Methods and compositions for resolving multi-bead partitions

Abstract

Methods and compositions for deconvoluting partitions comprising multiple bead-specific barcodes are provided. The methods can involve for example in vitro transcription of detection oligonucleotides within partitions, which can be linked to bead-specific barcodes and sequenced.

Description

PATENT Attorney Docket No.094868-1447664-121410PC Client Ref. No. BRP01247-WO METHODS AND COMPOSITIONS FOR RESOLVING MULTI-BEAD PARTITIONS CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No.63 / 527,772, filed July 19, 2023, the entirety of which is hereby incorporated by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] Beads conjugated to oligonucleotides are used in microfluidic detection applications such as high-throughput sequencing having many different partitions (e.g., droplets). The beads for example deliver many copies of the same oligonucleotide to a partition. In order to uniquely identify each partition, the oligonucleotide copies linked to a bead will have barcode sequences unique or nearly unique for the bead to which the oligonucleotides are linked. However, if one wants to ensure that partitions have only one bead and thus are uniquely labeled by the barcode, bead concentrations are typically adjusted so that only about 1 out of 10 partitions are occupied by a bead. This results in low utilization of the partitions and increases the amount of sample and reagents that is needed for detection of samples. Increasing bead concentrations would result in higher partition occupancy and greater utilization of partitions. The amount of sample and reagents that are needed for detection of samples would be decreased. Conversely, higher bead concentrations would lead to a greater number of partitions having more than one bead. Thus, some partitioned samples would be labeled by more than one barcode. In instances where a partition contains two beads or more beads, for example, the sample is split between the different barcodes from different beads, resulting in a loss of sensitivity per barcode unless one can identify this issue. BRIEF SUMMARY OF THE INVENTION

[0003] 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) a different dsDNA detection oligonucleotide, wherein the detection oligonucleotide comprises 5’-3’: a promoter, a partition barcode sequence specific for the partition in which the detection oligonucleotide resides, and a poly A 3’ sequence; (ii) an RNA polymerase that recognizes the promoter; (iii) a reverse transcriptase; (iv) a single lysed cell or single lysed nucleus and cellular RNA; and (v) a bead linked to multiple copies of a barcoding oligonucleotide comprising 5’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a poly T 3’ end sequence; wherein at least one partition contains (a) a first bead linked to a first barcoding oligonucleotide comprising a first bead-specific barcode sequence and (b) a second bead linked to a second barcoding oligonucleotide comprising a second bead-specific barcode sequence; in the partitions, generating a plurality of detection RNAs comprising the partition barcode sequence and a poly A tail sequence by transcribing the detection oligonucleotides with the RNA polymerase.

[0004] In some embodiments, the methods further comprise in the partitions, releasing the barcoding oligonucleotides from the beads; in the partitions performing reverse transcription, wherein the performing comprises: (i) annealing some of the detection RNA poly A tail sequences to some copies of the barcoding oligonucleotides and forming first strand detection cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the detection RNAs as a template, wherein the first strand detection cDNAs comprise a bead-specific barcode sequence and a partition barcode sequence, wherein in the at least one partition (a) a first first strand cDNA in the partition comprises the first bead-specific barcode sequence and a specific partition barcode sequence and (b) a second first strand cDNA in the partition comprises the second bead-specific barcode sequence and the specific partition barcode sequence, and(ii) annealing cellular RNAs to some copies of the barcoding oligonucleotides and forming first strand cellular cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template; performing second strand synthesis to form second strand detection cDNAs and second strand cellular cDNAs using first strand detection cDNAs and first strand cellular cDNAs, respectively, as templates, thereby forming double-stranded detection cDNAs and double- stranded cellular cDNAs; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the double-stranded detection cDNAs and double- stranded cellular cDNAs, wherein if two bead bead-specific barcode sequences are linked to the same partition barcode sequence, then sequencing reads comprising either of the two bead bead-specific barcode sequences are from the same partition.

[0005] In some embodiments, at least some of the different partitions comprise two or more different dsDNA detection oligonucleotides having different partition barcode sequences specific for the partition in which the detection oligonucleotide resides.

[0006] In some embodiments, the detection oligonucleotide comprises a sequencing adapter sequence between the promoter and the barcode sequence.

[0007] In some embodiments, the providing comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions.

[0008] In some embodiments, the intact cells are fixed cells.

[0009] In some embodiments, the providing comprises providing partitions comprising isolated cell nuclei comprising cellular RNA and subsequently lysing the nuclei in the partitions.

[0010] In some embodiments, after performing second strand synthesis: inactivating the polymerase and reverse transcriptase in the partitions. In some embodiments, the inactivating comprises applying heat to the partitions. In some embodiments, the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

[0011] In some embodiments, the method further comprises size selecting nucleic acids in the bulk mixture to separate detection cDNAs from cellular cDNAs.

[0012] In some embodiments, after generating the bulk mixture and before determining the nucleotide sequence: further comprising generating random breaks in the double-stranded cellular cDNAs and randomly inserting at the breaks adaptor oligonucleotides, thereby forming cellular cDNA fragments comprising at least one 5’ end linked to an adaptor oligonucleotide, wherein the adaptor oligonucleotide comprises a second universal sequence, wherein some of the cellular cDNA fragments also comprise a bead bead-specific barcode sequence and the first universal sequence. In some embodiments, the random breaks are induced by a transposase that inserts the adaptor oligonucleotides.

[0013] In some embodiments, the dsDNA detection oligonucleotide further comprises a second universal adaptor sequence 5’ of the partition barcode sequence and wherein the double-stranded detection cDNAs comprise the first universal adaptor sequence and the second universal adaptor sequence, and the method further comprises amplifying the double- stranded detection cDNAs and the cellular cDNA fragments that comprise the first universal adaptor sequence and the second universal adaptor sequence with a DNA polymerase and a first primer that anneal to the first universal adaptor sequence and a second primer that anneals to the second universal primer sequence.

[0014] In some embodiments, the promoter is a T7 or SP6 promoter and the RNA polymerase is T7 or SP6 RNA polymerase, respectively.

[0015] In some embodiments, the partitions are droplets in an emulsion or microwells.

[0016] In some embodiments, the cell is a mammalian cell.

[0017] In some embodiments, the bead is a hydrogel bead.

