Methods and compositions for unvoliding point area barcodes

Abstract

The invention relates to a method and a composition for uncoiling a point bar code. Methods of assessing the presence or absence of a plurality of differential barcode primers in a partition are provided.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980011307.2.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 624,400, filed on January 31, 2018, which is incorporated herein by reference for all purposes. Background Art

[0004] The beads of coupled oligonucleotides are used for microfluidic detection applications, such as high-throughput sequencing with many different partitions (e.g., droplets). In order to uniquely identify each partition, a unique barcode sequence can be used to mark beads. However, in order to ensure that the partition has only one bead and is therefore uniquely marked by a barcode, the bead concentration is usually adjusted so that only about one-tenth of the partition is occupied by beads. This results in the low utilization of the partition, and increases the reagent required for the detection sample and the amount of the sample. Increasing the bead concentration will result in higher partition occupancy and larger partition utilization. The amount of the sample and reagent required for the detection sample will be reduced. On the contrary, a higher bead concentration will result in a greater number of partitions having more than one bead. Therefore, some divided samples will be marked by more than one barcode. In some cases, the sample is segmented between more than one barcode, which results in the loss of the sensitivity of each barcode. SUMMARY OF THE INVENTION

[0006] In some embodiments, a method for detecting the presence or absence of multiple barcodes in a partition is provided. In some embodiments, the method comprises,

[0007] forming partitions, the partitions comprising: a forward primer comprising a barcode and a capture sequence, the capture sequence being complementary to: a 3′ sequence of the target nucleic acid, or a reverse complement thereof, wherein different partitions comprise different forward primers having different barcode sequences, and a partition ID tag oligonucleotide comprising a reverse complement of the capture sequence and a variable partition ID tag sequence;

[0008] in the partition, hybridizing at least one forward primer to the partition ID tag oligonucleotide to form a hybridization product;

[0009] amplifying the hybridization products to form amplicons, wherein at least some of the amplicons are formed from the forward primer and the partition ID tag oligonucleotide; and

[0010] The amplicons are sequenced, wherein if different forward primers form amplicons with the same variable partition ID tag sequence, then the different forward primers are considered to be from the same partition, wherein

[0011] The forward primer and the partition ID tag oligonucleotide are ligated to the same substrate when delivered to the partition; or

[0012] The partition ID tag oligonucleotide has a blocked 3' end such that a polymerase cannot extend the blocked 3' end during amplification; or

[0013] The partition ID tag oligonucleotide comprises a double-stranded variable partition ID tag sequence and one or two single-stranded 3' ends, wherein the single-stranded 3' ends comprise the reverse complement sequence of the capture sequence.

[0014] In some embodiments, the forward primer, the partition ID tag oligonucleotide, or both comprise a universal adaptor sequence.

[0015] In some embodiments, at least some of the partitions contain the target nucleic acid.In some embodiments, amplifying further amplifies the target nucleic acid using a forward primer and a target reverse primer to form an amplification product, and the sequencing comprises sequencing the amplification product.

[0016] In some embodiments, amplifying comprises extending a forward primer with a polymerase using the partition ID tag oligonucleotide as a template to form an amplicon comprising the reverse complement of the partition ID tag oligonucleotide and the forward primer.

[0017] In some embodiments, in the partition forming step, the forward primer is connected to a bead. In some embodiments, multiple copies of a partition ID tag oligonucleotide are connected to each other. In some embodiments, multiple copies are connected to each other through a bead. In some embodiments, during the partition formation, the forward primer and the partition ID tag oligonucleotide are connected to each other through a cleavable joint, and the method further comprises cutting the cleavable joint before the amplification. In some embodiments, the bead comprises at least 10 (e.g., 100, 1000, 10000) times the number of forward primers compared to the partition ID tag oligonucleotides connected to the bead.

[0018] In some embodiments, primers are cleaved from the beads prior to amplification.

[0019] In some embodiments, the partition ID tag oligonucleotide is single stranded.

[0020] In some embodiments, the variable partition ID tag sequence is double-stranded. In some embodiments, the 3' end of each strand of the double-stranded variable partition ID tag sequence is linked to the complement of the capture sequence.

[0021] In some embodiments, during the partition forming step, a partition ID tag oligonucleotide is attached to a solid support.

[0022] In some embodiments, the partition ID tag oligonucleotide has a blocked 3' end such that a polymerase cannot extend the blocked 3' end during amplification.

[0023] In some embodiments, the partition ID tag oligonucleotide comprises an affinity tag. In some embodiments, the affinity tag is biotin, an antibody, or an aptamer.

[0024] In some embodiments, the partitions are droplets.

[0025] In some embodiments, the partitions contain an average of 2-100000 copies of a partition ID tag oligonucleotide per partition. In some embodiments, the partitions contain an average of 10-1000 or 100-10,000 copies of a partition ID tag oligonucleotide per partition.

[0026] In some embodiments, the contents of the partitions are pooled prior to amplification.

[0027] In some embodiments, a target reverse primer is present in a partition.

[0028] In some embodiments, the target reverse primer is introduced after the contents of the partitions are combined.

[0029] In some embodiments, amplification occurs while the hybridization products are within the partitions.

