequence, the first and second deconvolution oligonucleotides comprise a bead capture sequence. The first bead capture sequence (exemplary sequence shown as Sequence A; SEQ ID NO: 2) of the first deconvolution oligonucleotide is reverse complementary' to the second bead capture sequence (exemplary sequence shown as Sequence B; SEQ ID NO: 3) of the second deconvolution oligonucleotide.

[0037] FIGS. 1B-1D depict steps involved in an exemplary bead deconvolution method. FIG. IB depicts two different beads, as described in FIG. 1A, present in a single partition. The first bead is show n at the left and includes capture and deconvolution oligonucleotides with a first bead-specific barcode sequence (shown as CBC), and the second bead is shown on the right and includes capture and deconvolution oligonucleotides with a second beadspecific barcode sequence (shown as CBC*). Because the first bead includes some deconvolution oligonucleotides having the first or second bead capture sequence and the second bead includes some deconvolution oligonucleotides having the first or second bead capture sequence, the first bead capture sequence on one bead may bind to the second bead capture sequence on the second bead (shown with dotted lines on FIG. IB). FIG. 1C depicts hypothetical products from the method described herein when two beads are present in a single partition. If two different bead-specific barcode sequences are linked on some of the resulting double-stranded nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences are from the same partition.

[0038] FIG. ID is a different schematic depicting the beads and steps involved in this exemplary bead deconvolution method. Two different beads, as described in FIG. 1A, are shown present in a single partition. The first bead is shown at the left and includes capture and deconvolution oligonucleotides with a first bead-specific barcode sequence (shown as BC1), and the second bead is shown on the right and includes capture and deconvolution oligonucleotides with a second bead-specific barcode sequence (shown as BC2). The target capture sequence is shown as “‘Cell NA capture.” and the first and second bead capture sequences are shown as “Bead capture 1” and “Bead capture 2.” The middle panel depicts how the first capture sequence of the first bead binds to the second bead capture sequence of the second bead. The bottom panel depicts hypothetical products from the method when the two different beads are present in a single partition.

[0039] FIGS. 2A-2D are schematic drawings depicting compositions and steps involved in another exemplary embodiment of methods for detecting multiple barcodes in a partition. FIG. 2A depicts a bead linked to first and second barcoding oligonucleotides, each including a universal adapter sequence (e.g., linker sequence shown as UUU, and a PCR priming or sequencing adapter sequence PCR R1 (e.g., sequencing handle to amplify and/or facilitate sequencing of the nucleic acids resulting from the steps depicted)), optionally a replication identifier sequence (UMI), and a bead-specific barcode sequence (shown as CBC). The bead includes more copies of the first barcoding oligonucleotide than copies of the second barcoding oligonucleotide. In addition to the universal adapter sequence, the optional UMI, and the bead-specific barcode sequence, the first barcoding oligonucleotide also includes a first target capture sequence. In the exemplified embodiment, the first target capture sequence is a poly T sequence (SEQ ID NO: 1). FIG. 2A includes at the top of the figure an alternate schematic for depicting the first barcoding oligonucleotide on a first bead (Bead A), with the universal adapter sequence, optionally the UMI. and bead-specific barcode sequence being shown as “Barcode A/UMI.” In addition to the universal adapter sequence, optional UMI, and the bead-specific barcode sequence, the second barcoding oligonucleotide includes a second target capture sequence. In the exemplified embodiment, the GAPDH gene is the target gene, and the second target capture sequence is a portion of the GAPDH second strand sequence (show n as GAPDH SS). The second target capture sequence also may be referred to as a cDNA capture sequence.

[0040] FIGS. 2B-2D depict steps involved in an exemplary bead deconvolution method, using beads as described in FIG. 2A. The bottom panel of FIG. 2C (which is also the bottom strand of the middle panel) depicts a hypothetical product from the method described herein when two beads are present in a single partition. If two different bead-specific barcode sequences or the reverse complements (e.g.. Barcode A, Barcode B, or the reverse complement of Barcode A or Barcode B) are linked to the target sequence (e.g., GAPDH in this example) on a single nucleic acid, then sequencing reads comprising either of the tw o bead-specific barcode sequences are from the same partition.

[0041] FIG. 2D is a different schematic depicting the beads and steps involved in this exemplary bead deconvolution method. Two different beads, as described in FIG. 2A, are shown present in a single partition. The first bead is shown at the left and includes first and second barcoding oligonucleotides with a first bead-specific barcode sequence (shown as BC1), and the second bead is shown on the right and includes first and second barcoding oligonucleotides with a second bead-specific barcode sequence (shown as BC2). The first target capture sequence is shown as '"Cell NA capture,” and the second target capture sequence is shown as “cDNA capture.” The second and third panels depict the binding of the first capture sequence of the first bead to the cellular RNA and the formation of the cDNA. The second from the bottom panel depicts the binding of the second capture sequence of the second bead to the cDNA formed in this method. The bottom panel depicts the resulting hypothetical products from the method when the two different beads are present in a single partition.

[0042] FIGS. 3A-3F are schematic drawings of compositions and method steps for another embodiment of methods for detecting multiple barcodes in a partition. FIG. 3 A depicts a bead comprising a barcoding oligonucleotide that includes a universal adapter sequence, optional UMI, and bead-specific barcode sequence (together shown as “Barcode A/UMI”) and a first target capture sequence. In the exemplified embodiment, the first target capture sequence is a poly T sequence. FIG. 3A also depicts a detection oligonucleotide at the top right. The detection oligonucleotide includes a universal adapter sequence (PCR priming sequence or sequencing handle), a unique molecular identifier (UM1/D0). and a second target capture sequence. In the exemplified embodiment, the second target capture sequence is a portion of the GAPDH second strand sequence (GAPDH SS). The second target capture sequence may be referred to as a cDNA capture sequence.

[0043] FIGS. 3B-3D depict steps involved in an exemplary bead deconvolution method, using beads and detection oligonucleotides as described in FIG. 3A. The bottom panel of FIG. 3D depicts a hypothetical product from the method described herein when two beads are present in a single partition. If two different bead-specific barcode sequences or reverse complement sequences (e.g.. Barcode A, Barcode B, or reverse complement of Barcode A or Barcode B) are linked to the target sequence (e.g., GAPDH) and UMI or reverse complement on a single nucleic acid, then sequencing reads comprising either of the two bead-specific barcode sequences are from the same partition.

[0044] FIGS. 3E-3F are different schematics depicting the same beads and steps involved in this exemplary bead deconvolution method. Two different beads, as described in FIG. 3A, are shown present in a single partition (FIG. 3E). The first bead is shown at the left and includes a barcoding oligonucleotide with a first bead-specific barcode sequence (shown as BC1), and the second bead is shown on the right and includes a barcoding oligonucleotide with a second bead-specific barcode sequence (shown as BC2). The target capture sequence is shown as “Cell NA capture/’ and the second target capture sequence, on the detection oligonucleotide at the top of the figure, is shown as “cDNA capture.” The second and third panels of FIG. 3E depict the binding of the first capture sequence of the first bead to the cellular RNA and the formation of the cDNA. The bottom panel of FIG. 3E depicts the binding of the detection oligonucleotide to the cDNA formed in this method. FIG. 3F depicts the strand synthesis, strand separation, and additional binding and synthesis steps. The bottom panel of FIG. 3F depicts the resulting hypothetical products from the method when the tw o different beads are present in a single partition.

[0045] FIGS. 4A-4E are schematic drawings of compositions and method steps for another embodiment of methods for detecting multiple barcodes in a partition. FIG. 4A depicts a bead comprising a barcoding oligonucleotide that includes a universal adapter sequence, an optional UMI, and a bead-specific barcode sequence (together shown as “Barcode A/UMI”). The barcoding oligonucleotide further includes a palindromic sequence (shown as “P” and backwards “P”)) and a target gene capture sequence. In the exemplified embodiment, the target gene capture sequence is a poly T sequence. [0046] FIGS. 4B-4C depict steps involved in an exemplary bead deconvolution method, using the beads as described in FIG. 4A. If two different bead-specific barcode sequences (e.g., Barcode A and Barcode B) or bead-specific barcode reverse complement sequences (e.g., Barcode A comp and Barcode B comp) are linked to the palindromic sequence in some of the double-stranded nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences or bead-specific barcode reverse complement sequences are from the same partition.

[0047] FIGS. 4D-4E are different schematics depicting the same beads and steps involved in this exemplary bead deconvolution method. Tw o different beads, as described in FIG. 4A, are shown present in a single partition (FIG. 4D). The first bead is shown at the left and includes a barcoding oligonucleotide with a first bead-specific barcode sequence (shown as BC1), and the second bead is shown on the right and includes a barcoding oligonucleotide with a second bead-specific barcode sequence (shown as BC2). The target capture sequence is shown as “Cell NA capture.” The steps of the exemplary method are depicted below the beads in FIG. 4D and continue in FIG. 4E. The bottom panel of FIG. 4E depicts exemplary resulting hypothetical products from the method when the two different beads are present in a single partition.

[0048] FIG. 5 is a graph showing the analysis of single cell behavior by a kneedie (cumulative fraction) knee plot.

[0049] FIGS. 6A-6E are graphs showing the expected versus observed lambda. The expected bead loading distributions were generated by a Poisson Distribution with a lambda of 1.9, comparing to the observed lambdas generated via barcode deconvolution for 1.000 cell loading (FIGS. 6A and 6B) and 10,000 cell loading (FIGS. 6C-6E). In each of FIGS. 6A-6E, the left bar represents the percentage of total droplets expected, and the right bar represents the percentage of droplets observed.

DEFINITIONS

[0050] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry' and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document.

[0051] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a.” “an.” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a bead” includes a plurality of such beads and reference to “the sequence” includes reference to one or more sequences known to those skilled in the art, and so forth.

[0052] An “oligonucleotide” is a polynucleotide. Generally, oligonucleotides will have fewer than 250 nucleotides, in some embodiments, between 4-200, e.g., 10-150 nucleotides.

[0053] A “primer” refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety’ of lengths and are often less than 50 nucleotides in length, for example 12-30 nucleotides, in length. The length and sequences of primers for use in PCR can be designed based on principles known to those of skill in the art, see, e.g., PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990). Primers can be DNA, RNA, or a chimera of DNA and RNA portions. In some cases, primers can include one or more modified or nonnatural nucleotide bases. In some cases, primers are labeled.

[0054] ‘ ‘Primer extension” refers to any method in which a primer is extended in a template-specific manner. Examples of primer extension include, for example, methods in which a primer hybridizes to a template nucleic acid and a polymerase extends the primer in a template-specific manner. In some embodiments, the method is referred to as “strand synthesis.” In some embodiments, the template is DNA, and the polymerase is a DNA polymerase. In some embodiments, the template is RNA, and the polymerase is a reverse transcriptase. As used herein, “reverse transcription” is a method that copies RNA into DNA. Primer extension can also include, for example, template switching (see, e.g., Zhu YY, Machleder EM et al. (2001) Biotechniques, 30(4):892-897; Ramskold D, Luo S. et al. (2012) Nat. Biotechnol. 30(8): 777-78, and nick polymerization (also referred to as nick translation), the latter involving nicking one strand of a nucleic acid duplex and using the nicked strand as a primer that is extended using the other strand as a template (see, e.g., Leonard G. Davis Ph.D., et al, in Basic Methods in Molecular Biology, 1986).