[0018] In some embodiments, generating the plurality of detection RNAs occurs at a temperature of between 35-50 degrees Celsius; performing the reverse transcription occurs at a temperature of between 37-55 degrees Celsius; and / or performing the second strand synthesis occurs at a temperature of between 60-70 degrees Celsius.

[0019] Also provided is a plurality of partitions, for example as described herein. In some embodiments, different partitions comprise (i) a different dsDNA detection oligonucleotide, wherein the detection oligonucleotide comprises 5’-3’: a promoter, a partition barcode sequence specific for the partition in which the detection oligonucleotide resides, and a poly A 3’ sequence;(ii) an RNA polymerase that recognizes the promoter; (iii) a reverse transcriptase; (iv) a lysed cell and cellular RNA; and (v) a bead linked to multiple copies of a barcoding oligonucleotide comprising 5’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a poly T 3’ end sequence; wherein at least one partition contains (a) a first bead linked to a first barcoding oligonucleotide comprising a first bead-specific barcode sequence and (b) a second bead linked to a second barcoding oligonucleotide comprising a second bead-specific barcode sequence.

[0020] In some embodiments, at least some of the different partitions comprise two or more different dsDNA detection oligonucleotides having different partition barcode sequences specific for the partition in which the detection oligonucleotide resides.

[0021] In some embodiments, the detection oligonucleotide comprises a sequencing adapter sequence between the promoter and the barcode sequence.

[0022] In some embodiments, the promoter is a T7 or SP6 promoter and the RNA polymerase is T7 or SP6 RNA polymerase, respectively.

[0023] In some embodiments, the partitions are droplets in an emulsion or microwells.

[0024] In some embodiments, the cell is a mammalian cell.

[0025] In some embodiments, the bead is a hydrogel bead. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG.1 depicts barcode deconvolution as described herein. “CBC” refers to bead- specific barcode sequences. “DO ID” refers to detection oligonucleotide ID, also referred herein as the partition barcode. “R1” and “R2” refer to universal sequences that can be used for example as “PCR handles” to amplify the nucleic acids resulting from the steps depicted. cDNAs formed by extending the bead-specific oligonucleotide using the detection RNAs as a template are depicted at the bottom of the Figure and show how various combinations can occur and be interpretated to deconvolute later sequencing reads. The bottom of the figure depicts hypothetical products from the method described herein. Bottom right productsrepresent cellular cDNAs formed in partitions from cellular RNA and a barcoding oligonucleotide that was delivered to the partition linked to a bead. Different cellular cDNAs have different bead-specific barcodes (CBC1, CBC2, CBC3, CBC4, and CBC5 referring to bead-specific barcodes having different sequences). This information alone indicates barcoding oligonucleotides from different beads were used to prime the different products, but does not indicate whether the beads were in the same or different partitions. Products depicted at the bottom left represent detection cDNAs formed in partitions from detection RNAs and a barcoding oligonucleotide that was delivered to the partition linked to a bead. Because there are multiple copies of detection RNAs in a partition (formed from transcription in the partition), the same detection oligonucleotide ID (partition barcode) will become associated with any bead-specific barcodes in the same partition. The sequencing reads depicted at the left bottom show that DO1 is linked to CBC1, CBC2 and CBC5 and DO2 is linked to CBC3 and CBC4. This indicates that sequencing reads having either CBC3 or CBC4 came from the same partition and similarly sequencing reads having any of CBC1, CBC2 or CBC5 came from the same partition different from the partition having CBC3 and CBC4. This is indicated by dotted lines showing common partition origin of the products at the bottom left and the bottom right.

[0027] FIG.2A and 2D depict an exemplary workflow for performing the methods described herein.

[0028] FIG.2B-2C depicts reactions within partitions. In an initial step the partitions are exposed to conditions to allow for in vitro transcription to form detection RNAs from detection oligonucleotides in the partitions. Optionally simultaneously, cellular RNAs are released into the partition, for example by cell lysis. Barcoding oligonucleotides linked to beads in the partitions can also be released from the beads. In a second step, reverse transcription is performed to generate first strand cDNAs from (i) the detection RNAs primed from the barcoding oligonucleotides and (ii) the cellular RNAs primed from the barcoding oligonucleotides. In a third step (not depicted) second strand synthesis is performed.

[0029] FIG.2D depicts optional sample preparation reactions following combination of contents of the partitions into a “bulk” reaction, allowing for subsequent generation of sequencing reads. Optionally (not depicted) detection cDNAs can be separated from cellular cDNAs, for example based on nucleic acid length as the detection cDNAs will in generally be shorter than cellular cDNAs, and already of a length useful for sequencing. As depicted,cellular cDNAs are contacted with a tagmentase that fragments the cellular cDNAs to generate fragments of appropriate size for sequencing. As depicted, fragmentation results in fragments comprising the ends of cellular cDNAs that include the bead-specific barcode and universal or other sequences introduced in the RT reaction by the bead-specific barcode. While specific adapter (p5 and p7) and index (i5 and i7) sequences are indicated in the figures any adapter and / or index sequences can be used, for example as PCR handle sequences.

[0030] FIG.3 depicts exemplary alternative options for configurations of the detection oligonucleotide. As can be seen in the figure, in some embodiments, the barcode sequence is discontinuous, separated by constant sequences.

[0031] FIG.4A-B depicts data regarding bead merging as discussed in the Example.

[0032] FIG.5 depicts data for bead distribution as discussed in the Example. DEFINITIONS

[0033] 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 invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical 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 and peptide 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.

[0034] The term “amplification reaction” refers to any in vitro method 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 (LCR); Qbeta RNA replicase and RNA transcription-based 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); single-primer isothermal amplification (SPIA), loop mediated isothermal amplification (LAMP), strand displacementamplification (SDA); multiple displacement amplification (MDA); rolling circle amplification (RCA); as well as others known to those of skill in the art. See, e.g., Fakruddin et al., J. Pharm Bioallied Sci.20135(4):245-252.

[0035] “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.

[0036] “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 known 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.

[0037] A “primer” refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and optionally serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety of lengths. In some embodiments, a primer is less than 100 or 50 nucleotides in length, e.g., from about 10 to about 900, from about 15 to about 80, or from about 30-85 to about 30 nucleotides in length. The length and sequences of primers for use in an amplification reaction (e.g., 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. The primer can include or be completely formed from DNA, RNA or non-natural nucleotides. In some embodiments, a primer comprises one or more modified and / or non- natural nucleotide bases. In some embodiments, a primer comprises a label (e.g., a detectable label).