[0030] In some embodiments, a method for detecting the presence or absence of a plurality of barcodes in a partition is provided. In some embodiments, the method comprises

[0031] A partition is formed, the partition comprising:

[0032] a forward primer comprising a barcode and a capture sequence, the capture sequence being complementary to: a 3′ sequence of the target nucleic acid, or a reverse complement thereof, wherein different partitions comprise different forward primers having different barcode sequences,

[0033] a partition ID tag oligonucleotide comprising a 5′ binding sequence and a 3′ variable partition ID tag sequence;

[0034] ligating the target nucleic acid to the forward primer and the target reverse primer, the target reverse primer having a 3' sequence identical to the 5' sequence of the target nucleic acid, or a reverse complement thereof, wherein the ligation also results in some products in which the forward primer is linked to the partition ID tag oligonucleotide; and

[0035] The products are sequenced, wherein if different forward primers form products with the same variable partition ID tag sequence, then the different forward primers are considered to be from the same partition, wherein

[0036] The forward primer and the partition ID tag oligonucleotide are attached to the same bead when delivered to the partition; or

[0037] The partition ID tag oligonucleotide has a blocked 3' end such that a polymerase cannot extend the blocked 3' end during amplification; or

[0038] The partition ID tag oligonucleotide comprises a double-stranded variable partition ID tag sequence and one or two single-stranded 3' ends, wherein the single-stranded 3' ends comprise the reverse complement sequence of the capture sequence.

[0039] In some embodiments, at least some of the partitions contain a target nucleic acid.

[0040] In some embodiments, the method also includes providing a bridge single-stranded nucleic acid in the partition, wherein the bridge single-stranded nucleic acid comprises a 3′ sequence of the target nucleic acid and a reverse complementary sequence of a 5′ binding sequence of the partition ID tag oligonucleotide from 3′ to 5′, so that hybridization of the forward primer and the partition ID tag oligonucleotide causes the 3′ end of the forward primer to be adjacent to the 5′ end of the partition ID tag oligonucleotide, and wherein the 3′ end of the forward primer is connected to the 5′ end of the partition ID tag oligonucleotide.

[0041] In some embodiments, in the partition forming step, the forward primer is connected to a bead. In some embodiments, multiple copies of a partition ID tag oligonucleotide are connected to each other. In some embodiments, multiple copies are connected to each other through a bead. In some embodiments, during the partition formation, the forward primer and the partition ID tag oligonucleotide are connected to each other through a cleavable joint, and the method further comprises cutting the cleavable joint before the connection. In some embodiments, the bead comprises at least 10 (e.g., 100, 1000, 10000) times the number of forward primers compared to the partition ID tag oligonucleotides connected to the bead.

[0042] In some embodiments, the primers are cleaved from the beads prior to ligation.

[0043] In some embodiments, the partition ID tag oligonucleotide is single stranded.

[0044] In some embodiments, the variable partition ID tag sequence is double-stranded.

[0045] In some embodiments, during the partition forming step, the partition ID tag oligonucleotide is attached to a solid support.

[0046] In some embodiments, the partition ID tag oligonucleotide has a blocked 3' end such that a polymerase in the amplification cannot extend the blocked 3' end.

[0047] In some embodiments, the partition ID tag oligonucleotide comprises an affinity tag. In some embodiments, the affinity tag is biotin, an antibody, or an aptamer.

[0048] In some embodiments, the partitions are droplets.

[0049] In some embodiments, the partitions contain an average of 2-100,000 copies of a partition ID tag oligonucleotide per partition. In some embodiments, the partitions contain an average of 10-1000 or 100-10,000 copies of a partition ID tag oligonucleotide per partition.

[0050] In some embodiments, a plurality of partitions are provided. In some embodiments, the partitions include:

[0051] one or more forward primers having a barcode and a capture sequence that is complementary to: a 3' sequence of the target nucleic acid, or a reverse complement thereof, wherein different partitions contain different forward primers having different barcode sequences, and

[0052] A partition ID tag oligonucleotide comprising a complementary sequence to the capture sequence and a variable partition ID tag sequence.

[0053] In some embodiments, at least some of the partitions contain a target nucleic acid.

[0054] In some embodiments, the partition further comprises a target reverse primer having a 3' sequence identical to the 5' sequence of the target nucleic acid, or a reverse complement thereof.

[0055] In some embodiments, the forward primer is attached to a bead.

[0056] In some embodiments, a plurality of partition ID tag oligonucleotides are connected to each other. In some embodiments, the plurality of partition ID tag oligonucleotides are connected to each other by beads. In some embodiments, the forward primer and the partition ID tag oligonucleotide are connected to each other by a cleavable joint, and the method further comprises cutting the cleavable joint before amplification. In some embodiments, the beads comprise at least 10 (e.g., 100, 1000, 10000) times the number of forward primers compared to the partition ID tag oligonucleotides connected to the beads.

[0057] In some embodiments, primers are cleaved from the beads prior to the amplifying.

[0058] In some embodiments, the partition ID tag oligonucleotide is single stranded.

[0059] In some embodiments, the variable partition ID tag sequence is double-stranded.