[0055] A nucleic acid, or a portion thereof, “hybridizes,” “binds,” or “anneals” to another nucleic acid under conditions such that non-specific hybridization is minimal at a defined temperature in a physiological buffer (e.g., pH 6-9, 25-150 mM chloride salt). In some cases, a nucleic acid, or portion thereof, hybridizes to a conserved sequence shared among a group of target nucleic acids. In some cases, a primer, or portion thereof, can hybridize to a primer binding site if there are at least about 6, 8, 10, 12, 14, 16, or 18 contiguous complementary nucleotides, including “universal” nucleotides that are complementary to more than one nucleotide partner. Alternatively, a primer, or portion thereof, can hybridize to a primer binding site if there are fewer than 1 or 2 complementarity mismatches over at least about 12, 14. 16, or 18 contiguous complementary nucleotides. In some embodiments, the defined temperature at which specific hybridization occurs is room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is higher than room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37. 40. 42. 45, 50, 55, 60, 65, 70, 75, or 80 °C. In some embodiments, the defined temperature at which specific hybridization occurs is 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80 °C.

[0056] A “template” refers to a polynucleotide sequence that comprises the polynucleotide to be copied or amplified, flanked by or a pair of primer hybridization sites. Thus, a “target template” comprises the target polynucleotide sequence adjacent to at least one hybridization site for a primer. In some cases, a “target template” comprises the target polynucleotide sequence flanked by a hybridization site for a “forward” primer and a “reverse” primer. In some embodiments, a “PCR priming sequence” refers to a site to which a forward primer or reverse primer binds for PCR amplification.

[0057] As used herein, “nucleic acid” means DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, points of attachment and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to. peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2’ -position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine. substitution of 5-bromo or 5-iodo-uracik backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like. Nucleic acids can also include non-natural bases, such as, for example, nitroindole. Modifications can also include 3' and 5' modifications including but not limited to capping with a fluorophore (e.g., quantum dot) or another moiety. As used herein, the terms ‘"reverse complement’⁷ or “reverse complementary sequence” of a particular nucleic acid refer to a sequence that has the complementary' nucleotide at all or substantially all positions of the nucleic acid and, therefore, specifically binds to the nucleic acid. As used herein, a “capture sequence” refers to a sequence that is the reverse complement of a sequence on a target nucleic acid, and therefore specifically binds the target nucleic acid.

[0058] A “polymerase” refers to an enzyme that performs template-directed synthesis of polynucleotides, e.g., DNA and/or RNA. The term encompasses both the full-length polypeptide and a domain that has polymerase activity'. DNA polymerases are well-known to those skilled in the art. including but not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. Additional examples of commercially available polymerase enzymes include, but are not limited to: Klenow fragment (New England Biolabs® Inc.), Taq DNA polymerase (QIAGEN), 9 °N™ DNA polymerase (New England Biolabs® Inc.), Deep Vent™ DNA polymerase (New England Biolabs® Inc.), Manta DNA polymerase (Enzymatics®), Bst DNA polymerase (New England Biolabs® Inc.), and phi29 DNA polymerase (New England Biolabs® Inc.).

[0059] Polymerases include both DNA-dependent polymerases and RNA-dependent polymerases such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A. B and C. Other types of DNA polymerases include phage polymerases. Similarly, RNA polymerases typically include eukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerases as well as phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

[0060] As used herein, the term “partitioning” or “partitioned” refers to separating a sample into a plurality of portions, or “partitions.” Partitions are generally physical, such that a sample in one partition does not, or does not substantially, mix with a sample in an adjacent partition. Partitions can be solid or fluid. In some embodiments, a partition is a solid partition, e.g., a microchannel. In some embodiments, a partition is a fluid partition, e.g, a droplet. In some embodiments, a fluid partition (e.g., a droplet) is a mixture of immiscible fluids (e.g., water and oil). In some embodiments, a fluid partition (e.g., a droplet) is an aqueous droplet that is surrounded by an immiscible carrier fluid (e.g., oil).

[0061] As used herein, “universal adapter sequence” refers to a short heterologous nucleotide sequence, linked to a set of diverse nucleic acids, which is not specific for one sample, cell, or partition. In some embodiments, a universal adapter sequence allows for use of a common (universal) primer to amplify and/or facilitate sequencing of the set. In some embodiments, the universal adapter sequence includes a sequencing handle. As used herein, a “sequencing handle” or a “sequencing adapter sequence” refers to a sequence that is used to amplify’ and/or facilitate sequencing of the nucleic acids. In some embodiments, the sequencing handle may be referred to as a PCR priming sequence (e.g., a sequence to which a PCR primer binds).

[0062] As used herein a “barcode” is a short nucleotide sequence (e.g. , at least about 4, 6, 8, 10, or 12, nucleotides long) that identifies a molecule to which it is conjugated. Barcodes can be used, e.g.. to identify molecules in a partition. Such a partition-specific barcode should be unique for that partition as compared to barcodes present in other partitions. For example, partitions containing target RNA from single-cells can be subjected to reverse transcription conditions using primers that contain a different partition-specific barcode sequence in each partition, thus incorporating a copy of a unique “cellular barcode” into the reverse transcribed nucleic acids of each partition. Thus, nucleic acid from each cell can be distinguished from nucleic acid of other cells due to the unique “cellular barcode.” In some cases, the cellular barcode is provided by a “bead-specific barcode” or “bead barcode” that is present on oligonucleotides conjugated to a bead, wherein the bead-specific barcode is shared by (e.g, identical or substantially identical amongst) all. or substantially all. of the oligonucleotides conjugated to that bead. Thus, cellular and bead-specific barcodes can be present in a partition, attached to a bead, or bound to cellular nucleic acid as multiple copies of the same barcode sequence. Cellular or bead-specific barcodes of the same sequence can be identified as deriving from the same cell, partition, or bead. Such partition-specific, cellular, or beadspecific barcodes can be generated using a variety of methods, which methods result in the barcode conjugated to or incorporated into a solid or hydrogel support (e.g., a solid bead or particle or hydrogel bead or particle). In some cases, the partition-specific, cellular, or bead- specific barcode is generated using a split and mix (also referred to as split and pool) synthetic scheme as described herein. A partition-specific barcode can be a cellular barcode and/or a bead-specific barcode. Similarly, a cellular barcode can be a partition specific barcode and/or a bead-specific barcode. Additionally, a bead-specific barcode can be a cellular barcode and/or a partition-specific barcode.

[0063] In some embodiments, barcodes uniquely identify the molecule to which it is conjugated and are referred to as a unique molecular identifier (UMI). The number of nucleotides of the UMI, which can be continuous, or discontinuous, will depend on the number of UMI sequences required. In some embodiments, the number of UMIs available are many times (e.g., 2X, 10X, 100X, etc.) higher than possible conjugation partners, thereby reducing the chance of rare duplicates being linked to different molecules. In some embodiments, pools of different UMIs are present in a partition and the composition of the pool acts as an identifier for the partition, with some UMIs being in common with some other partitions but the total pool of UMIs being unique or substantially unique between partitions. UMI sequences can be generated for example as random sequences of a set length, and in some embodiments is identified by a flanking known sequence.

[0064] The length of the barcode sequence determines how many unique samples can be differentiated. For example, a 1 nucleotide barcode can differentiate 4, or fewer, different samples or molecules; a 4 nucleotide barcode can differentiate 4⁴ or 256 samples or less; a 6 nucleotide barcode can differentiate 4096 different samples or less; and an 8 nucleotide barcode can index 65,536 different samples or less. Additionally, barcodes can be attached to both strands, for example, through barcoded primers for both first and second strand synthesis.

[0065] Barcodes are typically synthesized and/or polymerized (e.g., amplified) using processes that are inherently inexact. Thus, barcodes that are meant to be uniform (e.g., a cellular, bead-specific, or partition-specific barcode shared amongst all barcoded nucleic acid of a single partition, cell, or bead) can contain various N-l deletions or other mutations from the canonical barcode sequence. Thus, barcodes that are referred to as "identical" or “substantially identical” copies refer to barcodes that differ due to one or more errors in, e.g., synthesis, polymerization, or purification errors, and thus contain various N-l deletions or other mutations from the canonical barcode sequence. Moreover, the random conjugation of barcode nucleotides during synthesis using e.g., a split and pool approach and/or an equal mixture of nucleotide precursor molecules as described herein, can lead to low probability events in which a barcode is not absolutely unique (e.g., different from all other barcodes of a population or different from barcodes of a different partition, cell, or bead). However, such minor variations from theoretically ideal barcodes do not interfere with the high-throughput sequencing analysis methods, compositions, and kits described herein. Therefore, as used herein, the term “unique” in the context of a bead-specific, cellular, partition-specific, or molecular barcode encompasses various inadvertent N-l deletions and mutations from the ideal barcode sequence. In some cases, issues due to the inexact nature of barcode synthesis, polymerization, and/or amplification, are overcome by oversampling of possible barcode sequences as compared to the number of barcode sequences to be distinguished (e.g., at least about 2-, 5-, 10-fold or more possible barcode sequences). For example, 10,000 cells can be analyzed using a cellular barcode having 9 barcode nucleotides, representing 262,144 possible barcode sequences. The use of barcode technology is well know n in the art, see for example Katsuyuki Shiroguchi, et al., Proc. Natl. Acad. Sci. U S A., 2012 Jan

24;109(4): 1347-52; and Smith, AM et al., Nucleic Acids Research Can 11, (2010). Further methods and compositions for using barcode technology include those described in U.S. 2016/0060621.

[0066] The term “bead” refers to any solid support that can be in a partition, e.g., a small particle or other solid support. Exemplary beads can include hydrogel beads. In some cases, the hydrogel is in sol form. In some cases, the hydrogel is in gel form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, e.g., U.S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U.S. Patent Appl. Nos. 2002/0,009,591; 2013/0,022,569; 2013/0,034,592; and International Patent Publication Nos. WO/1997/030092; and WO/2001/049240.

[0067] The term “sample” refers to a biological composition, such as a cell, comprising a target nucleic acid.

[0068] The term “amplification reaction” refers to any in vitro means for multiplying the copies of a target sequence of nucleic acid in a linear or exponential manner. Such methods include but are not limited to polymerase chain reaction (PCR); DNA ligase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inms et al., eds.. 1990)) (LCR); QBeta RNA replicase and RNA transcriptionbased amplification reactions (e.g., amplification that involves T7, T3, or SP6 primed RNA polymerization), such as the transcription amplification system (TAS), nucleic acid sequence based amplification (NASBA), and self-sustained sequence replication (3SR); isothermal amplification reactions (e.g., single-primer isothermal amplification (SPIA)); as well as others known to those of skill in the art.