[0038] A nucleic acid, or portion thereof, “hybridizes” to another nucleic acid under conditions such that non-specific hybridization is minimal at a defined temperature in a physiological buffer. In some cases, a nucleic acid, or portion thereof, hybridizes to aconserved 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.

[0039] As used herein, “nucleic acid” refers to 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-uracil; 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.

[0040] 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 or microwell. 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).Exemplary array of wells and well descriptions can be found for example in U.S. Patent No. 9,103,754 and 10,391,493. The array of wells (set of nanowells, microwells, wells) can function to capture the solid supports, optionally in addressable, known locations. As such, the array of wells can be configured to facilitate bead capture in at least one of a single-solid support format or optionally in small groups of solid supports. Exemplary microwell arrays and methods of delivery of beads to the microwells and analysis thereof is described in, e.g., PCT / US2021 / 034152.

[0041] 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.

[0042] As used herein, a “barcode” is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30 or more nucleotides long) that identifies a molecule to which it is conjugated. In some embodiments, a barcode is used to identify molecules in a partition. Such a partition-specific barcode (or multiple different partition-specific barcodes) can 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 different (e.g., each) partition, 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 embodiments, a barcode is present on oligonucleotides conjugated to a particle, wherein the “particle barcode” or “bead barcode” is shared by (e.g., identical or substantially identical amongst) all, or substantially all, of the oligonucleotides conjugated to that particle or bead. In some embodiments, the barcode is discontinuous. See, e.g., FIG.3.

[0043] “Clonal” copies of a polynucleotide means the copies are identical in sequence. In some embodiments, there are at least 100, 1000, 104or more clonal copies of oligonucleotides in linked to a bead.

[0044] The term “deconvolution” refers to the assignment of 2 or more barcodes (and optionally the beads to which they were attached) as being from the same partition or originally occupying the same partition. Deconvolution can be determined as described herein by the detection of the same detection oligonucleotide barcode associated with two different bead-specific barcodes during sequencing. DETAILED DESCRIPTION OF THE INVENTION

[0045] The inventors have discovered that in-partition transcription and reverse transcription can be used to generate a plurality of copies of a partition-specific barcode. Linkage of the partition-specific barcodes with bead-specific barcodes delivered to partitions linked to beads can be detected using sequencing to determine whether and which bead- specific barcodes originated in the same partition. Because bead-specific barcodes are also linked to sample nucleic acids in partitions, one can use whether bead-specific barcodes where linked to partition-specific barcodes to tell whether multiple bead-specific barcodes were present in a single partition, and thus sample nucleic acid sequences having such bead- specific barcodes (linked to the same partition-specific barcode) are identified as being from the same partition in spite of a portion of the sequences being linked to different bead- specific barcodes. Said another way, the presence of more than one differently-bead barcoded oligonucleotide primer (e.g., two differently bead-barcoded primers 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 (for example as a function of a Poisson distribution). By being able to detect which bead barcodes occurred in the same partitions, one can either deconvolute (i.e., determine that multiple bead barcodes are from the same partition and account for that in the sequencing analysis) and use the data from such combinations or disregard (e.g., discard) data from such partitions, leaving the remaining data more “clean” from background effects.

[0046] The methods described herein provide for providing partitions comprising different double-stranded (ds) DNA detection oligonucleotides. For example, partitions can be provided such that different partitions contain different dsDNA detection oligonucleotides that differ at least by having different partition barcode sequences. For example, in some embodiments, the dsDNA detection oligonucleotides comprise 5’-3’: a promoter operably linked to a sequence comprising a partition barcode sequence specific for the partition in which the detection oligonucleotide resides and optionally a poly A 3’ sequence. Thepartitions further comprise an RNA polymerase that can recognizes the promoter and that generates RNA transcripts comprising reverse complements of the partition barcode sequences. The partitions can also contain a reverse transcriptase and one or more bead linked to a barcoding oligonucleotide as a primer comprising a bead-specific barcode sequence such that the RNA transcripts can be converted to cDNAs comprising the partition barcode sequences and the bead-specific barcode sequence, thereby providing a plurality of copies of the partition barcode sequences in the partition itself. In parallel, cellular RNA (from a biological sample) will also be converted to cDNAs using a barcoding oligonucleotide as a primer such that the resulting cDNAs comprise a bead-specific barcode sequence.

[0047] The various reagents described, as provided in partitions, can be present when the partitions are formed or can be added to partitions after the partitions are formed. While one can inject reagents into droplets for example, it may be more convenient when the partitions are droplets to have the reagents at a desired concentration in a bulk solution and then form droplets from the bulk solution. In general, reagents such as buffers, nucleotides, and enzymes should be provided so most or all partitions receive a sufficient concentration to allow for the reactions described herein, e.g., transcription and reverse transcription.

[0048] The number or concentration of DNA detection oligonucleotides, which differ at least by their partition barcode sequences, can be selected such that a majority (e.g., at least 60, 70, 80, 90% or more) of the partitions contain at least one DNA detection oligonucleotide. Due to Poisson distribution, the average number of detection oligonucleotides per partition will be more than one copy per partition to avoid an excess of partitions lacking a detection oligonucleotide. In some embodiments, the number or concentration of detection oligonucleotides is selected such that the average number of detection oligonucleotides in a partition is for example between 1-100, e.g., 5-50, 10-40, 10- 30, e.g., 25 or a different range or value therein. Each of the different detection oligonucleotides in a partition can have a different sequence, for example differing by their barcode sequence. The barcode sequence for each will be unique or substantially unique to the partition in which the detection oligonucleotide resides, but there can be different barcodes within a particular partition wherein each is different from each other and unique to the partition, i.e., the barcode sequence(s) do not occur in other partitions.

[0049] The detection oligonucleotides are composed of DNA (e.g., dsDNA) or a nucleotide analog that can be recognized by the RNA polymerase to form RNA transcripts. The detection oligonucleotide will at least have a promoter operably linked a sequence comprising a partition barcode sequence. In some embodiments, a sequencing adapter sequence can occur between the promoter and the barcode sequence, allowing for the user to better identify the start position of the barcode sequence. Optionally the sequencing adapter sequence comprises a phase-shift region, for example as described in PCT / US2017 / 013205. Some options for the detection oligonucleotide configurations are depicted in FIG.3.