[0060] In some embodiments, the 3' end of each strand of the double-stranded variable partitioned ID tag sequence is linked to a complementary sequence of the capture sequence.

[0061] In some embodiments, the partition ID tag oligonucleotide is attached to a solid support.

[0062] In some embodiments, the partition ID tag oligonucleotide has a blocked 3' end such that a polymerase in the amplification cannot extend the blocked 3' end.

[0063] In some embodiments, the partition ID tag oligonucleotide comprises an affinity tag. In some embodiments, the affinity tag is biotin, an antibody, or an aptamer.

[0064] In some embodiments, the partition further comprises a target reverse primer.

[0065] In some embodiments, the partitions are droplets.

[0066] In some embodiments, the partitions contain an average of 2-100,000 copies of a partition ID tag oligonucleotide per partition.

[0067] In some embodiments, the partitions contain an average of 10-1000 or 100-10,000 copies of a partition ID tag oligonucleotide per partition.

[0068] In some embodiments, the plurality of partitions comprises at least 100 (e.g., at least 1,000, 10,000, 100,000) partitions.

[0069] definition

[0070] Unless otherwise indicated, all scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, nucleic acid chemistry, and hybridization described below are well known and commonly used in the art. Nucleic acid and peptide synthesis are performed using standard techniques. These techniques and procedures are performed according to conventional methods described in the art and various general references (generally referring to Sambrook et al., "Molecular Cloning: A Laboratory Manual" (MOLECULAR CLONING: A LABORATORY MANUAL), 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (Cold Spring Harbor, NY), which is incorporated herein by reference), and these references are provided in full.

[0071] A "partition ID tag" refers to a unique combination of nucleic acid sequences or sequences in a particular partition. Partition ID tags can be introduced into each partition as part of a longer polynucleotide sequence, for example, with a capture sequence or its complement at the 3' end of the polynucleotide for subsequent connection to a forward primer. The partition ID tag sequence can be any length desired. As a non-limiting example, the length of the partition ID tag can be, for example, 4-50 or more nucleotides and can be continuous or non-continuous (for example, in two or more parts separated by a non-tag (e.g., spacer) sequence). Each partition will typically contain a different partition ID tag, for example, which can be generated randomly or non-randomly.

[0072] The term "amplification reaction" refers to any in vitro method for multiplying copies of a nucleic acid target sequence in a linear or exponential manner. These 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 involving RNA polymerization initiated by T7, T3 or SP6), such as 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 displacement amplification (SDA); multiple displacement amplification (MDA); rolling circle amplification (RCA); and other methods known to those skilled in the art. See, for example, Fakruddin et al., J. Pharm Bioallied Sci. 20135(4): 245-252.

[0073] "Amplification" refers to the step of placing a solution under conditions sufficient to amplify a polynucleotide (if all components of the reaction are intact). The components of an amplification reaction include, for example, primers, a polynucleotide template, a polymerase, nucleotides, etc. The term "amplification" generally refers to the "exponential" growth of a target nucleic acid. However, as used herein, "amplification" may also refer to the linear growth of the number of a selected target sequence of a nucleic acid, such as obtained by cycle sequencing or linear amplification.

[0074] "Polymerase chain reaction" or "PCR" refers to a method by which a specific segment or subsequence of a target double-stranded DNA is amplified exponentially. PCR is well known to those skilled in the art; see, for example, U.S. Patent 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 generally include two or three-step cycles. A two-step cycle has a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step followed by a separate extension step.

[0075] "Primer" refers to a polynucleotide sequence that hybridizes with a sequence on a target nucleic acid, and optionally, is used as a starting point for nucleic acid synthesis. Primers can have a variety of lengths. In some embodiments, the primer length is less than 100 or 50 nucleotides, for example, a length of about 10 to about 900, about 15 to about 80, or about 30-85 to about 30 nucleotides. The length and sequence of primers for amplification reactions (e.g., PCR) can be designed based on principles known to those skilled in the art, see, for example, Innis et al., 1990, PCR Protocols: A Guide to Methods and Applications. Primers can include or be formed entirely of DNA, RNA, or non-natural nucleotides. In some embodiments, a primer comprises one or more modified and / or non-natural nucleoside bases. In some embodiments, a primer comprises a marker (e.g., a detectable marker).

[0076] A nucleic acid or a portion thereof "hybridizes" with another nucleic acid under certain conditions so that non-specific hybridization is minimized in a physiological buffer at a defined temperature. In some cases, a nucleic acid or a portion thereof hybridizes with a conserved sequence common between a group of target nucleic acids. In some cases, if there are at least about 6, 8, 10, 12, 14, 16 or 18 consecutive complementary nucleotides including "universal" nucleotides complementary to more than one nucleotide partner, a primer or a portion thereof can hybridize to a primer binding site. Alternatively, if there are less than 1 or 2 complementary mismatches in at least about 12, 14, 16 or 18 consecutive complementary nucleotides, a primer or a portion thereof can hybridize to a primer binding site. In some embodiments, the defined temperature for specific hybridization is room temperature. In some embodiments, the defined temperature for specific hybridization is higher than room temperature. In some embodiments, the defined temperature for specific hybridization is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75 or 80°C.