[0069] “Amplifying” refers to a step of submitting a solution to conditions sufficient to allow for amplification of a polynucleotide if all of the components of the reaction are intact. Components of an amplification reaction include, e.g., primers, a polynucleotide template, polymerase, nucleotides, and the like. The term “amplifying” typically refers to an “exponential” increase in target nucleic acid. However, “amplifying” as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid, such as is obtained with cycle sequencing or linear amplification. In an exemplary’ embodiment, amplify ing refers to PCR amplification using a first and a second amplification primer. In some embodiments, an amplification primer introduces a heterologous sequence to the amplification product. In an exemplary’ embodiment, the amplification primer introduces a universal adapter sequence to the amplification product to facilitate sequencing of the amplification product.

[0070] “Polymerase chain reaction” or “PCR” refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well knoyvn to those of skill in the art (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990). Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization/elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

[0071] The term “deconvolution” refers to the assignment of two barcodes and the beads they were attached to as being from the same partition or originally occupying the same partition. Deconvolution can be determined by the detection of the two barcodes on a single nucleic acid fragment during sequencing.

[0072] The term “about” refers to the usual error range for the respective value that is known by a person of ordinary skill in the art for this technical field, for example, a range of ± 10%. ± 5%, or ± 1% can encompass the recited value, even if the recited value is not modified by the term “about.” [0073] It will be understood that any range of numerical values disclosed herein can include the endpoints of the range, and any values or sub-ranges in between the endpoints. For example, a range of 1 to 10 includes a range from 2 to 9, 3 to 8, 4 to 7, 5 to 6, 1 to 5, 2 to 5, 2 to 10, 3 to 10, and so on. The values typically include one significant digit.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

[0074] The disclosed methods and compositions provide a solution to problems associated with more than one barcoded bead being present in a single partition. In some embodiments, the disclosed methods involve use of beads having a plurality of copies of barcoding oligonucleotides including the same bead-specific barcode as w ell as sequences for the capture of nucleic acids from a cell or sequences for the capture of other beads present in a partition in various ways. Following the labeling of nucleic acids in a library preparation method with the beads of these embodiments, linkage of two different bead-specific barcode sequences or reverse complement sequences on the nucleic acid products can be used to determine whether and which bead-specific barcodes originated from the same partition. In other embodiments, the disclosed methods involve the use of beads having a single barcoding oligonucleotide in combination with a detection oligonucleotide having a UMI. Following the labeling of nucleic acids in a library preparation method with the beads of these embodiments, linkage of two different bead-specific barcode sequences or reverse complement sequences to a UMI on the nucleic acid products can be used to determine whether and which bead-specific barcodes originated from the same partition. In yet other embodiments, the disclosed methods involve the use of a single barcoding oligonucleotide that includes a palindromic sequence that facilitates the detection of multiple barcodes in a partition. Following the labeling of nucleic acids in a I i bran- preparation method with the beads of these embodiments, linkage of tw o different bead-specific barcode sequences or reverse complement sequences with the palindromic sequence on the nucleic acid products can be used to determine whether and which bead-specific barcodes originated from the same partition.

[0075] In each of the disclosed methods, linkage of two different bead-specific barcodes or reverse complement sequences on a single nucleic acid (e.g., linked to a sample nucleic acid or to a palindromic sequence or to a UMI) can be detected using sequencing to determine whether and which bead-specific barcodes originated in the same partition. This determination is important because the presence of more than one bead-specific barcode in a partition (e.g.. two different bead-specific-barcoded oligonucleotides linked to different beads in the same partition) can interfere with sequence analysis and quantification because different barcodes are assumed to be from different partitions when in fact some fraction of the barcodes occur together. The disclosed methods enable deconvolution (i.e.. determination that multiple bead barcodes are from the same partition and accounting for that in the sequencing analysis) and appropriate use of the data from such combinations or disregarding (e.g., discarding) data from such partitions, leaving the remaining data with reduced background.

A. Methods and Compositions for Detecting Multiple Barcodes in a Partition Using Beads with Three Bead-Specific Barcoding Oligonucleotides

[0076] In some embodiments, the disclosure provides methods of detecting multiple barcodes in a partition using beads having three bead-specific barcoding oligonucleotides with different functions (i.e., one for target capture, two for bead capture). This approach also may be referred to as a mirror oligo approach because the two bead capture sequences mirror or are reverse complementary to one another. The use of these beads to barcode nucleic acids from cells in a partition produces some double stranded nucleic acids having a bead capture sequence flanked by two bead-specific barcode sequences or bead-specific barcode reverse complement sequences. If two different beads (each having a different bead-specific barcode sequence) are present in a partition, at least some of the double stranded nucleic acids will include two different bead-specific barcode sequences or bead-specific barcode reverse complement sequences (FIG. 1C and bottom panel of FIG. ID).

[0077] These methods include a plurality of partitions (e.g., droplets in an emulsion or microwells). In some embodiments, the different partitions include different beads (e.g., hydrogel beads) linked to a plurality of capture oligonucleotides and first and second deconvolution oligonucleotides, each including the same bead-specific barcode sequence (see, e.g., FIGS. 1A and ID); a DNA polymerase; a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; and optionally, a reverse transcriptase.

[0078] In some embodiments, the cell or nucleus is obtained from a biological sample. Biological samples can be obtained from any biological organism, e.g., an animal, plant, fungus, pathogen (e.g., bacteria or virus), or any other organism. In some embodiments, the biological sample is from an animal, e.g., a mammal (e.g., a human or a non-human primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g, chicken), or a fish. A biological sample can be any tissue or bodily fluid obtained from the biological organism, e.g., blood, a blood fraction, or a blood product (e.g., serum, plasma, platelets, red blood cells, and the like), sputum or saliva, tissue (e.g., kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue); cultured cells, e.g, primary cultures, explants, and transformed cells, stem cells, stool, urine, etc. In some embodiments, the sample is a single-cell sample. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cell is a mammalian cell.

[0079] In some embodiments, the capture oligonucleotide includes 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence. In some embodiments, the first deconvolution oligonucleotide includes 5 ’-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a first bead capture sequence. In some embodiments, the second deconvolution oligonucleotide includes 5^?-3’: a third PCR priming sequence, the bead-specific barcode sequence, and a second bead capture sequence that is reverse complementary to the first bead capture sequence; and wherein the bead has more copies of the first barcoding oligonucleotide than copies of the second and third barcoding oligonucleotides. In some embodiments, two or more of the first, second, and third PCR priming sequences may be the same. In other embodiments, the first, second, and third PCR priming sequences are different.

[0080] Universal adapter sequences are sequences that are used to end-label nucleic acids from cells or nuclei in a partition in each of two or more partitions for use, for example, in sequencing applications known in the art. In some embodiments, the universal adapter sequence may include a PCR priming or sequencing adapter sequence when the adapter sequence facilitates sequencing methods. In some embodiments, the cDNAs from two or more partitions are end-labelled for use with Illumina, Ion Torrent, Element Biosciences, or BGI sequencing technology. Any known adapter sequences may be suitable for use as the universal adapter sequence in the disclosed methods. For example, in some embodiments, the universal adapters may comprise a P5 adapter sequence (5’ AAT GAT ACT GCG ACC GA 3’ (SEQ ID NO: 4)), a P7 adapter sequence (5’ CAA GCA GAA GAC GGC ATA CGA GAT 3’ (SEQ ID NO: 5)) (Illumina), an Ion Torrent Pl adapter sequence (5’ CCA CTA CGC CTC CGC TTT CCT CTC TAT GGG CAG TCG GTG AT 3’ (SEQ ID NO: 6)). an Ion Torrent A adapter sequence (5’ CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG 3’ (SEQ ID NO: 7)), an Element Adept surface primer sequence, or a BGI adapter sequence. In some embodiments, the primers used in the amplification reaction include an index sequence, thereby forming double stranded sequencing cDNAs comprising the index sequence.

[0081] In some embodiments of the disclosed methods, the capture oligonucleotide binds to a DNA from the cell or nucleus in the partition. In other embodiments, the capture oligonucleotide binds to an RNA from the cell or nucleus in the partition.

[0082] In some embodiments, the capture oligonucleotide includes a capture sequence that comprises a poly T sequence (also referred to as an oligo dT sequence) for the capture of mRNA sequences from a cell or nucleus in the partition. This capture oligonucleotide also may be referred to as an mRNA capture oligonucleotide as it may bind to the poly A tail sequence of mRNAs. In some embodiments, the poly T is a single stranded sequence of deoxythymine (dT). The length of the poly T sequence can vary, for example, from about 6 bases to about 40 bases, or any number within that range, and may be a mixture of lengths. For example, in some embodiments, the capture sequence comprises a poly T sequence of about 6, 10, 15, 20, 25. 30. 35. or 40 Ts. In certain embodiments, the capture sequence comprises 22-36 Ts. In other embodiments of the disclosed methods, the capture oligonucleotide includes a capture sequence that is a DNA sequence that is reverse complementary to a portion of the target RNA (e.g.. the capture sequence may comprise a portion of a target gene sequence to capture a corresponding target mRNA). In some embodiments of the disclosed methods, the binding site for the capture sequence is between about 50 and 600 nucleotides downstream of the 3 ’ end of the RNA. Therefore, the capture sequence may bind to a sequence that is located between about 50, 100, 150, 200, 250, 300, 350, 400. 450, 500, 550, or 600 nucleotides downstream of the 3‘ end of the RNA, or any length in between. In certain embodiments, the capture sequence binds to a sequence that is 180 to 220 nucleotides downstream of the 3’ end of the RNA.

[0083] In some embodiments of these methods, the step of providing a plurality of partitions includes providing intact cells in partitions and subsequently lysing the cells in the partitions. The cells may be lysed using any method known in the art. For example, in certain embodiments, the cells are lysed with a buffer containing a detergent. In other embodiments. the step of providing a plurality' of partitions includes providing partitions including fixed cells.

[0084] In some embodiments, the methods include performing several steps of the methods within the partitions. In other embodiments, only nucleic acid capture (annealing sequences of cellular nucleic acids to some copies of the capture oligonucleotides) and bead capture (annealing of some bead capture sequences to other bead capture sequences) are performed within the partitions. In some embodiments, the steps performed in the partitions include releasing the barcoding oligonucleotides from the beads; performing strand synthesis by annealing sequences of cellular nucleic acids from the single cell or nucleus to some copies of the capture oligonucleotides and extending the capture oligonucleotides with the polymerase using the cellular nucleic acids as a template; and annealing some copies of the first bead capture sequence of the first deconvolution oligonucleotide to some copies of the second bead capture sequence of the second deconvolution oligonucleotide (see, e.g., FIGS. IB and ID), extending the first deconvolution oligonucleotide with the DNA polymerase using the second deconvolution oligonucleotide as a template, and extending the second deconvolution oligonucleotide with the DNA polymerase using the first deconvolution oligonucleotide as a template, thereby generating double-stranded nucleic acids, the first strand including the PCR priming sequence, the first bead-specific barcode sequence, the bead capture sequence, the bead-specific barcode reverse complement sequence, and the second PCR priming reverse complement sequence (see FIGS. 1C and ID).