[0050] The partition barcode sequence length and complexity can be selected based on the number of unique sequences desired. For example, for a given number of partitions desired, one could select an excess number (e.g., 2X, 5X, 10X, 20X, etc.) of unique detection oligonucleotides. In some embodiments, the barcode sequence is 15-30, e.g., 18-22, e.g., 19, 20 or 21 nucleotides long and can be continuous or discontinuous (e.g., composed of two or more segments). In some embodiments, the detection oligonucleotides are between 150-200 nucleotides long. Optionally the barcode sequence can be discontinuous, for example two or more sub-sequences separated by a constant sequence. In preferred embodiments, the detection oligonucleotides also comprise a poly thymine (poly-T) sequence at the 3’ end of the detection oligonucleotides. Poly-T sequence are single stranded sequences of deoxythymine (dT). The length of the poly-T sequence can vary, for example from 6 bases to 30 bases and may be a mixture of poly-T sequences with different lengths.

[0051] The promoter in the detection oligonucleotides can be any promoter recognized by the RNA polymerase to be used in the partitions. Exemplary RNA polymerases can include but are not limited to the T7 polymerase (see, e.g., Cheetham, G. M., et al. Nature 399, 80–83 (1999) and the SP6 polymerase (see, e.g., Stump et al., Nucleic Acids Res.21(23): 5480– 5484 (1993). Promoter sequences recognized by the T7 polymerase include but not limited to a sequence comprising TAATACGACTCACTATAG. Promoter sequences recognized by the SP6 polymerase include but not limited to a sequence comprising ATTTAGGTGACACTATAGAAGNG.

[0052] In vitro transcription can be initiated in the partitions with the RNA polymerase using the detection oligonucleotides as a template(s). Any desired in vitro transcription conditions can be selected that are compatible with the stability of the partitions and allow for activity by the RNA polymerase. Exemplary conditions can include, for example,performing in vitro transcription at 37 degrees C or for example between 35-39 degrees C with the appropriate free ribonucleotides to form RNA transcripts. The in vitro transcription generates detection RNAs in the partitions comprising a reverse complement of the partition barcode sequence and a poly A tail sequence. While partitions may contain more than one detection oligonucleotide (indeed, in some embodiments, the partitions on average contain more than one detection oligonucleotide as discussed herein), detection oligonucleotides between partitions will differ thereby allowing the partition barcode(s) in a droplet to be unique for that partition and thus the resulting detection RNAs are also unique for the partition in which they are produced.

[0053] After, or simultaneous with, the production of the detection RNAs, reverse transcription can be performed in the partitions. By using a barcoding oligonucleotide primer (delivered to the partition linked to a bead and optionally released from the bead) that has a 3’ poly T sequence, reverse transcription can occur in the partition to generate first strand cDNAs based on the (i) detection RNAs and (ii) sample mRNAs as templates. In some embodiments, the reverse transcriptase has an optimal temperature that is higher than the RNA polymerase. In these embodiments, the partitions are incubated at a lower temperature (e.g., 37 degrees C) to generate the detection RNAs and then the partitions are incubated at a higher temperature (e.g., 50 degrees C) to generate first strand cDNAs from the detection RNAs and sample mRNAs as templates.

[0054] Reverse transcription (RT) is an amplification method that copies RNA into DNA. A variety of reagents can be included in the partitions for the reverse transcription reaction. RT reactions can be performed with reaction mixtures as desired. Components and conditions for RT reactions are generally known. Reverse transcriptases (RTs) can be selected based on the conditions of the reaction and user preference. In some embodiments, the reverse transcriptase is an MMLV RT or a variant thereof (for example but not limited to variants with improved thermostability compared to native MMLV RT). See, e.g., WO2018200867, Arezi et al., Nucleic Acids Res.37(2): 473–481 (2009); Baba et al., Protein Engineering, Design and Selection, Volume 30, Issue 8, August 2017, Pages 551–557.

[0055] Partitions will comprise cellular RNA from single cells or isolated nuclei. In some embodiments, the partitions are formed with cells or nuclei are a sufficiently low concentration such that a majority of the partitions containing a cell or nuclei contain only one (a single) cell or nuclei. In some embodiments, the cells are fixed cells. For example insome embodiments, the cells are formalin-fixed, paraffin-embedded (FFPE) samples. In some embodiments, the cells are not fixed. In either case, in some embodiments, the cells are permeabilized to allow for entry of reagents while the cells themselves remain substantially intact. In some embodiments, once the cells or nuclei are present in the partitions, the cells or nuclei are lysed to release cellular RNA in cells or nuclei into the partitions, allowing for the RNA to take part in the reverse transcription reaction. In some embodiments, the cells or nuclei in the partitions have been permeabilized such that the RNA in the permeabilized cell or nuclei can diffuse from the cells or nuclei into the remaining inside portion of the partitions. Permeabilization can remove cellular membrane lipids to allow large molecules such as enzymes to enter the cell. In some embodiments, a detergent is used for permeabilization. Exemplary detergents can include, for example, Triton X-100 and NP-40 are used for permeabilization (for example, at 0.1–0.5% (v / v, in PBS). In some embodiments, a steroidal saponin (or saraponin) is used to solubilize lipid, resulting in permeabilization. An exemplary saraponin is Digitonin. The appropriate permeabilization reagent can be selected to be compatible with the integrity of the partition used. In some embodiments, the cells or isolated nuclei are lysed so that their contents, including RNA, are released into the partition in which the cell or nucleus is present.

[0056] Any type of cells can be used according to the methods and compositions described herein. In some embodiments, the cells are mammalian, for example human cells. In some embodiments, the cells are 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, or cells found in stool, urine, etc.

[0057] Methods and compositions for partitioning are described, for example, in published patent applications WO 2010 / 036352, US 2010 / 0173394, US 2011 / 0092373, and US 2011 / 0092376. The plurality of partitions can be, for example, a plurality of emulsion droplets, or a plurality of microwells, etc.