[0077] 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 modifications of chemical groups that provide for the introduction of other charges, polarizability, hydrogen bonds, electrostatic interactions, connection points and action points with nucleic acid ligand bases or nucleic acid ligands as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNA), phosphodiester group modifications (e.g., phosphorothioate, methylphosphonate), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at the exocyclic amine, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, methylation, unusual base pairing combinations such as isobases, isocytidines and isoguanidines, etc. Nucleic acids may also contain non-natural bases, such as nitroindole. Modifications may also include 3' and 5' modifications, including but not limited to capping with fluorophores (e.g., quantum dots) or other moieties.

[0078] As used herein, the term "partition" or "partitioned" refers to dividing a sample into multiple parts or multiple "partitions". Partitions can be solid or fluid. In some embodiments, partitions are solid partitions, for example, microchannels or holes (i.e., in a porous microtiter dish). In some embodiments, partitions are fluid partitions, for example, droplets. In some embodiments, fluid partitions (e.g., droplets) are mixtures of immiscible fluids (e.g., water and oil). In some embodiments, fluid partitions (e.g., droplets) are aqueous droplets surrounded by an immiscible carrier fluid (e.g., oil).

[0079] 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 in length) that identifies the molecule to which it is coupled. In some embodiments, a barcode is used to identify molecules in a partition. Such a partition-specific barcode should be unique to the partition relative to the barcodes of other partitions. For example, a partition containing target RNA from a single cell can be subjected to reverse transcription conditions, and primers containing different partition-specific barcode sequences are used in each partition, thereby incorporating a copy of a unique "cell barcode" into the reverse transcribed nucleic acid of each partition. Thus, nucleic acids from each cell can be distinguished from nucleic acids of other cells by a unique "cell barcode". In some embodiments, a barcode is present on an oligonucleotide coupled to a particle, wherein the "particle barcode" is shared by all or substantially all oligonucleotides coupled to the particle (e.g., identical or substantially identical between them).

[0080] In some embodiments, a barcode is provided in a substrate. In some embodiments, the substrate includes a single barcode sequence. In some embodiments, the substrate includes a repeated clonal barcode sequence. As used herein, "a substrate including a repeated clonal barcode sequence" refers to a composition comprising a plurality of identical barcode sequences, the identical barcode sequences being physically connected to each other (e.g., a tandem repeat barcode sequence separated by a cleavable joint between each repeat sequence in a hairpin molecule, a linear nucleic acid polymer, or a circular nucleic acid polymer) or isolated from other components when delivered to a partition (e.g., encapsulated in a droplet delivered to a partition). In some embodiments, individual barcode sequences are released from substrates or from repeated clonal barcode sequences in partitions (e.g., released from a hairpin, a linear nucleic acid polymer, or a circular nucleic acid polymer by cutting a polymer at a cleavable joint, or released from a droplet by breaking a droplet), and are associated with particles in a partition, for example, by annealing a cloned barcode sequence to an oligonucleotide on a partition or by connecting a cloned barcode sequence to an oligonucleotide on a partition. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Aspects of using label tags to detect multiple barcodes in partitions are described. The above figure describes the barcoding of target sequences, wherein the barcode (also referred to as "forward primer" in this article) is associated with the target nucleic acid sequence through a capture sequence. For example, the capture sequence connected to the barcode can be complementary to the capture sequence of the target. The resulting product comprises a barcode, a capture sequence, and a target sequence. The box below describes the label tag barcode sequence. In this aspect, the forward primer barcode is associated with the partition ID tag through the interaction between the barcode oligonucleotide (forward primer) and the capture sequence on the label tag oligonucleotide. The resulting product of the reaction is a barcode covalently connected to the partition ID tag through the capture sequence. The depicted reaction can occur in parallel in the same partition with the same or varying workflows. The products of these reactions can be further prepared for simultaneous analysis using the same workflow, or for separation and extraction by different workflows. Although the accompanying drawings show oligonucleotides without PCR manipulation sequences, it should be understood that in some embodiments, such sequences may be present. PCR processing sequences can be included, for example, as part of the forward primer, target reverse primer, and reverse tag primer, thereby allowing universal sequences to be used for subsequent amplification of the resulting products.

[0082] Figure 2Another configuration of the partition ID tag delivered to the partition is described. In all embodiments described, the capture sequence is connected to the partition ID tag sequence. The upper part of the figure shows that the tag sequence can be single-stranded or double-stranded (for example, partially double-stranded at the partition ID tag sequence and single-stranded at the capture sequence). The middle part of the figure shows that the tag sequence can be delivered to the partition connected to the solid support or connected to another molecule. The lower part of the figure shows that the barcode sequence and the tag sequence can be delivered as part of the same molecule (separated later), and in some embodiments, a solid support is optionally further connected.

[0083] Figure 3 Describes aspects of single-stranded partition ID tags and multiple barcode beads in solution. Circles represent partitions where the oligonucleotide capture and sample preparation steps shown result in barcodes covalently bound to target and tag molecules. This partition is then combined with the contents of other partitions (e.g., by breaking the emulsion) for downstream sample preparation and / or analysis. Figure 3 Having described a single TAG1 oligonucleotide interacting first with BC1 and then with BC2, it will be appreciated that in some embodiments multiple copies of the same partition ID tag will be present in a partition, such that different copies of the partition ID tag interact with different bead forward primers.