[0085] The surface of the beads can be modified to include a linker for attaching barcoding oligonucleotides (e.g., capture oligonucleotides or deconvolution oligonucleotides). The linkers may comprise a cleavable moiety, which may be cleaved in the partitions. Nonlimiting examples of cleavable moieties include a disulfide bond, a dioxyuridine moiety⁷, and a restriction enzyme recognition site. The cleavable sequence can be any cleavable sequence that can be targeted enzymatically or otherw ise while leaving the rest of the nucleic sequences in the mixture intact. In some embodiments, the cleavable sequence comprises one or more uracils. For example, the cleavable sequence can include 1, 2, 3, 4, or more uracils, which can be contiguous. Uracils can be selectively removed, and the backbone cleaved (nicked), by contacting with uracil DNA glycosylase and endonuclease VIII. which excises the one or more uracil. Uracil DNA glycosylase and endonuclease VIII is available commercially, for example from New England Biolabs as “USER™’’ (Uracil-Specific Excision Reagent). In some embodiments, the cleavable sequence comprises one or more ribonucleotide(s). For example, the cleavable sequence can include 1, 2, 3, 4 or more ribonucleotides, which can be contiguous. This allows one to use an enzyme that selectively cleaves ribonucleotides and does not substantially cleave deoxyribonucleotides. For example, in some embodiments, RNase H is used to specifically cleave at a ribonucleotide in the cleavable sequence. In some embodiments, the cleavable sequence comprises a restriction enzyme recognition or cleavage site (collectively referred to as a “restriction site”) located between the first oligonucleotide and the second oligonucleotide. In these embodiments, the long oligonucleotide can be cleaved with a restriction enzyme that cleaves the restriction site on the long oligonucleotide without cleaving the linking oligonucleotide. Examples of such enzymes nicking endonuclease. Preferably, the restriction enzyme is selected such that its recognition and/or cleavage site only occurs in the cleavable sequence and does not occur elsewhere in the oligonucleotides in the mixture. In some other embodiments, the beadspecific barcoding oligonucleotides are released by dissolving the bead.

[0086] In some embodiments of these methods, sequences of cellular mRNAs from the single cell or nucleus bind to some copies of the capture oligonucleotide and form first strand gene-specific cDNAs by extending the capture oligonucleotides with a reverse transcriptase using the cellular mRNAs as a template. In these embodiments, second strand synthesis is performed using the DNA polymerase to form second strand cDNAs using first strand genespecific cDNAs as templates and the cellular mRNAs as primers, thereby forming doublestranded gene-specific cDNAs from a plurality of different RNAs.

[0087] Reverse transcription can be performed using any suitable reverse transcriptases, such as, but not limited to Maxima RNase⁺ (Thermo), Maxima RNase" (Thermo), murine leukemia virus (MLV) reverse transcriptase (Gerard and Grandgenett, Journal of Virology 15:785-797, 1975; Verma, Journal of Virolog’ 15:843-854, 1975), feline leukemia virus (FLV) reverse transcriptase (Rho and Gallo. Cancer Lett., 10:207-221, 1980, bovine leukemia virus (BLV) (Demirhan et al.. Anticancer Res., 16:2501-5, 1996; Drescher et al.. Arch Geschwulstforsch., 49:569-79, 1979), Avian Myeloblastosis Virus (AMV) reverse transcriptase, Respiratory⁷ Syncytial Virus (RSV) reverse transcriptase, Equine Infectious Anemia Virus (EIAV) reverse transcriptase, Rous-associated Virus-2 (RAV2) reverse transcriptase. SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT III reverse transcriptase (US8541219, US7056716, US7078208), THERMOSCRIPT reverse transcriptase, MMLV RNase H" reverse transcriptase, and Sensiscript (Qiagen). [0088] In some embodiments, the RNA in the RNA:DNA heteroduplex is degraded by an enzyme having RNase H⁺ activity. Ribonuclease H (RNase H or RNH) refers to a family of non-sequence-specific endonuclease enzymes that cleave RNA in an RNA/DNA heteroduplex. These enzymes cleave RNA backbone phosphodiester bonds to leave a 3’ hydroxyl and a 5’ phosphate group. In some embodiments of the disclosed methods, the reverse transcription and the RNA:DNA duplex degradation are performed by a single enzyme having RNase H⁺ activity. In some embodiments, the enzyme is Maxima Reverse Transcriptase (RNase H⁺⁾ (Thermo), M-MLV RT (Thermo), or iScript (Bio-Rad).

[0089] In some embodiments, after performing strand synthesis, the methods can include inactivating the polymerase and reverse transcriptase in the partitions. In certain embodiments, the inactivating comprises applying heat to the partitions and raising the temperature sufficiently high to inactivate the enzymes. In some embodiments, the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

[0090] In some embodiments, the methods include performing any remaining steps outside the individual partitions. In some embodiments, these steps include generating a bulk mixture by combining contents of the partitions; and performing an amplification (e.g., PCR) reaction wi th primers that bind to the PCR priming sequence or reverse complement sequence. In some embodiments, the amplification reaction is performed with primers that introduce heterologous sequences. In some embodiments, the primers used in the amplification reaction include an index sequence, thereby forming double stranded sequencing cDNAs comprising the index sequence. In some embodiments, the methods also include determining the nucleotide sequence of the double-stranded nucleic acids and double-stranded cDNAs and performing bead deconvolution. If two different bead-specific barcode sequences or reverse complement sequences are linked on some of the double-stranded nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences or reverse complement sequences are from the same partition.

[0091] Also provided are a plurality of partitions (e.g., droplets in emulsion or microwells). In some embodiments, the different partitions include at least one bead (e.g., a hydrogel bead) linked to a plurality' of copies of a capture oligonucleotide, a first deconvolution oligonucleotide, and a second deconvolution oligonucleotide, each including the same beadspecific barcode sequence, and wherein the bead comprises more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides; a polymerase; a single fixed, lysed, or permeabilized cell (e.g., a mammalian cell), single nucleus; or nucleic acids from a single cell or nucleus; and optionally, a reverse transcriptase. In some embodiments, the capture oligonucleotide includes 5’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence; the first deconvolution oligonucleotide includes 5’-3’: a second PCR priming sequence, the beadspecific barcode sequence, and a first bead capture sequence; and the second deconvolution oligonucleotide includes 5^?-3’: a third PCR priming sequence, the bead-specific barcode sequence, and a second bead capture sequence that is reverse complementary to the first bead capture sequence. Also provided are the beads as described in this paragraph.

B. Methods and Compositions for Detecting Multiple Barcodes in a Partition Using Beads with Two Bead-Specific Barcoding Oligonucleotides

[0092] Also provided are methods of detecting multiple barcodes in a partition using beads having two bead-specific barcoding oligonucleotides with different target capture sequences. The use of these beads to barcode cDNAs from cells in a partition produces nucleic acids having a portion of a target gene sequence flanked by a bead-specific barcode sequence and a bead-specific reverse complement sequence. If two different beads (each having a different bead-specific barcode sequence) are present in a partition, at least some of the nucleic acids will include two different bead-specific barcode or reverse complement sequences (middle and bottom panel of FIG. 2C; bottom panel of FIG. 2D). This approach also may be referred to as a cellular content bridged mirror approach because in the resulting nucleic acids, the target gene sequence (or cellular content) may bridge two different bead-specific barcode or reverse complement sequences if two different beads are present in the partition. The description of various components and steps described for the methods in Section A. above (e.g., partitions, beads, cells, universal adapter sequences, PCR priming sequences, target capture sequences, reverse transcription, RNA degradation, inactivation of polymerase and reverse transcriptase) also applies to the methods described in this section unless otherwise noted in this section.

[0093] In some embodiments of the disclosed methods, the bead is linked to a plurality of copies of a first barcoding oligonucleotide and a plurality of copies of a second barcoding oligonucleotide (see. e.g., FIGS. 2A and 2D). The beads include more copies of the first barcoding oligonucleotide than copies of the second barcoding oligonucleotide. In some embodiments, the first barcoding oligonucleotide includes 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target capture sequence. In some embodiments, the first barcoding oligonucleotide may be referred to as an RNA or cell nucleic acid capture oligonucleotide as the first target capture sequence binds to RNAs from the cell or nucleus in the partition. In some embodiments, the first target capture sequence is a poly T sequence that binds to a poly A tail sequence of a cellular mRNA. In other embodiments, the first barcoding oligonucleotide or RNA or cell nucleic acid capture oligonucleotide includes a first capture sequence that binds to a portion of a target RNA other than a poly A sequence.

[0094] In some embodiments, the second barcoding oligonucleotide includes 5’-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a second target capture sequence. The second barcoding oligonucleotide also may be referred to as a cDNA capture oligonucleotide as the second target capture sequence comprises a sequence that binds to a cDNA of the cellular RNA. In some embodiments, the second target capture sequence binds to the target gene cDNA about 50-600 bp away from the first target capture sequence. Therefore, the second target capture sequence may bind to a sequence that is located between about 50, 60, 70, 80, 90, 100. 150, 200, 250, 300, 350. 400, 450. 500, 550, or 600, nucleotides upstream of the first capture sequence, or any length in between. In some embodiments, the second target capture sequence binds to a sequence that is 100 to 400 nucleotides base pairs away from the first capture sequence. In some embodiments, the second target capture sequence binds to a sequence that is 180 to 220 nucleotides base pairs away from the first capture sequence.

[0095] In some embodiments, the methods include performing several steps of the library preparation within the partitions (see, e.g., FIGS. 2B-2D). In other embodiments, only RNA capture (annealing sequences of cellular RNA to some copies of the first barcoding oligonucleotides) is performed within the partitions (top panel of FIGS. 2B and 2D). In some embodiments, the steps performed in the partitions include releasing the barcoding oligonucleotides from the beads; performing reverse transcription by annealing sequences of cellular RNAs from the single cell or nucleus to some copies of the first barcoding oligonucleotides and forming first strand cDNAs by extending the first barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template; RNA degradation (top and middle panels of FIG. 2B; top two panels of FIG. 2D); annealing some copies of the second target capture sequence of the second barcoding oligonucleotide to the first strand cDNAs (top panel of FIG. 2C; fourth panel of FIG. 2D), extending the second barcoding oligonucleotide with the DNA polymerase using the first strand gene-specific cDNA as a template (middle panel of FIG. 2C; bottom panel of FIG. 2D), thereby generating nucleic acids comprising 5 ’-3’: the second PCR priming sequence, the bead-specific barcode sequence, a portion of the target gene sequence, the reverse complement of the first target capture sequence, the reverse complement of the bead-specific barcode sequence, and the reverse complement of the first PCR priming sequence (see bottom two panels of FIG. 2C; bottom panel of FIG. 2D).