[0058] In some embodiments, one or more reagents are added during droplet formation or to the droplets after the droplets are formed. Methods and compositions for delivering reagents to one or more partitions include microfluidic methods as known in the art; droplet or microcapsule combining, coalescing, fusing, bursting, or degrading (e.g., as described in U.S.2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g., as described in WO 2010 / 151,776); and combinations thereof.

[0059] The partitions can be picowells, nanowells, or microwells. The partitions can be pico-, nano-, or micro- reaction chambers, such as pico, nano, or microcapsules. The partitions can be pico-, nano-, or micro- channels. The partitions can be droplets, e.g., emulsion droplets. In some embodiments, a droplet comprises an emulsion composition, i.e., a mixture of immiscible fluids (e.g., water and oil). In some embodiments, a droplet is an aqueous droplet that is surrounded by an immiscible carrier fluid (e.g., oil). In some embodiments, a droplet is an oil droplet that is surrounded by an immiscible carrier fluid (e.g., an aqueous solution). In some embodiments, the droplets described herein are relatively stable and have minimal coalescence between two or more droplets. In some embodiments, less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of droplets generated from a sample coalesce with other droplets. The emulsions can also have limited flocculation, a process by which the dispersed phase comes out of suspension in flakes. In some cases, such stability or minimal coalescence is maintained for up to 4, 6, 8, 10, 12, 24, or 48 hours or more (e.g., at room temperature, or at about 0, 2, 4, 6, 8, 10, or 12 °C). In some embodiments, the droplet is formed by flowing an oil phase through an aqueous sample or reagents.

[0060] The oil phase of an emulsion can comprise a fluorinated base oil which can additionally be stabilized by combination with a fluorinated surfactant such as a perfluorinated polyether. Exemplary oil phase compositions along these lines are described in, e.g., PCT WO 2020 / 247950 and US Patent Publication No.2017 / 0175179.

[0061] In some embodiments, the sample is partitioned into, or into at least, 500 partitions, 1000 partitions, 2000 partitions, 3000 partitions, 4000 partitions, 5000 partitions, 6000 partitions, 7000 partitions, 8000 partitions, 10,000 partitions, 15,000 partitions, 20,000 partitions, 30,000 partitions, 40,000 partitions, 50,000 partitions, 60,000 partitions, 70,000 partitions, 80,000 partitions, 90,000 partitions, 100,000 partitions, 200,000 partitions, 300,000 partitions, 400,000 partitions, 500,000 partitions, 600,000 partitions, 700,000partitions, 800,000 partitions, 900,000 partitions, 1,000,000 partitions, 2,000,000 partitions, 3,000,000 partitions, 4,000,000 partitions, 5,000,000 partitions, 10,000,000 partitions, 20,000,000 partitions, 30,000,000 partitions, 40,000,000 partitions, 50,000,000 partitions, 60,000,000 partitions, 70,000,000 partitions, 80,000,000 partitions, 90,000,000 partitions, 100,000,000 partitions, 150,000,000 partitions, or 200,000,000 partitions.

[0062] In some embodiments, the droplets that are generated are substantially uniform in shape and / or size. For example, in some embodiments, the droplets are substantially uniform in average diameter. In some embodiments, the droplets that are generated have an average diameter of about 0.001 microns, about 0.005 microns, about 0.01 microns, about 0.05 microns, about 0.1 microns, about 0.5 microns, about 1 microns, about 5 microns, about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 300 microns, about 400 microns, about 500 microns, about 600 microns, about 700 microns, about 800 microns, about 900 microns, or about 1000 microns. In some embodiments, the droplets that are generated have an average diameter of less than about 1000 microns, less than about 900 microns, less than about 800 microns, less than about 700 microns, less than about 600 microns, less than about 500 microns, less than about 400 microns, less than about 300 microns, less than about 200 microns, less than about 100 microns, less than about 50 microns, or less than about 25 microns. In some embodiments, the droplets that are generated are non-uniform in shape and / or size.

[0063] In some embodiments, the droplets that are generated are substantially uniform in volume. For example, the standard deviation of droplet volume can be less than about 1 picoliter, 5 picoliters, 10 picoliters, 100 picoliters, 1 nL, or less than about 10 nL. In some cases, the standard deviation of droplet volume can be less than about 10-25% of the average droplet volume. In some embodiments, the droplets that are generated have an average volume of about 0.001 nL, about 0.005 nL, about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5 nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL, about 10 nL, about 11 nL, about 12 nL, about 13 nL, about 14 nL, about 15 nL, about 16 nL, about 17nL, about 18 nL, about 19 nL, about 20 nL, about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, or about 50 nL.

[0064] As noted above, one or more beads linked to clonal copies of a barcoding oligonucleotides are present when the partitions are formed or are added to the partitions. The barcoding oligonucleotides can comprise, for example, 5’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a poly T 3’ end sequence. As explained above, the barcoding oligonucleotides are used as RT primers by annealing to RNA in a partition to form cDNAs that include the bead-specific barcode sequence. Additional barcodes, such as but not limited to, unique molecule identifiers (UMIs) or sample-specific barcodes can also be included in the oligonucleotide sequence.

[0065] Any bead of useful size and composition for delivery to partitions can be used. The particle or bead can be any particle or bead having a solid support surface. Solid supports suitable for particles include controlled pore glass (CPG)(available from Glen Research, Sterling, Va.), oxalyl-controlled pore glass (See, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527), TentaGel Support—an aminopolyethyleneglycol derivatized support (See, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373), polystyrene, Poros (a copolymer of polystyrene / divinylbenzene), or reversibly cross-linked acrylamide. Many other solid supports are commercially availa—e and amenable to the methods described herein. In some embodiments, the bead material is a polystyrene resin or poly(methyl methacrylate) (PMMA). The bead material can be metal.

[0066] In some embodiments, the particle or bead comprises hydrogel or another similar composition. 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.20020009591; 20130022569; 20130034592; and International Patent Publication Nos. WO1997030092; and WO2001049240. Additional compositions and methods for making and using hydrogels, such as barcoded hydrogels, include those described in, e.g., Klein et al., Cell, 2015 May 21;161(5):1187-201.

[0067] The solid support surface of the bead can be modified to include a linker for attaching barcode oligonucleotides. The linkers may comprise a cleavable moiety, which may be cleaved before reverse transcription is performed in the partitions. Non-limiting examples of cleavable moieties include a disulfide bond, a dioxyuridine moiety, and arestriction enzyme recognition site. The cleavable sequence can be any cleavable sequence that can be targeted enzymatically or otherwise 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 deoxribonucleotides. For example, in some embodiments, RNAseH is used to specifically cleave at a ribonucleotide in the cleavable sequence. Preferably, a 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.