[0084] Figure 4 The first part of an example workflow for single-stranded partition ID tags involving multiple PCR cycles is described (allowing the same partition ID tag sequence to be combined with multiple forward primer barcode sequences). As shown, the partition ID tag has a 3' dideoxynucleotide that prevents it from being extended by the polymerase. The workflow shows two beads with different barcodes (BC1 and BC2) in the same partition (such as the droplet). The forward primer attached to the bead is cleaved to release the barcoded forward primer.

[0085] Figure 5 yes Figure 4 Continuation of the workflow. Section 2) describes the extension of a forward primer with BC1 using the partition ID tag as a template. Section 3) describes the extension of a forward primer with BC2 using the partition ID tag as a template. As shown in section 4), two different forward barcodes are associated with the same partition ID tag (denoted as TAG1) indicating that the two forward primer sequences are in the same partition.

[0086] Figure 6 The use of partition ID tags in single-stranded solutions without chain terminators is described. This aspect is similar to Figure 4-5 , but in some cases the partition ID tag itself may be extended. However, because the BC1 and BC2 forward primers have the same capture zone, BC1 and BC2 amplicons will be generated that each contain TAG1.

[0087] Figure 7 Partially double-stranded partitioned ID tags and multiple barcode beads in solution are described.

[0088] Figure 8 Part 1 of an example workflow for double-stranded partition ID tags is described. As shown, the partition ID tag is double-stranded at the tag sequence and in the constant regions flanking the tag sequence. The separated 3′ end contains a single-stranded capture region. The barcoded forward primer is described directly below the partition ID tag. The workflow shows two beads with different barcodes (BC1 and BC2) in the same partition (such as the droplet described). The forward primer attached to the beads is cut to release the barcoded forward primer. This workflow does not use multiple PCR steps, so different barcodes appear for the forward and reverse tag sequences. Only a single oligonucleotide extension step occurs in the partition, rather than multiple cycles of melting, annealing, and extension as in PCR. The various tagging molecules in this workflow involve merging the tag sequence with the barcode of one bead, and combining the reverse complement of the tag sequence with the barcode of the second bead during the extension step. This will be described in detail in the next section. Fig. 9 is further described in the following description of .

[0089] Fig. 9 yes Figure 8 Continuation of workflow. In part 2 of this workflow, the barcoded forward primer hybridizes through the capture sequence of the partitioned ID tag, while extension in part 3 generates amplicons in which two different barcodes (BC1 and BC2) are each associated with the tag sequence or its reverse complement.

[0090] Fig.10 Described is the delivery of a partitioned ID tag attached to a solid particle having a plurality of barcode beads.

[0091] Fig.11 Such aspects are described, wherein the partition ID tag and the forward primer of barcoding are connected to the same bead. "SINGLE BEAD" workflow shows the situation of only one bead in the partition. "TWO BEADS" workflow shows the situation of two beads in the partition. In either aspect, the oligonucleotide is released from the bead in the partition and then replicated. In the latter case, the resulting product will include sequencing reads, wherein the tag sequence and the barcode are mismatched compared to the initial bead, which shows that the two beads are located in the same partition.

[0092] Fig.12Described are partition ID tags and barcodes that are established on the same bead and also serve as part of a single cleavable molecule. In the aspect, the partition ID tag and barcoded forward primer sequence are parts of an oligonucleotide separated by a cleavable sequence (which may be present in multiple copies), thereby allowing the subsequent separation of the partition ID tag and forward primer in the partition. The workflow will also continue as described in other parts of this article.

[0093] Fig.13 Shows the use Figure 6 Barcode detection data for the partition ID label scheme shown in . DETAILED DESCRIPTION

[0094] Introduction

[0095] A finite number of entities can be distributed among a certain number of partitions. This means that you may encounter partitions with 0, 1, 2, 3, etc. entities in a particular partition. Especially at lower concentrations (e.g., <20 entities per partition on average), the distribution of what percentage of partitions contain what number of entities follows a Poisson distribution. In some applications, the entities to be partitioned are beads, and the partitions themselves are droplets.

[0096] Partition barcodes or mixtures thereof can be used to label molecules of interest (e.g., mRNA, cDNA, DNA, etc.) within a particular partition. Ideally, it is desirable to have only one barcode per partition. Because this is not always feasible, the methods described herein provide methods for determining what partition barcodes or barcode combinations exist within each partition. The methods described herein provide for adding partition ID tags to partitions. These partition ID tags label ("label") the barcodes. Therefore, barcodes occupying the same partition will be labeled with the same partition ID or a combination of partition IDs.