[0096] In some embodiments, the methods include performing the remaining steps outside the individual partitions. These steps include generating a bulk mixture by combining contents of the partitions; and performing an amplification (e.g., a PCR reaction) with primers that bind to the first or second PCR priming sequence or reverse complement sequence. In some embodiments, the amplification reaction is performed with primers that introduce heterologous sequences. In some embodiments, the primers used in the amplification reaction include an index sequence, thereby forming double stranded sequencing cDNAs comprising the index sequence. In some embodiments, the methods include determining the nucleotide sequence of the nucleic acids and performing bead deconvolution. If two different bead-specific barcode sequences or bead-specific reverse complement sequences are linked to the portion of the target gene sequence in some of the nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences are from the same partition.

[0097] Also provided are pluralities of partitions (e.g., droplets in an emulsion or microwells) for use in this cellular content bridged mirror approach for bead deconvolution. In some embodiments, the different partitions include different beads, each bead (e.g., a hydrogel bead) linked to a plurality of copies of a first barcoding oligonucleotide and a plurality of copies of a second barcoding oligonucleotide, wherein the bead comprises more copies of the first barcoding oligonucleotide than copies of the second barcoding oligonucleotide; a reverse transcriptase; and a single fixed, lysed, or permeabilized cell or a single nucleus or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads (i.e., the first bead linked to first and second barcoding oligonucleotides having a first bead-specific barcode sequence, and the second bead linked to first and second barcoding oligonucleotides having a second beadspecific barcode sequence). In some embodiments, the first barcoding oligonucleotide includes 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target capture sequence; and the second barcoding oligonucleotide comprises 5’-3‘: a second PCR priming sequence, the bead-specific barcode sequence, and a second target capture sequence. Also provided are the beads as described in this paragraph.

C. Methods and Compositions for Deconvolution Using a Single Barcoding Oligonucleotide and a Detection Oligonucleotide

[0098] The disclosure also provides methods of detecting multiple barcodes in a partition using a bead with a single barcoding oligonucleotide in combination with a detection oligonucleotide having a unique molecule identifier (UMI) (FIGS. 3A and 3E). The use of these beads to barcode cDNAs from cells in a partition produces nucleic acids having a portion of a target gene sequence flanked by the UMI from the detection oligonucleotide and either a bead-specific barcode sequence or a bead-specific reverse complement sequence. In some embodiments, this approach may be referred to as a housekeeping gene secondary UMI merging approach, where the detection oligonucleotide includes a capture sequence for a housekeeping gene (e.g., GAPDH). If two different beads (each having a different beadspecific barcode sequence) are present in a partition, at least some of the nucleic acids will include two different bead-specific barcode or reverse complement sequences (bottom panel of FIGS. 3D and 3F). The description of various components and steps described for the methods in Sections A.-B. above (e.g., partitions, beads, cells, universal adapter sequences, PCR priming sequences, target capture sequences, reverse transcription, RNA degradation, inactivation of polymerase and reverse transcriptase) also applies to the methods described in this section unless otherwise noted in this section.

[0099] In some embodiments of the disclosed methods, the bead is linked to a plurality of copies of a barcoding oligonucleotide that includes 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence. In some embodiments, the barcoding oligonucleotide may be referred to as an RNA or cell nucleic acid capture oligonucleotide as the first target capture sequence binds to RNAs from the cell or nucleus in the partition. In some embodiments, the first target capture sequence is a poly T sequence that binds to a poly A tail sequence of a cellular mRNA. In other embodiments, the barcoding oligonucleotide or RNA or cell nucleic acid capture oligonucleotide includes a first capture sequence that binds to a portion of a target RNA other than a poly A sequence.

[0100] In some embodiments, the detection oligonucleotide includes 5 ’-3’: a second PCR priming sequence, a unique molecular identifier (UMI), and a second target gene capture sequence. The detection oligonucleotide also may be referred to as a cDNA capture oligonucleotide as the second target capture sequence comprises a sequence that binds to a cDNA of the cellular RNA. The detection oligonucleotide also may be referred to as a genespecific primer. In some embodiments, the second target capture sequence binds to the target gene cDNA, about 50-600 bp away from the first target capture sequence. Therefore, the second target capture sequence may bind to a sequence that is located between about 50, 60, 70. 80. 90, 100, 150. 200, 250. 300, 350, 400. 450, 500. 550, or 600, nucleotides upstream of the first capture sequence, or any length in between. In some embodiments, the second target capture sequence binds to a sequence that is 100 to 400 nucleotides base pairs away from the first capture sequence. In some embodiments, the second target capture sequence binds to a sequence that is 180 to 220 nucleotides base pairs away from the first capture sequence. In some embodiments, the target gene may be a ‘'housekeeping” gene (e.g., a gene required for basic and ubiquitous cellular functions). For example, in certain embodiments, the housekeeping gene is glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

[0101] In some embodiments, the methods include performing several steps of the librarypreparation within the partitions (see, e.g.. FIGS. 3B-3F). In other embodiments, only RNA capture (annealing sequences of cellular RNA to some copies of the first barcoding oligonucleotides) is performed within the partitions (top panel of FIGS. 3B and 3E). In some embodiments, the steps performed in the partitions include releasing the barcoding oligonucleotides from the beads: performing reverse transcription by annealing sequences of cellular RNAs from the single cell or nucleus to some copies of the barcoding oligonucleotides and forming first strand cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template (top and middle panels of FIG. 3B and 3E); RNA degradation (middle and bottom panels of FIG. 3B); annealing some copies of the second target capture sequence of the detection oligonucleotide to the first strand cDNAs (top panel of FIG. 3C: bottom panel of FIG. 3E), extending the detection oligonucleotide with the DNA polymerase using the first strand cDNA as a template (second panel of FIG. 3C; top panel of FIG. 3F); separating the two strands (bottom panel of FIG. 3C; second panel of FIG. 3F); and annealing some copies of the barcoding oligonucleotide to some copies of the second strand cDNA (top panel of FIG. 3D; third panel of FIG. 3F), extending the barcoding oligonucleotide using the second strand cDNA as a template, thereby generating double-stranded nucleic acids comprising the first PCR priming sequence, the bead-specific barcode sequence, the portion of the target gene sequence, the UMI, and the second PCR primer reverse complement sequence (bottom panels of FIGS. 3D and 3F). Two different barcoding oligonucleotides and their reverse complement sequences present in the second strand nucleic acids produced in the previous step of this method may bind to one another at some percentage because most of the barcoding oligonucleotide, other than the bead-specific barcode sequence portion, is complementary.

[0102] In some embodiments, the methods include performing the remaining steps outside the individual partitions. These steps include generating a bulk mixture by combining contents of the partitions; and performing an amplification (e.g., a PCR reaction) with primers that bind to the first or second PCR priming sequence or reverse complement sequence. In some embodiments, the amplification reaction is performed with primers that introduce heterologous sequences. In some embodiments, the primers used in the amplification reaction include an index sequence, thereby forming double stranded sequencing cDNAs comprising the index sequence. In some embodiments, the methods include determining the nucleotide sequence of the nucleic acids and performing bead deconvolution. If two different bead-specific barcode sequences or bead-specific reverse complement sequences are linked to the UMI in the nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences are from the same partition.

[0103] Also provided are pluralities of partitions (e.g., droplets in an emulsion or microwells) for use in this approach for bead deconvolution. In some embodiments, the different partitions include different beads, each bead (e.g., a hydrogel bead) linked to a plurality of copies of a barcoding oligonucleotide, comprising 5’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence; a detection oligonucleotide comprising 5 ’-3’: a second PCR priming sequence, a unique molecular identifier (UMI), and a second target gene capture sequence; a reverse transcriptase; a DNA polymerase; and a single fixed, lysed, or permeabilized cell or a single nucleus or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads (i.e., the first bead linked to a first barcoding oligonucleotide having a first bead-specific barcode sequence, and the second bead linked to a second barcoding oligonucleotide having a second bead-specific barcode sequence). Also provided are the beads as described in this paragraph. D. Methods and Compositions for Deconvolution Using Barcoding Oligonucleotides with Palindromic Sequences

[0104] The disclosure also provides methods of detecting multiple barcodes in a partition using a bead with a single barcoding oligonucleotide having a palindromic sequence at its 5 ’ end (FIGS. 4A and 4D). The use of these beads to barcode cDNAs from cells in a partition results in some nucleic acids having a palindromic sequence flanked on each side by at least a PCR priming sequence or PCR priming reverse complement sequence, and a bead-specific barcode sequence or a bead-specific reverse complement sequence (bottom panels of FIGS. 4C and 4E). In some embodiments, the resulting nucleic acids include the palindromic sequence flanked by a PCR priming sequence or PCR priming reverse complement sequence, a bead-specific barcode sequence or a bead-specific reverse complement sequence, and a portion of a target gene sequence or target gene reverse complement sequence (bottom panel of FIG. 4E) If two different beads (each having a different bead-specific barcode sequence) are present in a partition, at least some of the nucleic acids will include two different beadspecific barcode or reverse complement sequences (bottom panel of FIGS. 4C and 4E). The description of various components and steps described for the methods in Sections A.-C. above (e.g., partitions, beads, cells, universal adapter sequences, PCR priming sequences, target capture sequences, reverse transcription, RNA degradation, inactivation of polymerase and reverse transcriptase) also applies to the methods described in this section unless otherwise noted in this section.

[0105] In some embodiments of the disclosed methods, the bead is linked to a plurality of copies of a barcoding oligonucleotide that includes 5 ’-3’: a palindromic sequence, a PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence. In some embodiments, the barcoding oligonucleotide may be referred to as an RNA or cell nucleic acid capture oligonucleotide as the first target capture sequence binds to RNAs from the cell or nucleus in the partition. In some embodiments, the first target capture sequence is a poly T sequence that binds to a poly A tail sequence of a cellular mRNA. In other embodiments, the barcoding oligonucleotide or RNA or cell nucleic acid capture oligonucleotide includes a first capture sequence that binds to a portion of a target RNA other than a poly A sequence.

[0106] In some embodiments, the partitions also include a detection oligonucleotide. In some embodiments, the detection oligonucleotide includes a second target gene capture sequence. The detection oligonucleotide also may be referred to as a cDNA capture oligonucleotide as the second target capture sequence comprises a sequence that binds to a cDNA of the cellular RNA (FIG. 4C). In some embodiments, the second target capture sequence binds to the target gene cDNA, about 50-600 bp away from the first target capture sequence. Therefore, the second target capture sequence may bind to a sequence that is located between about 50, 60, 70, 80, 90, 100. 150, 200, 250, 300, 350, 400, 450. 500, 550, or 600, nucleotides upstream of the first capture sequence, or any length in between. In some embodiments, the second target capture sequence binds to a sequence that is 100 to 400 nucleotides base pairs away from the first capture sequence. In some embodiments, the second target capture sequence binds to a sequence that is 180 to 220 nucleotides base pairs away from the first capture sequence.

[0107] In some embodiments, the methods include performing several steps of the library preparation within the partitions (see, e g., FIGS. 4A-4E). In other embodiments, only RNA capture (annealing sequences of cellular RNA to some copies of the first barcoding oligonucleotides) is performed within the partitions (top panel of FIGS. 4B and 4D).