[0068] Oligonucleotides can be linked to beads as desired. Methods of linking oligonucleotides to beads are described in, e.g., WO 2015 / 200541. In some embodiments, the oligonucleotide configured to link a hydrogel bead to the barcode is covalently linked to the hydrogel. Numerous methods for covalently linking an oligonucleotide to one or more hydrogel matrices are known in the art. As but one example, aldehyde derivatized agarose can be covalently linked to a 5’-amine group of a synthetic oligonucleotide.

[0069] First strand cDNA synthesis is performed as described herein followed by second strand synthesis in the partitions to form double-stranded cDNAs, i.e., using first strand detection cDNAs and first strand cellular cDNAs, respectively, as templates, thereby forming double-stranded detection cDNAs and double-stranded cellular cDNAs. Optionally, the enzymes in the bulk solution can be inactivated after second strand synthesis, for example by applying heat to the partitions. In some cases, the inactivating comprises incubating the partitions at 75-90 degrees Celsius.

[0070] As noted herein, due to a Poisson distribution of beads and associate oligonucleotides within partitions, some partitions will not contain any beads, some partitions will contain one bead, some two beads, and some more than two beads. Thus, for example,following reverse transcription in the partitions, at least some partitions that contain two or more beads and associated barcoding oligonucleotides will comprise: (i)(a) a first cDNA in the partition comprises the first bead-specific barcode sequence and a specific partition barcode sequence (formed by extending a first barcoding oligonucleotide from a first bead in the partition with the reverse transcriptase using the detection RNAs as a template) and (i)(b) a second cDNA in the partition comprises the second bead-specific barcode sequence and the specific partition barcode sequence (formed by extending a second barcoding oligonucleotide from a second bead in the partition with the reverse transcriptase using the detection RNAs as a template), and (ii) cellular cDNAs (formed by extending the first and / or second barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template).

[0071] The above pattern of bead-specific and partition barcode sequences can then be used to identify those partitions having more than one bead-specific barcode. While the example above provides for two bead-specific barcodes in a partition, the same approach can be used for a higher number of beads in the partition (e.g., 3, 4, 5, or more), for example using different bead-specific barcodes associated with the same partition barcode sequence to determine that those different bead-specific barcodes resided in the same partition. See, e.g., FIG.1.

[0072] Once the cDNAs are linked to bead-specific barcode sequences, the contents of the partitions can be combined into a bulk solution. In some embodiments, for example, the droplets are burst such that their contents are combined in form a bulk aqueous solution.

[0073] The fragments in the resulting bulk solution can be nucleotide sequenced as desired. In some embodiments, a certain size of fragments is desired for the sequencing reaction. In some embodiments, the detection cDNAs are sufficiently short to be processed for sequencing directly, whereas it can be desirable to fragment the cellular cDNAs, which are longer than the detection cDNAs. Thus in some embodiments, one can size select cDNAs from the bulk solution that are sufficiently short to be processed for sequencing. Exemplary size selection methods can include but are not limited to solid-phase reversible immobilization (SPRI) technology. While the smaller (primarily detection cDNAs) can be sequenced directly, the resulting larger cDNAs can be fragmented and sequenced.

[0074] Fragmentation of the longer (primarily cellular cDNAs) can be fragmented by tagmentation. FIG.2D for example depicts embodiments in which tagmentation is used to fragment the cellular cDNAs and add adapter sequences to the ends of the fragments. As shown in FIG.2D, some of the fragments will include the end of the cellular cDNA having at one end the bead-specific barcode and universal sequence introduced by the barcoding oligonucleotide and an adapter sequence on the fragment’s other end introduced by the tagmentase. In some embodiments, the universal sequence introduced from the barcoding oligonucleotide and the adapter sequence introduced by the tagmentase is subsequently used to amplify the desired fragments. See, e.g., FIG.2D, bottom panel. The number of cycles of amplification can be determined by the user depending on the amount of amplified product desired for sequencing.

[0075] The action of some transposases sometimes referred to as “tagmentation” and can involve introduction of different adapter sequences on different sides of a DNA breakage point or the adapter sequences added can be identical. In either case, the adapter sequences are common adapter sequences in that the adapter sequences are the same across a diversity of DNA fragments. Adapter loaded tagmentases are further described, e.g., in U.S. Patent Publication Nos: 2010 / 0120098; 2012 / 0301925; and 2015 / 0291942 and U.S. Patent Nos: 5,965,443; U.S.6,437,109; 7,083,980; 9,005,935; and 9,238,671, the contents of each of which are hereby incorporated by reference in the entirety for all purposes.

[0076] A tagmentase is an enzyme that is capable of forming a functional complex with a transposon end-containing composition and catalyzing insertion or transposition of the transposon end-containing composition into the double-stranded target DNA with which it is incubated in an in vitro transposition reaction. Exemplary transposases include but are not limited to modified Tn5 transposases that are hyperactive compared to wildtype Tn5, for example can have one or more mutations selected from E54K, M56A, or L372P. Wild-type Tn5 transposon is a composite transposon in which two near-identical insertion sequences (IS50L and IS50R) are flanking three antibiotic resistance genes (Reznikoff WS. Annu Rev Genet 42: 269–286 (2008)). Each IS50 contains two inverted 19-bp end sequences (ESs), an outside end (OE) and an inside end (IE). However, wild-type ESs have a relatively low activity and were replaced in vitro by hyperactive mosaic end (ME) sequences. A complex of the transposase with the 19-bp ME is thus all that is necessary for transposition to occur, provided that the intervening DNA is long enough to bring two of these sequences close together to form an active Tn5 transposase homodimer (Reznikoff WS., Mol Microbiol 47:1199–1206 (2003)). Transposition is a very infrequent event in vivo, and hyperactive mutants were historically derived by introducing three missense mutations in the 476 residues of the Tn5 protein (E54K, M56A, L372P), which is encoded by IS50R (Goryshin IY, Reznikoff WS.1998. J Biol Chem 273: 7367–7374 (1998)). Transposition works through a “cut-and- paste” mechanism, where the Tn5 excises itself from the donor DNA and inserts into a target sequence, creating a 9-bp duplication of the target (Schaller H. Cold Spring Harb Symp Quant Biol 43: 401–408 (1979); Reznikoff WS., Annu Rev Genet 42: 269–286 (2008)). In current commercial solutions (Nextera™ DNA kits, Illumina), free synthetic ME adapters are end-joined to the 5′-end of the target DNA by the transposase (tagmentase).