[0097] The inventor has found how to detect the presence of multiple beads loaded with bead-specific barcode primers in a partition. The presence of more than one differential barcoded primer (e.g., two differential barcoded forward primers connected to different beads in the same partition) can interfere with sequence analysis and quantification, because it is assumed that different barcodes come from different partitions, and in fact some parts of the barcode appear together (e.g., as a function of Gaussian distribution). By being able to detect what kind of barcode appears in the same partition, deconvolution can be performed (i.e., determining that multiple barcodes are from the same partition and explaining it in sequencing analysis), and using data from such combinations or not considering (e.g., discarding) data from such partitions, making the remaining data more "clean" compared to background effects. The inventor has found that partition ID tag sequences can be introduced into partitions, and they can be associated with some parts of forward primers in the partitions. By sequencing these associated products, these partitions with multiple differential barcoded forward primers can be detected, because they will be associated with the same partition ID tag (or its complementary sequence). Various ways of delivering and associating partition ID tags and forward primers are described herein.

[0098] Figure 1 An embodiment is described in which a target sequence is barcoded by a partition barcoding primer present in a partition (top of the figure). In the same partition, a partition ID tag oligonucleotide is linked to several copies of the partition barcoding primer to link the partition ID tag sequence of the partition ID tag oligonucleotide to the partition barcode, thereby allowing detection of multiple barcodes in a partition (if present).

[0099] Figure 3 An aspect is described in which two forward primers with different barcodes are in the same partition. Although the forward primers are described as being connected to beads, this aspect is not required. Included in the partitions are some copies of single-stranded partition ID tag oligonucleotides (having capture and partition ID tag sequences) and target nucleic acids (having capture and test (e.g., sequence to be determined) sequences). Some copies of the forward primers are connected to the target nucleic acid via the capture sequence, while other copies of the forward primers are connected to the partition ID tag oligonucleotides via the capture sequence. The connection of different barcodes (BC1 and BC2) to the same partition ID tag sequence (TAG1) indicates the presence of two different forward primers (associated with BC1 and BC2) in the same partition.

[0100] Such partitions are provided, which include forward primers, and in some embodiments also include reverse primers for amplifying target nucleic acids. In many cases, it is convenient to deliver a solid support (e.g., beads) connected to a barcoded forward primer for the partition, or form a partition around a solid support (e.g., beads) connected to a barcoded forward primer, wherein the target is a 1:1 distribution of beads and partitions connected to the forward primer. However, due to the Gaussian distribution, some partitions will include more than one forward primer bead. The method and composition for partitioning are described in, for example, disclosed patent applications WO 2010 / 036,352, US 2010 / 0173,394, US 2011 / 0092,373 and US 2011 / 0092,376, the entire contents of which are incorporated herein by reference. Multiple partitions can be multiple emulsion droplets, or multiple micropores, etc.

[0101] In some embodiments, one or more reagents are added during droplet formation, or one or more reagents are added to the droplet after droplet formation. Methods and compositions for delivering reagents to one or more partitions include microfluidic methods known in the art; droplet or microcapsule merging, coalescence, fusion, rupture or degradation (e.g., as described in US2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g., as described in WO2010 / 151,776); and combinations thereof.

[0102] As described herein, a partition can be a picometer, nanometer, or micrometer hole. A partition can be a picometer, nanometer, or micrometer reaction chamber, such as a picometer, nanometer, or micrometer capsule. A partition can be a picometer, nanometer, or micrometer channel. A partition can be a droplet, such as an emulsion droplet.

[0103] In some embodiments, the partition is a droplet. In some embodiments, the droplet comprises an emulsion composition, i.e., a mixture of immiscible fluids (such as water and oil). In some embodiments, the droplet is an aqueous droplet, which is surrounded by an immiscible carrier fluid (such as oil). In some embodiments, the droplet is an oily droplet, which is surrounded by an immiscible carrier fluid (such as an aqueous solution). In some embodiments, the droplet described herein is relatively stable and has 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 the droplets generated by the sample coalesce with other droplets. These emulsions can also have limited flocculation, a process in which a dispersed phase is produced as a suspension in a thin sheet. In some cases, this stability or minimal aggregation can be maintained for up to 4, 6, 8, 10, 12, 24, or 48 hours or longer (e.g., at room temperature, or at about 0, 2, 4, 6, 8, 10, or 12° C.). In some embodiments, the oil phase is flowed over the aqueous phase or reagent to form droplets.

[0104] The oil phase may include a fluorinated base oil, which may be further stabilized by being used in conjunction with a fluorinated surfactant such as a perfluoropolyether. In some embodiments, the base oil includes one or more of: HFE 7500, FC-40, FC-43, FC-70 or other common fluorinated oils. In some embodiments, the oil phase includes an anionic fluorosurfactant. In some embodiments, the anionic fluorosurfactant is an ammonium salt of Ammonium Krytox (Krytox-AS), Krytox FSH, or a morpholino derivative of Krytox FSH. The concentration of Krytox-AS may be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% or 4.0% (w / w). In some embodiments, the concentration of Krytox-AS is about 1.8%. In some embodiments, the concentration of Krytox-AS is about 1.62%. The concentration of the morpholino derivative of Krytox FSH can be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% or 4.0% (w / w). In some embodiments, the concentration of the morpholino derivative of Krytox FSH is about 1.8%. In some embodiments, the concentration of the morpholino derivative of Krytox FSH is about 1.62%.