[0108] In some embodiments of the methods that include a detection oligonucleotide, the steps performed in the partitions include releasing the barcoding oligonucleotides from the beads; performing reverse transcription by annealing sequences of cellular RNAs from the single cell or nucleus to some copies of the barcoding oligonucleotides and forming first strand cDNAs (by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template (top and middle panels of FIG. 4B); second strand synthesis (i.e., synthesis of the complement of the barcoding oligonucleotide) using the barcoding oligonucleotide (or the barcoding oligonucleotide linked to the first strand cDNA) as a template and extending from the cellular RNAs, thereby producing double stranded molecules having an RNA:DNA heteroduplex, wherein the second strand comprises the palindromic sequence at its 3’ end as part of the complement of the barcoding oligonucleotide (middle panel of FIG. 4B); RNA degradation (middle and bottom panels of FIG. 4B); separating the two strands (i.e., each strand including the palindromic sequence (either on the 5’ end of the barcoding oligonucleotide which will not prime synthesis, or on the 3’ end of the complement of the barcoding oligonucleotide which is capable of priming synthesis), the PCR priming sequence or reverse complement sequence, and the bead-specific barcode sequence or reverse complement sequence) by annealing some copies of the second target capture sequence of the detection oligonucleotide to the first strand cDNAs (top panel of FIG. 4C) and extending the detection oligonucleotide with a strand displacing DNA polymerase using the first strand cDNA with barcoding oligonucleotide as a template (second panel of FIG. 4C); and performing additional strand synthesis with the second strand nucleic acids and the displaced strand nucleic acids as templates, wherein the palindromic sequences of some of the displaced strands (which are located on the 3 ' end and are complimentary to the original 5’ end palindromic sequence) anneal to the palindromic sequences of different displaced stands (also located on the 3’ end and complimentary to the original 5’ end palindromic sequence) (bottom panel of FIG. 4C), and wherein strand synthesis uses the bead-specific barcodes and PCR priming sequences as template and extends from the palindromic sequence, thereby producing double-stranded nucleic acids comprising 5 ’-3’: the PCR priming reverse complement sequence, the bead-specific barcode reverse complement sequence, the palindromic sequence, the bead-specific barcode sequence, and the PCR priming sequence.

[0109] In some embodiments that do not include a detection oligonucleotide, the steps performed in the partitions include releasing the barcoding oligonucleotides from the beads; performing reverse transcription by annealing sequences of cellular RNAs from the single cell or nucleus to some copies of the barcoding oligonucleotides (first panel of FIG. 4D) and forming first strand cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template (second panel of FIG. 4D); second strand synthesis (i.e., synthesis of the complement of the barcoding oligonucleotide) using the barcoding oligonucleotide (or the barcoding oligonucleotide linked to the first strand cDNA) as a template and extending from the cellular RNAs, thereby producing double stranded molecules having an RNA:DNA heteroduplex, wherein the second strand comprises the palindromic sequence at its 3’ end as part of the complement of the barcoding oligonucleotide (third panel of FIG. 4D); separating the two strands (e.g., strand separation by heating) (bottom panel of FIG. 4D); and performing additional strand synthesis, wherein the palindromic sequences of some of the second strand nucleic acids (which are located on the 3’ end of the second strand and are complimentary' to the original 5’ end palindromic sequence) anneal to the palindromic sequences of different second strand nucleic acids (which are located on the 3’ end of the different second strand and are complimentary to the original 5’ end palindromic sequence) (top panel of FIG. 4E), and wherein strand synthesis uses the complement of the barcoding oligonucleotides of the second strand nucleic acids as template and extends from the 3’ palindromic sequence, thereby producing double-stranded nucleic acids comprising 5’-3‘: the target gene reverse complement sequence, the beadspecific barcode reverse complement sequence, the PCR priming reverse complement sequence, the palindromic sequence, the PCR priming sequence, the bead-specific barcode sequence, and target gene sequence (bottom panel of FIG. 4E).

[0110] In some embodiments, the first strand cDNAs are separated from the second strand cDNAs by heating. In some embodiments, the defined temperature at which the strand separation occurs is at least about 60, 65, 70, 75, 80, 85, 90. 95. or 100 °C, or any temperature within this range. In some embodiments, the defined temperature at which strand separation occurs is 75-98 °C.

[OHl] In some embodiments, the methods include performing the remaining steps outside the individual partitions. These steps include generating a bulk mixture by combining contents of the partitions; and performing an amplification (e.g., a PCR reaction) wi th primers that bind to the first or second PCR priming sequence or reverse complement sequence. In some embodiments, the amplification reaction is performed with primers that introduce heterologous sequences. In some embodiments, the primers used in the amplification reaction include an index sequence, thereby forming double stranded sequencing cDNAs comprising the index sequence. In some embodiments, the methods include determining the nucleotide sequence of the nucleic acids and performing bead deconvolution. If two different bead-specific barcode sequences or bead-specific reverse complement sequences are linked to the palindromic sequence in some of the nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences are from the same partition.

[0112] Also provided are pluralities of partitions (e.g., droplets in an emulsion or microwells) for use in this palindromic sequence approach for bead deconvolution. In some embodiments, the different partitions include different beads, each bead (e.g., a hydrogel bead) linked to a plurality of copies of a barcoding oligonucleotide, comprising 5 ‘-3’: a palindromic sequence, a PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence; optionally, a detection oligonucleotide comprising a second target gene capture sequence; a reverse transcriptase; a DNA polymerase; and a single fixed, lysed, or permeabilized cell or a single nucleus or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a first bead-specific barcode sequence, and wherein the second bead is linked to a second bead-specific barcode sequence. Also provided are the beads as described in this paragraph.

Kits

[0113] In some embodiments, the disclosure provides kits for use in any of the disclosed methods for detecting more than one barcode in a partition. In certain embodiments, the kits include at least one bead having at least one bead-specific barcoding oligonucleotide; optionally, at least one detection oligonucleotide; a DNA polymerase; and optionally, a reverse transcriptase, for example as described above or elsewhere herein.

[0114] In certain embodiments, the kits include a DNA polymerase and at least one bead linked to a plurality of copies of a capture oligonucleotide, a plurality of copies of a first deconvolution oligonucleotide, and a plurality of copies of a second deconvolution oligonucleotide, wherein the bead has more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides. In these embodiments, the capture oligonucleotide includes 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence; the first deconvolution oligonucleotide includes 5’- 3’: a second PCR priming sequence, the bead-specific barcode sequence, and a first bead capture sequence; and the second deconvolution oligonucleotide includes 5 '-3’: a third PCR priming sequence, the bead-specific barcode sequence, and a second bead capture sequence that is reverse complementary to the first bead capture sequence.

[0115] In other embodiments, the kits include a reverse transcriptase; a DNA polymerase; and at least one bead linked to a plurality of copies of a first barcoding oligonucleotide and a plurality of copies of a second barcoding oligonucleotide, wherein the bead has more copies of the first barcoding oligonucleotide than copies of the second barcoding oligonucleotide. In these embodiments, the first barcoding oligonucleotide includes 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target capture sequence; and the second barcoding oligonucleotide includes 5’-3’: a second PCR priming sequence, the beadspecific barcode sequence, and a second target capture sequence.

[0116] In additional embodiments, the kits include a reverse transcriptase; a DNA polymerase; at least one bead linked to a plurality of copies of a barcoding oligonucleotide; and a detection oligonucleotide. In these embodiments, the barcoding oligonucleotide includes 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence. In these embodiments, the detection oligonucleotide includes 5’-3’: a second PCR priming sequence, a unique molecular identifier (UMI), and a second target gene capture sequence.

[0117] In other embodiments, the kits include a reverse transcriptase; a DNA polymerase; at least one bead linked to a plurality of copies of a barcoding oligonucleotide; and optionally, a detection oligonucleotide. In these embodiments, the barcoding oligonucleotide includes 5 ’-3’: a palindromic sequence, a PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence. When included in these kits, the detection oligonucleotide includes a second target gene capture sequence.

EXAMPLES

[0118] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1 - Materials and Methods

[0119] The droplet generating instrument (ddSEQ) and SureCell WTA library prep kit reagents, including hydrogel beads synthesized with deconvolution oligonucleotides (as described in this application), were acquired from Bio-Rad Laboratories. Human (HEK 293) and Mouse (NIH 3T3) cell lines were acquired from ATCC. Enzymes used were Maxima RNase H Minus Reverse Transcriptase (Thermo). ddTaq (Bio-Rad), and USER (NEB). The ddSEQ platform and droplet generating reagents, including cell suspension buffer, bead suspension buffer, and cell encapsulation oil were acquired from Bio-Rad. Magnetic bead cleanups were performed using SPRIselect beads (Beckman Coulter). Sequencing was performed on the NextSeq 2000 (Illumina) using manufacturer protocols and reagents.

[0120] Human and mouse cells were washed in PBS and counted using a TC20 Automated Cell Counter (Bio-Rad Laboratories). Cells were then added (1,000, 5,000 and 9,000 per reaction, human and mouse mixed) to the cell suspension buffer (Bio-Rad Laboratories) and loaded onto the ddSEQ chip. Beads were loaded at X = 2 (2 beads per total droplets generated) in bead suspension buffer onto the ddSEQ chip. Droplets were generated using the ddSEQ and incubated at 50 °C for 45 minutes (with 80 °C heat inactivation for 20 minutes) using a CFX96 Touch Deep Well thermocycler (Bio-Rad Laboratories). Droplets were then processed, and library preparation was completed according to manufacturer protocols. Libraries were sequenced using aNextSeq 2000 (Illumina) at -50,000 reads per cell and analyzed using a bioinformatics pipeline. Reads of 2 sets of barcodes linked on the same oligonucleotide were used to merge data from multiple beads to a same droplet. Merging efficacy was assessed via sequencing analysis, through single cell behavior of a knee plot and examining barcode-barcode dimers that had the deconvolution sequence between them.

Example 2 -Analysis of Single Cell Behavior

[0121] Single cell behavior was analyzed by a kneedie (cumulative fraction) knee plot (FIG. 5) after including deconvolution sequences to the bead oligo and merging droplets using unique barcode dimer connections. For example, a 9,000 cell input, 7,045 cells were detected, and the knee plot (FIG. 5) showed single cell behavior. Additionally, sequencing through the deconvolution sequence determined the sequence was correlated with the formation of at least 2.5 million sequenced barcode dimers across the theoretical -100,000 droplets produced by the ddSEQ instrument, an average of 25 connections per droplet.

[0122] Cross-talk between mouse and human cells was used to assess inappropriate merging behavior. More cross-talk after merging would suggest droplets are artificially being merged. The average measured cross-talk for merged (4.0%) was not higher and was comparable to unmerged (4.3%) across 10 samples. In addition, there is a slight but reproducible decrease in overall cross talk in the samples. This may suggest that droplets containing two cells and more than one bead are observed as multiple droplets when bead barcode connections are not used to merge droplets. However, after merging droplets with bead barcode connections, these droplets are properly resolved to one droplet with two cells.