[0077] Sequencing platforms can be selected as desired. In some embodiments, Illumina™-supported sequencing methods are employed. See, e.g., U.S. Patent Nos 11,029,513; US 11,150,179; 11,308,640; and 11,473,067 and citations therein. Exemplary DNA sequencing techniques include fluorescence-based sequencing methodologies (See, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety). In some embodiments, automated sequencing techniques understood in that art are utilized. In some embodiments, the present technology provides parallel sequencing of partitioned amplicons (PCT Publication No. WO 2006 / 0841,32, herein incorporated by reference in its entirety). In some embodiments, DNA sequencing is achieved by parallel oligonucleotide extension (See, e.g., U.S. Pat. Nos. 5,750,341; and 6,306,597, both of which are herein incorporated by reference in their entireties). Additional examples of sequencing techniques include the Church polony technology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; and U.S. Pat. Nos.6,432,360; 6,485,944; 6,511,803; herein incorporated by reference in their entireties), the 454 picotiter pyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380; U.S. Publication No.2005 / 0130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Pat. Nos.6,787,308; and 6,833,246; herein incorporated by reference in their entireties), the Lynx massively parallel signature sequencing technology (Brenner et al. (2000). Nat. Biotechnol.18:630-634; U.S. Pat. Nos.5,695,934; 5,714,330; herein incorporated by reference in their entireties), and the Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res.28, E87; WO 2000 / 018957; herein incorporated by reference in its entirety).EXAMPLE

[0078] This example describes the production of barcode oligonucleotide chimeras in single-cell 3’ RNA library preparation workflow. Cell preparation. HEK293 cells (ATCC, CRL-1573) and NIH3T3 cells (ATCC, CRL-1658) were used for ddSEQ reagents setup. Cells were prepared according to Illumina SureCell WTA 3’ Reference Guide. HEK293 and NIH3T3 cells were mixed in a 1:1 ratio for the experiment. Droplet generation and incubation. Bead and Cell Suspension mixes were prepared prior to loading into the ddSEQ M cartridge (Biorad, PN12008720). The Bead suspension mix includes Tris buffer, salts, detergents, density gradient media, T7 RNA polymerase, and 5950 barcoded beads / µl. The Cell suspension mix contains Tris buffer, salts, detergent, density gradient media, NTPs, dNTPs, enzyme mix, detection oligos, and 400 cells / µl mixed HEK293 / NIH3T3 cells. Assembled bead and cell suspension mixes were loaded and run in the ddSEQ Single-Cell Isolator as instructed in Illumina SureCell WTA 3’ Reference Guide. The droplet emulsions were transferred to a Bio-Rad’s ddPCR 96-well plate, sealed with an 8-cap strip, and incubated as follows: 37°C for 60 minutes, 50°C for 15 minutes, 70°C for 45 minutes, 80°C for 5 minutes, and hold at 4°C. Library preparation. Droplet contents were released by adding 40 µl droplet disruptor to each sample. Cell cDNA was cleaned up and separated from detection cDNA was by performing two rounds of 0.68X ampure XP. Detection cDNA was extracted from the supernatant of the first ampure round and purified by double-sided size selection. The purified cell cDNA was then tagmented according to protocol in the Illumina SureCell WTA 3’ Reference Guide. Next, the tagmented cell cDNA and detection cDNA were amplified and indexed separately. The index PCR reaction consisted of Kapa HiFi HotStart Ready Mix (Roche, PN 9420398001), custom P5-Read 1 primer, and Nextera P7 index primer. The reactions were thermal cycled in Bio-Rad C1000 thermal cycler using separate protocols for cell cDNA and detection cDNA. Thermal profile for cell cDNA: 95°C for 3 min, 13 cycles of 98°C for 20 s, 60°C for 45 s, 72°C for 60 s, and 72°C for 5 min in the last cycle, hold at 4°C. Thermal profile for detection cDNA: 95°C for 3 min, 15 cycles of 98°C for 20 s, 60°C for 45 s, 72°C for 30 s, and 72°C for 5 min in the last cycle, hold at 4°C. Final DNA libraries were cleaned up using two rounds of 0.6X ampure XP for the cell library and two rounds of 0.8Xampure XP for the DO library. Purified final DNA libraries were QC’ed on an Agilent Bioanalyzer before sequencing. Sequencing and analysis. The DNA libraries were sequenced in the Illumina Nextseq 500 / 550 system using the high output 150 cycles kit. Sequencing was set up for paired-end reads with 54 cycles for Read 1, 75 cycles for Read 2, and 8 cycles for index 1. Sequencing data was analyzed using the Omnition analysis software, development version, from Bio-Rad. Results. Three metrics were employed to assess data for effective bead merging; they are correlation of observed and expected values for cell recovery, cell crosstalk, and bead distribution. FIG.4A showed 4242 cells recovered from an initial 8000 cell input (400 cells / µl x 20 µl). The 53% yield is consistent with the expected 64% cell recovery based on double poisson calculation of bead 1.19λ and cell 0.08λ. The lower observed yield can be explained by loss in dead volume and during droplet transfer. FIG.4B displayed 4.01% crosstalk between HEK293 and NIH3T3 cells and matched with the 4.1% expected crosstalk at 8000 cell input. This indicated cell data were of single-cells and confirmed that the observed lower cell yield was not the result over-merged beads. Lastly, the observed and expected bead distribution in FIG 5 also closely matched each other and supported the effectiveness of bead deconvolution. In all, the data demonstrated the feasibility of using the method and composition described herein to co-localize beads to discrete droplets.