[0105] In some embodiments, the oil phase also includes additives for adjusting oil properties (such as vapor pressure, viscosity or surface tension). Non-limiting examples include perfluorooctanol and 1H, 1H, 2H, 2H-perfluorodecanol. In some embodiments, 1H, 1H, 2H, 2H-perfluorodecanol is added to a concentration of about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.9%, 0.3%, 0.4%, 05%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75% or 3.0% (w / w). In some embodiments, 1H, 1H, 2H, 2H-perfluorodecanol is added to a concentration of about 0.18% (w / w).

[0106] In some embodiments, the emulsion is formulated to generate highly monodisperse droplets with a liquid-like interface membrane, which can be converted into microcapsules with a solid-like interface membrane by heating; such microcapsules can serve as bioreactors to maintain their contents through a period of incubation. Conversion to microcapsules can occur upon heating. For example, such conversion can occur at a temperature greater than about 40°, 50°, 60°, 70°, 80°, 90°, or 95°C. During the heating process, a fluid or mineral oil cover can be used to prevent evaporation. Excess continuous phase oil can be removed or left in place before heating. These microcapsules can resist aggregation and / or flocculation under a wide range of thermal and mechanical treatments.

[0107] After the droplets are converted into microcapsules, the microcapsules can be stored at about -70°C, -20°C, 0°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C. In some embodiments, the microcapsules can be used to store or transport the partition mixture. For example, a sample can be collected at one location, partitioned into droplets containing enzymes, buffers, and / or primers or other probes, optionally one or more polymerization reactions can be performed, the partitions can then be heated for microencapsulation, and the microcapsules can be stored or transported for further analysis.

[0108] In some embodiments, the sample is divided 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, 700,000 partitions, partitions, 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.

[0109] In some embodiments, the droplets 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 average diameter of the droplets generated is about 0.001 micron, about 0.005 micron, about 0.01 micron, about 0.05 micron, about 0.1 micron, about 0.5 micron, about 1 micron, 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 average diameter of the generated droplets is 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 generated droplets are non-uniform in shape and / or size.

[0110] In some embodiments, the generated droplets are substantially uniform in volume. For example, the standard deviation of the 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 the droplet volume can be less than about 10-25% of the average droplet volume. In some embodiments, the average volume of the generated droplets is 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 2.5nL, about 3nL, about 3.5nL, about 4nL, about 4.5nL, about 5nL, about 5.5nL, about 6nL, about 6.5nL, about 7nL, about 7.5nL, about 8nL, about 8.5nL, about 9nL, about 9.5nL, about 10nL, about 11nL, about 12nL, about 13nL, about 14nL, about 15nL, about 16nL, about 17nL, about 18nL, about 19nL, about 20nL, about 25nL, about 30nL, about 35nL, about 40nL, about 45nL or about 50nL.

[0111] In some embodiments, the method includes dividing the sample including one or more target nucleic acids into multiple partitions. In some embodiments, the sample including the target nucleic acid includes DNA, RNA or its combination or hybrid. In some embodiments, the sample including the target nucleic acid includes DNA or genomic DNA from a subgroup of the genome (for example, may include a selected gene of a mutation of a specific population (such as an individual susceptible to a specific type of cancer)). In some embodiments, the sample includes the genomic DNA retained by adjacency, which has been fragmented but retains adjacency by connection with the DNA fragment ends by protein (for example, Tn5 transposase (label enzyme)). In some embodiments, the sample including the target nucleic acid includes cDNA. In some embodiments, the sample including the target nucleic acid includes exon DNA (i.e., a subgroup of the whole genome DNA rich in transcribed sequences, which includes the exon group in the genome) or transcriptome DNA (i.e., all mRNAs or "transcripts" produced in a cell or cell group). In some embodiments, the sample comprising the target nucleic acid comprises a long fragment DNA (e.g., a DNA having a base pair length of at least about 300, 400, 500, 600, 700, 800, 1000 or more bases, or for double-stranded DNA). In some embodiments, the sample comprising the target nucleic acid comprises RNA, e.g., mRNA or lncRNA. In some embodiments, the target nucleic acid is double-stranded. In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the sample comprises a target nucleic acid isolated from a tissue, a cell, or a single cell sample.

[0112] In some embodiments, the sample comprising the target nucleic acid is a biological sample. The biological sample can be obtained from any organism, such as an animal, a plant, a fungus, a pathogen (such as a bacterium or a virus) or any other organism. In some embodiments, the biological sample is from an animal, such as a mammal (such as a human or non-human primate, a cow, a horse, a pig, a sheep, a cat, a dog, a mouse or a rat), a bird (such as a chicken) or a fish. The biological sample can be any tissue or body fluid obtained from an organism, such as blood, blood components or blood products (such as serum, plasma, platelets, red blood cells, etc.), sputum or saliva, tissue (such as kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage or bone tissue); cultured cells, such as primary cultures, explants, and transformed cells, stem cells, feces, urine, etc. In some embodiments, the sample is a sample comprising cells. In some embodiments, the sample is a single cell sample.