Example 3 -Analysis of Merging in Barcode Deconvolution

Materials and Methods

[0123] Human peripheral blood mononuclear cells (PBMCs) were acquired from ATCC. Single cell libraries were generated and prepared for sequencing using the ddSEQ Single-Cell 3’ RNA-Seq Kit & Omnition Analysis Software (Bio-Rad Laboratories), following manufacturer’s protocols. Magnetic bead cleanups were performed using SPRIselect beads (Beckman Coulter). Gel electrophoresis was done on the TapeStation instrument (Agilent) using manufacturer protocols and reagents (High Sensitivity’ D5000 dsDNA kit). [0124] The beads in the kit contain the enclosed deconvolution oligo that forms a barcode dimer with or without the presence of cells (FIGS. 1A-1D). To analyze the deconvolution, 3 samples had a cell loading of 10,000 and 2 samples had a cell loading of 1,000. Beads were loaded at a lambda of 1.9 (average of 1.9 beads per droplet) for all samples. Edges were detected after sequencing as connections between two barcodes formed by the barcode dimer. Cells were called using a Kneedle/Cumulative Fraction algorithm as per the manufacturer's Omnition Analysis Software.

Results

[0125] For all samples, the unique edges that were formed by the barcode dimers were between 4 million and 4.8 million. Filtering those connections for those associated with called cells resulted in 48,280 and 50,507 edges for the two samples of 1.000 cell loading, and 498,668, 505,562, and 546,989 edges for 10,000 cell input samples. For all samples, the average UMIs associated with each edge was 25, indicating 25 unique barcode dimers (before any amplifications) support each of the edge connections, with a UMI threshold of 3 to be considered an edge. The average UMIs associated with within bead edges was 15, indicating the barcode dimers are preferentially being generated and preserved in the workflow between beads rather than within beads. To assess if beads were being appropriately merged using the deconvolution oligos, the distribution of beads per droplet was compared to a statistical model of the expected distribution if beads were loaded at a 1.9 lambda (FIGS. 6A-6B (1,000 cell loading) and FIGS. 6C-6E (10,000 cell loading)). A merge score was calculated, comparing the deviations between the observed and expected bead lambdas. Those scores ranged from 11.37% to 25.28%, indicating minimal deviation from the statically modeled distributions.

[0126] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity' of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein, including patents, patent applications, non-patent literature, and GenBank accession numbers, is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting multiple barcodes in a partition, the method comprising: providing a plurality of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality of copies of a capture oligonucleotide, a first deconvolution oligonucleotide, and a second deconvolution oligonucleotide, wherein the capture oligonucleotide comprises 5’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence; wherein the first deconvolution oligonucleotide comprises 5 ’-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a first bead capture sequence; wherein the second deconvolution oligonucleotide comprises 5 ’-3’: a third PCR priming sequence, the bead-specific barcode sequence, and a second bead capture sequence that is reverse complementary to the first bead capture sequence; and wherein the bead comprises more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides;

(ii) a DNA polymerase; and

(iii) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a capture oligonucleotide and first and second deconvolution oligonucleotides that comprise a first bead-specific barcode sequence, and wherein the second bead is linked to a capture oligonucleotide and first and second deconvolution oligonucleotides that comprise a second bead-specific barcode sequence; in the partitions, performing strand synthesis, wherein the performing comprises annealing sequences of cellular nucleic acids from the single cell or nucleus to some copies of the capture oligonucleotide and extending the capture oligonucleotides using the cellular nucleic acids as a template, wherein the strands synthesized comprise the first PCR priming sequence, the bead-specific barcode sequence, the capture sequence, and a target gene sequence; in the partitions, annealing some copies of the first bead capture sequence of the first deconvolution oligonucleotide to some copies of the second bead capture sequence of the second deconvolution oligonucleotide, extending the first deconvolution oligonucleotide with the DNA polymerase using the second deconvolution oligonucleotide as a template, and extending the second deconvolution oligonucleotide with the DNA polymerase using the first deconvolution oligonucleotide as a template, thereby generating double-stranded nucleic acids comprising the second PCR priming sequence, the beadspecific barcode sequence, the first bead capture sequence, the bead-specific barcode reverse complement sequence, and the third PCR priming reverse complement sequence; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the double-stranded nucleic acids. wherein if two different bead-specific barcode sequences or reverse complement sequences are linked on some of the double-stranded nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences or reverse complement sequences are from the same partition.

2. The method of claim 1, wherein the target capture sequence comprises a poly T sequence.

3. The method of claim 2, wherein performing strand synthesis comprises: annealing sequences of cellular mRNAs from the single cell or nucleus to some copies of the capture oligonucleotide and forming first strand gene-specific cDNAs by extending the capture oligonucleotides with a reverse transcriptase using the cellular mRNAs as a template; and in the partitions, performing second strand synthesis using the DNA polymerase to form second strand cDNAs using first strand gene-specific cDNAs as templates and the cellular mRNAs as primers, thereby forming double-stranded gene-specific cDNAs from a plurality of different RNAs.

4. The method of any one of claims 1-3, wherein the providing a plurality of partitions comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions.

5. The method of any one of claims 1-3. wherein the providing a plurality of partitions comprises providing partitions comprising fixed cells.

6. The method of any one of claims 1 -5. further comprising releasing the barcoding oligonucleotides from the beads.

7. The method of any one of claims 1-6, further comprising, after performing strand synthesis: inactivating the polymerase and reverse transcriptase, if present, in the partitions.

8. The method of claim 7, wherein the inactivating comprises applying heat to the partitions.

9. The method of claim 8, wherein the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

10. The method of any one of claims 1-9, wherein the partitions are droplets in an emulsion or microwells.

11. The method of any one of claims 1-10, wherein the cell is a mammalian cell.

12. The method of any one of claims 1-11, wherein the bead is a hydrogel bead.

13. A plurality of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality⁷ of copies of a capture oligonucleotide, a first deconvolution oligonucleotide, and a second deconvolution oligonucleotide, wherein the capture oligonucleotide comprises 5’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence; wherein the first deconvolution oligonucleotide comprises 5 ’-3’: a second PCR priming sequence, the bead-specific barcode sequence and a first bead capture sequence; wherein the second deconvolution oligonucleotide comprises 5 ’-3’: a third PCR priming sequence, the bead-specific barcode sequence, and a second bead capture sequence that is reverse complementary to the first bead capture sequence; and wherein the bead comprises more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides;

(ii) optionally, a reverse transcriptase;

(iii) a DNA polymerase; and

(iv) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus, wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a capture oligonucleotide and first and second deconvolution oligonucleotides comprising a first bead-specific barcode sequence, and wherein the second bead is linked to a capture oligonucleotide and first and second deconvolution oligonucleotides comprising a second bead-specific barcode sequence.

14. The plurality’ of partitions of claim 13, wherein the target capture sequence is a poly T sequence.

15. The plurality of partitions of any one of claims 13-14, wherein the partitions are droplets in an emulsion or microwells.

16. The plurality of partitions of any one of claims 13-15, wherein the cell is a mammalian cell.

17. The plurality of partitions of any one of claims 13-16, wherein the bead is a hydrogel bead.

18. A bead, comprising: a plurality of copies of a capture oligonucleotide, wherein the capture oligonucleotide comprises 5’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a target capture sequence; a plurality of copies of a first deconvolution oligonucleotide, wherein the first deconvolution oligonucleotide comprises 5’-3^?: a second PCR priming sequence, the beadspecific barcode sequence, and a first bead capture sequence; and a plurality of copies of a second deconvolution oligonucleotide, wherein the second deconvolution oligonucleotide comprises 5’-3‘: a third PCR priming sequence, the bead-specific barcode sequence and a second bead capture sequence that is reverse complementary to the first bead capture sequence; wherein the bead comprises more copies of the capture oligonucleotide than copies of the first and second deconvolution oligonucleotides.

19. The bead of claim 18, wherein the target capture sequence is a poly T sequence.

20. The bead of claim 18 or 19, wherein the bead is a hydrogel bead.

21. A method of detecting multiple barcodes in a partition, the method comprising: providing a plurality' of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality of copies of a first barcoding oligonucleotide and a second barcoding oligonucleotide, wherein the first barcoding oligonucleotide comprises 5’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target capture sequence; wherein the second barcoding oligonucleotide comprises 5^?-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a second target capture sequence; and wherein the bead comprises more copies of the first barcoding oligonucleotide than the second barcoding oligonucleotide;

(ii) a reverse transcriptase;

(iii) a DNA polymerase; and

(iv) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to first and second barcoding oligonucleotides comprising a first bead-specific barcode sequence, and wherein the second bead is linked to first and second barcoding oligonucleotides comprising a second bead-specific barcode sequence; in the partitions, performing reverse transcription, wherein the performing comprises annealing sequences of cellular RNAs from the single cell or nucleus to some copies of the first barcoding oligonucleotide and forming first strand gene-specific cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template, thereby producing a DNA:RNA heteroduplex sequence, wherein the first strand gene-specific cDNAs comprise the first PCR priming sequence, the bead-specific barcode sequence, the first target capture sequence, and the target gene sequence; in the partitions, degrading the RNA in the DNA:RNA heteroduplex; in the partitions, annealing some copies of the second target capture sequence of the second barcoding oligonucleotide to the first strand gene-specific cDNAs, extending the second barcoding oligonucleotide with the DNA polymerase using the first strand genespecific cDNA as a template, thereby generating nucleic acids comprising 5 ’-3’: the second PCR priming sequence, the bead-specific barcode sequence, a portion of the target gene sequence, the reverse complement of the first target capture sequence, the reverse complement of the bead-specific barcode sequence, and the reverse complement of the first PCR priming sequence; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the nucleic acids, wherein if two different bead-specific barcode sequences or reverse complement of the bead-specific barcode sequences are linked to the portion of the target gene sequence in the nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences are from the same partition.

22. The method of claim 21, wherein the first target capture sequence comprises a poly T sequence.

23. The method of claim 21 or 22, wherein the second target capture sequence binds to a sequence that is 50-600 nucleotides away from the first target capture sequence.

24. The method of any one of claims 21-23, wherein the second target capture sequence binds to a cDNA of a target gene that is highly expressed in the cell.

25. The method of any one of claims 21-24, further comprising releasing the barcoding oligonucleotides from the beads.

26. The method of any one of claims 21-25, wherein the providing a plurality⁷ of partitions comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions.

27. The method of any one of claims 21-25, wherein the providing a plurality of partitions comprises providing partitions comprising fixed cells.

28. The method of any one of claims 21-27, further comprising, after performing the last strand synthesis: inactivating the polymerase and reverse transcriptase in the partitions.

29. The method of claim 28, wherein the inactivating comprises applying heat to the partitions.

30. The method of claim 29, wherein the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

31. The method of any one of claims 21-30, wherein the partitions are droplets in an emulsion or microwells.

32. The method of any one of claims 21-31, wherein the cell is a mammalian cell.

33. The method of any one of claims 21-32, wherein the bead is a hydrogel bead.

34. The method of any one of claims 21-33, wherein the second target capture sequence is the same as the first target capture sequence.