[0079] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

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) a different dsDNA detection oligonucleotide, wherein the detection oligonucleotide comprises 5’-3’: a promoter, a partition barcode sequence specific for the partition in which the detection oligonucleotide resides, and a poly A 3’ sequence; (ii) an RNA polymerase that recognizes the promoter; (iii) a reverse transcriptase; (iv) a single lysed cell or single lysed nucleus and cellular RNA; and (v) a bead linked to multiple copies of a barcoding oligonucleotide comprising 5’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a poly T 3’ end sequence; wherein at least one partition contains (a) a first bead linked to a first barcoding oligonucleotide comprising a first bead-specific barcode sequence and (b) a second bead linked to a second barcoding oligonucleotide comprising a second bead-specific barcode sequence; in the partitions, generating a plurality of detection RNAs comprising the partition barcode sequence and a poly A tail sequence by transcribing the detection oligonucleotides with the RNA polymerase; in the partitions, releasing the barcoding oligonucleotides from the beads; in the partitions performing reverse transcription, wherein the performing comprises: (i) annealing some of the detection RNA poly A tail sequences to some copies of the barcoding oligonucleotides and forming first strand detection cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the detection RNAs as a template, wherein the first strand detection cDNAs comprise a bead-specific barcode sequence and a partition barcode sequence, wherein in the at least one partition (a) a first first strand cDNA in the partition comprises the first bead-specific barcode sequence and a specific partition barcode sequence and (b) a second first strand cDNA in thepartition comprises the second bead-specific barcode sequence and the specific partition barcode sequence, and (ii) annealing cellular RNAs to some copies of the barcoding oligonucleotides and forming first strand cellular cDNAs by extending the barcoding oligonucleotides with the reverse transcriptase using the cellular RNAs as a template; performing second strand synthesis to form second strand detection cDNAs and second strand cellular cDNAs using first strand detection cDNAs and first strand cellular cDNAs, respectively, as templates, thereby forming double-stranded detection cDNAs and double-stranded cellular cDNAs; generating a bulk mixture by combining contents of the partitions; and determining the nucleotide sequence of the double-stranded detection cDNAs and double-stranded cellular cDNAs, wherein if two bead bead-specific barcode sequences are linked to the same partition barcode sequence, then sequencing reads comprising either of the two bead bead-specific barcode sequences are from the same partition.

2. The method of claim 1, wherein at least some of the different partitions comprise two or more different dsDNA detection oligonucleotides having different partition barcode sequences specific for the partition in which the detection oligonucleotide resides.

3. The method of claim 1, wherein the detection oligonucleotide comprises a sequencing adapter sequence between the promoter and the barcode sequence.

4. The method of claim 1, wherein the providing comprises providing partitions comprising intact cells and subsequently lysing the cells in the partitions.

5. The method of claim 1, wherein the intact cells are fixed cells.

6. The method of claim 1, wherein the providing comprises providing partitions comprising isolated cell nuclei comprising cellular RNA and subsequently lysing the nuclei in the partitions.

7. The method of claim 1, after performing second strand synthesis: inactivating the polymerase and reverse transcriptase 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 1-8, further comprising size selecting nucleic acids in the bulk mixture to separate detection cDNAs from cellular cDNAs.

11. The method of any one of claims 1-10, after generating the bulk mixture and before determining the nucleotide sequence: further comprising generating random breaks in the double-stranded cellular cDNAs and randomly inserting at the breaks adaptor oligonucleotides, thereby forming cellular cDNA fragments comprising at least one 5’ end linked to an adaptor oligonucleotide, wherein the adaptor oligonucleotide comprises a second universal sequence, wherein some of the cellular cDNA fragments also comprise a bead bead-specific barcode sequence and the first universal sequence.

12. The method of claim 11, wherein the random breaks are induced by a transposase that inserts the adaptor oligonucleotides.

13. The method of any one of claims 1-12, wherein the dsDNA detection oligonucleotide further comprises a second universal adaptor sequence 5’ of the partition barcode sequence and wherein the double-stranded detection cDNAs comprise the first universal adaptor sequence and the second universal adaptor sequence, and the method further comprises amplifying the double-stranded detection cDNAs and the cellular cDNA fragments that comprise the first universal adaptor sequence and the second universal adaptor sequence with a DNA polymerase and a first primer that anneal to the first universal adaptor sequence and a second primer that anneals to the second universal primer sequence.

14. The method of any one of claims 1-13, wherein the promoter is a T7 or SP6 promoter and the RNA polymerase is T7 or SP6 RNA polymerase, respectively.

15. The method of any one of claims 1-14, wherein the partitions are droplets in an emulsion or microwells.

16. The method of any one of claims 1-15, wherein the cell is a mammalian cell.

17. The method of any one of claims 1-16, wherein the bead is a hydrogel bead.

18. The method of any one of claims 1-17, wherein: generating the plurality of detection RNAs occurs at a temperature of between 35-50 degrees Celsius; performing the reverse transcription occurs at a temperature of between 37-55 degrees Celsius; and / or performing the second strand synthesis occurs at a temperature of between 60- 70 degrees Celsius.

19. A plurality of partitions, wherein different partitions comprise (i) a different dsDNA detection oligonucleotide, wherein the detection oligonucleotide comprises 5’-3’: a promoter, a partition barcode sequence specific for the partition in which the detection oligonucleotide resides, and a poly A 3’ sequence; (ii) an RNA polymerase that recognizes the promoter; (iii) a reverse transcriptase; (iv) a lysed cell and cellular RNA; and (v) a bead linked to multiple copies of a barcoding oligonucleotide comprising 5’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a poly T 3’ end sequence; wherein at least one partition contains (a) a first bead linked to a first barcoding oligonucleotide comprising a first bead-specific barcode sequence and (b) a second bead linked to a second barcoding oligonucleotide comprising a second bead-specific barcode sequence.

20. The plurality of partitions of claim 19, wherein at least some of the different partitions comprise two or more different dsDNA detection oligonucleotides having different partition barcode sequences specific for the partition in which the detection oligonucleotide resides.

21. The plurality of partitions of claim 19, wherein the detection oligonucleotide comprises a sequencing adapter sequence between the promoter and the barcode sequence.

22. The plurality of partitions of claim 19, wherein the promoter is a T7 or SP6 promoter and the RNA polymerase is T7 or SP6 RNA polymerase, respectively.

23. The plurality of partitions of any one of claims 19-22, wherein the partitions are droplets in an emulsion or microwells.

24. The plurality of partitions of any one of claims 19-23, wherein the cell is a mammalian cell.

25. The plurality of partitions of any one of claims 19-24, wherein the bead is a hydrogel bead.

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