35. A plurality of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality of copies of a first barcoding oligonucleotide and a second barcoding oligonucleotide, wherein the first barcoding oligonucleotide comprises 5'-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target capture sequence: wherein the second barcoding oligonucleotide comprises 5 ’-3’: a second PCR priming sequence, the bead-specific barcode sequence, a second target capture sequence; and wherein the bead comprises more copies of the first barcoding oligonucleotide than copies of the second barcoding oligonucleotide;

(ii) a reverse transcriptase; (iii) a DNA polymerase; and

(iv) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to first and second barcoding oligonucleotides comprising a first bead-specific barcode sequence, and wherein the second bead is linked to first and second barcoding oligonucleotides comprising a second bead-specific barcode sequence.

36. The plurality of partitions of claim 35, wherein the first target capture sequence comprises a poly T sequence.

37. The plurality of partitions of claim 35 or 36, wherein the second target capture sequence binds to a sequence that is 50-600 nucleotides away from the first target gene capture sequence.

38. The plurality of partitions of any one of claims 35-37, wherein the second target capture sequence binds to a cDNA of a gene that was highly expressed in the cell.

39. The plurality of partitions of any one of claims 35-38, wherein the partitions are droplets in an emulsion or microwells.

40. The plurality of partitions of any one of claims 35-39, wherein the cell is a mammalian cell.

41. The plurality of partitions of any one of claims 35-40, wherein the bead is a hydrogel bead.

42. The plurality' of partitions of any one of claims 35-41, wherein the second target capture sequence is the same as the first target capture sequence.

43. A bead, comprising: a plurality of copies of a first barcoding oligonucleotide, wherein the first barcoding oligonucleotide comprises 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target capture sequence; and a plurality of copies of a second barcoding oligonucleotide; wherein the second barcoding oligonucleotide comprises 5’-3’: a second PCR priming sequence, the bead-specific barcode sequence, and a second target capture sequence; wherein the bead comprises more copies of the first barcoding oligonucleotide than copies of the second barcoding oligonucleotide.

44. The bead of claim 43, wherein the first target capture sequence comprises a poly T sequence.

45. The bead of claim 43 or 44, wherein the bead is a hydrogel bead.

46. The bead of any one of claims 43-45, wherein the second target capture sequence is the same as the first target capture sequence.

47. A method of detecting multiple barcodes in a partition, the method comprising: providing a plurality⁷ of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality of copies of a barcoding oligonucleotide, comprising 5 ’-3’: a first PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence;

(ii) a detection oligonucleotide comprising 5'-3’: a second PCR priming sequence, a unique molecular identifier (UMI). and a second target gene capture sequence;

(iii) a reverse transcriptase;

(iv) a DNA polymerase; and

(v) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a first bead-specific barcode sequence, and wherein the second bead is linked to a second bead-specific barcode sequence; in the partitions, releasing the barcoding oligonucleotides from the beads; in the partitions, performing reverse transcription, wherein the performing comprises annealing sequences of cellular RNAs from the single cell or nucleus to some copies of the barcoding oligonucleotide and forming first strand gene-specific cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template, thereby producing an RNA:DNA heteroduplex, wherein the first strand gene-specific cDNAs comprise the first PCR priming sequence, the bead-specific barcode sequence, the first target gene capture sequence, and target gene sequence; in the partitions, degrading the RNA in the heteroduplex; in the partitions, performing second strand synthesis to form second strand cDNAs using first strand cDNAs as templates and the detection oligonucleotide as a primer, wherein the second strand cDNA comprises 5’-3’: the second PCR priming sequence, the UMI, the second target capture sequence, a portion of the target gene sequence, the beadspecific barcode reverse complement sequence, and the first PCR priming reverse complement sequence; in the partitions, annealing some copies of the barcoding oligonucleotide to some copies of the second strand cDNA, extending the barcoding oligonucleotide using the second strand cDNA as a template, thereby generating double-stranded nucleic acids, the first strand comprising the first PCR priming sequence, the bead-specific barcode sequence, the portion of the target gene sequence, the UMI reverse complement sequence, and the second PCR primer reverse complement sequence; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the nucleic acids, wherein if two different bead-specific barcode sequences or bead-specific barcode reverse complement sequences are linked to the UMI in the nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences or bead-specific barcode reverse complement sequences are from the same partition.

48. The method of claim 47, wherein the first target gene capture sequence comprises a poly T sequence.

49. The method of claim 47 or 48, wherein the providing a plurality of partitions comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions.

50. The method of claim 47 or 48, wherein the providing a plurality of partitions comprises providing partitions comprising fixed cells.

51. The method of any one of claims 47-50, further comprising, after performing the last strand synthesis: inactivating the polymerase and reverse transcriptase in the partitions.

52. The method of claim 51 , wherein the inactivating comprises applying heat to the partitions.

53. The method of claim 52 wherein the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

54. The method of any one of claims 47-53, wherein the partitions are droplets in an emulsion or microwells.

55. The method of any one of claims 47-54, wherein the cell is a mammalian cell.

56. The method of any one of claims 47-55, wherein the bead is a hydrogel bead.

57. A plurality of partitions, wherein different partitions comprise:

(iii) a reverse transcriptase;

(iv) a DNA polymerase; and

(v) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a first bead-specific barcode sequence, and wherein the second bead is linked to a second bead-specific barcode sequence.

58. The plurality’ of partitions of claim 57, wherein the first target gene capture sequence comprises a poly T sequence.

59. The plurality' of partitions of claim 57 or 58, wherein the partitions are droplets in an emulsion or microwells.

60. The plurality⁷ of partitions of any one of claims 57-59, wherein the cell is a mammalian cell.

61. The plurality of partitions of any one of claims 57-60, wherein the bead is a hydrogel bead.

62. A method of detecting multiple barcodes in a partition, the method comprising: providing a plurality of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality⁷ of copies of a barcoding oligonucleotide, comprising 5 ’-3 ’: a palindromic sequence, a PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence;

(iii) a reverse transcriptase;

(iv) a DNA polymerase; and

(v) a single fixed, lysed, or permeabilized cell; single nucleus; or nucleic acids from a single cell or nucleus; wherein at least one partition contains a first and second bead of the different beads, wherein the first bead is linked to a first bead-specific barcode sequence, and wherein the second bead is linked to a second bead-specific barcode sequence; in the partitions, performing reverse transcription, wherein the performing comprises annealing sequences of some cellular RNAs from the single cell or nucleus to some copies of the barcoding oligonucleotide and forming first strand cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template, thereby producing an RNA:DNA heteroduplex, wherein the first strand cDNAs comprise 5 ‘-3’: the palindromic sequence, the first PCR priming sequence, the bead-specific barcode sequence, the first target gene capture sequence, and the target gene sequence; in the partitions, performing second strand synthesis using the barcoding oligonucleotide of the first strand cDNAs as a template and extending from the cellular RNAs, thereby producing double stranded molecules having an RNA:DNA heteroduplex, wherein the second strand nucleic acid comprises the palindromic sequence at its 3’ end; in the partitions, separating the first strand cDNAs from the second strand nucleic acids; in the partitions, performing additional strand synthesis, wherein the palindromic sequences of some of the second strand nucleic acids anneal to the palindromic sequences of different second strand nucleic acids, and wherein strand synthesis uses the second strand nucleic acids as template and extends from the palindromic sequence, thereby producing double-stranded nucleic acids comprising 5’-3’: the target gene sequence, the bead-specific barcode reverse complement sequence, the PCR priming reverse complement sequence, the palindromic sequence, the PCR priming sequence, the bead-specific barcode sequence, and target gene reverse complement sequence; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the double-stranded nucleic acids, wherein if two different bead-specific barcode or bead-specific barcode reverse complement sequences are linked to the palindromic sequence in some of the double-stranded nucleic acids, then sequencing reads comprising either of the two bead-specific barcode sequences or bead-specific barcode reverse complement sequences are from the same partition.

63. The method of claim 62, wherein the separating comprises heating to separate the first strand cDNAs from the second strand nucleic acids.

64. The method of claim 62, wherein the separating and additional strand synthesis comprise: in the partitions, degrading the RNA from the heteroduplex; in the partitions, performing strand synthesis with a displacing DNA polymerase, using first strand cDNAs as templates and the detection oligonucleotide as a primer, wherein the second strand nucleic acid comprises 5 ‘-3’: a portion of the target gene sequence, a bead-specific barcode reverse complement sequence, and the palindromic sequence, and wherein the displaced strand comprises 5’-3’: the bead-specific barcode reverse complement sequence, and the palindromic sequence; in the partitions, performing additional strand synthesis with the second strand nucleic acids and the displaced strand nucleic acids as templates, wherein the palindromic sequences of some of the displaced strands anneal to the palindromic sequences of different displaced stands, and wherein strand synthesis uses the bead-specific barcodes and PCR priming sequences as template and extends from the palindromic sequence, thereby producing double-stranded nucleic acids comprising 5’-3’: the PCR priming reverse complement sequence, the bead-specific barcode reverse complement sequence, the palindromic sequence, the bead-specific barcode sequence, and the PCR priming sequence.

65. The method of claim 62 or 64, wherein the first target gene capture sequence comprises a poly T sequence.

66. The method of any one of claims 62-65, wherein the providing a plurality of partitions comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions.

67. The method of any one of claims 62-65, wherein the providing a plurality of partitions comprises providing partitions comprising fixed cells.

68. The method of any one of claims 62-66, further comprising releasing the barcoding oligonucleotides from the beads.

69. The method of any one of claims 62-68, further comprising, after the last strand synthesis: inactivating the polymerase and reverse transcriptase in the partitions.

70. The method of claim 69, wherein the inactivating comprises applying heat to the partitions.

71. The method of claim 70 wherein the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

72. The method of any one of claims 62-71, wherein the partitions are droplets in an emulsion or microwells.

73. The method of any one of claims 62-72, wherein the cell is a mammalian cell.

74. The method of any one of claims 62-73, wherein the bead is a hydrogel bead.

75. A plurality of partitions, wherein different partitions comprise:

(i) different beads, each bead linked to a plurality of copies of a barcoding oligonucleotide, comprising 5^?-3’: a palindromic sequence, a PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence;

(iii) a reverse transcriptase;

(iv) a DNA polymerase; and

76. The plurality of partitions of claim 75, wherein the first target gene capture sequence comprises a poly T sequence.

77. The plurality of partitions of claim 75 or 76, wherein the partitions are droplets in an emulsion or microwells.

78. The plurality of partitions of any one of claims 75-77, wherein the cell is a mammalian cell.

79. The plurality of partitions of any one of claims 75-78, wherein the bead is a hydrogel bead.

80. A bead, comprising: a plurality of copies of a barcoding oligonucleotide, comprising 5'-3’: a palindromic sequence, a PCR priming sequence, a bead-specific barcode sequence, and a first target gene capture sequence.

81. The bead of claim 80, wherein the first target gene capture sequence comprises a poly T sequence.

82. The bead of claim 80 or 81, wherein the bead is a hydrogel bead.