[0113] In some embodiments, the methods described herein are used for single cell analysis. Accordingly, in some embodiments, the target nucleic acid from a single cell is partitioned into a plurality of partitions. In some embodiments, the target nucleic acid from a biological sample comprising a plurality of cells is extracted and partitioned so that the individual partitions contain nucleic acid from less than one, one or more cells.

[0114] As shown above, the partitions will contain forward primers for amplification or other detection of target nucleic acids. In some embodiments, when provided in the partitions, the forward primers are connected to beads or other solid supports. Due to statistical distribution, although many partitions will contain only one forward primer (or multiple copies of only one forward primer), other partitions will contain forward primers with different sequences (e.g., different barcodes). In some embodiments, beads or other solid supports coupled to barcode-labeled forward primers are used in the methods, partition libraries, and kits described herein. In some embodiments, beads include a solid support surface having a plurality of oligonucleotide primers coupled thereto. In some embodiments, the beads include at least about 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 10 8 , 10 9 , 10 10 In some embodiments, the forward primer is double-stranded. In some embodiments, the forward primer is single-stranded.

[0115] In some embodiments, the forward primer includes a barcode sequence, wherein at least most of, substantially all or all of the multiple forward primers coupled to the solid support surface include the same barcode sequence. In some embodiments, the barcode is a sequence of about 6-about 400 nucleotides, for example, about 6-25, about 10-24, about 8-20, about 8-18, about 10-20, about 10-18 or about 12-20 nucleotides. In some embodiments, the barcode is a sequence of at least about 10 nucleotides. In some embodiments, the oligonucleotide primer coupled to a specific bead includes such a barcode sequence, which can be identical or substantially identical in a plurality of oligonucleotides on the bead, but is unique or substantially unique compared to a plurality of oligonucleotides on other beads.

[0116] The forward primer also includes a capture sequence, typically located at the 3' end of the forward primer, which hybridizes to the target sequence or its reverse complement under experimental conditions. The target sequence can be 100% complementary or partially (e.g., at least 95%, 90%, 80%, etc.) complementary, depending on the desired results and conditions. In some embodiments, the capture sequence is a sequence of about 6 to about 20 nucleotides, for example, about 6-16, about 6-14, about 8-20, about 8-18, about 10-20, about 10-18, or about 12-20 nucleotides. In some embodiments, the capture sequence is a sequence of at least about 6 or 10 nucleotides. For example, the forward primer may include a capture sequence that is complementary to the 3' sequence of the target nucleic acid or its reverse complement. In addition, the forward primer may include one or more other sequences. For example, in some embodiments, each forward primer includes a unique molecular identifier (UMI) sequence, so each copy can be tracked and counted separately.

[0117] The term "bead" refers to any solid support that may be present in a partition, e.g., a small particle or other solid support. In some embodiments, the bead comprises polyacrylamide. For example, in some embodiments, the bead incorporates the barcode oligonucleotide into a gel matrix by chemical modification of acrylamide (acrydite) attached to each oligonucleotide. Exemplary beads can also be hydrogel beads. In some cases, the hydrogel is in the form of a sol (sol). In some cases, the hydrogel is in the form of a gel (gel). An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, for example, U.S. Patent Nos. 4,438,258, 6,534,083, 8,008,476, 8,329,763; U.S. Patent Application Nos. 2002 / 0,009,591, 2013 / 0,022,569, 2013 / 0,034,592; and International Patent Application Nos. WO / 1997 / 030092 and WO / 2001 / 049240.

[0118] Methods for connecting oligonucleotides to beads are described in, for example, WO 2015 / 200541. In some embodiments, the oligonucleotides configured to connect the hydrogel and the barcode are covalently attached to the hydrogel. Many methods for covalently attaching oligonucleotides to one or more hydrogel matrices are known in the art. To name just one example, aldehyde-derivatized agarose can be covalently attached to the 5'-amine group of a synthetic oligonucleotide. In some embodiments, the forward primer is attached to a bead or solid support via a cleavable linker (as described below) and can be cleaved from the bead or solid support in the partition.

[0119] In some embodiments, a second oligonucleotide primer used as a reverse primer in combination with a first oligonucleotide primer on a target nucleic acid can be included in a partition, or optionally, after the partition is merged into a bulk reaction. For example, a target reverse primer will include a sequence that hybridizes with a reverse complementary sequence on the target under test conditions to allow, for example, an extension based on a polymerase. Therefore, for example, if a target sequence comprising a 5' sequence and a 3' sequence is present in a partition, the 3' end of the target reverse primer will be identical to the 5' sequence of the target. This will allow the 3' end of the target reverse primer to hybridize with an extension product of a forward primer using the target nucleic acid as a template. Alternatively, the target reverse primer can use the target nucleic acid as a template to initiate extension, in which case the target reverse primer 3' sequence will be the reverse complementary sequence of the target nucleic acid 5' sequence. For example, the target reverse primer may also have a universal sequence (also referred to as a "PCR handle" or "adapter" sequence) near its 5' end or its 5' end.

[0120] Also included in the partition is a partition ID tag oligonucleotide. The oligonucleotide comprises a variable partition ID tag sequence and a connection sequence (e.g., at the partition ID tag oligonucleotide 3' end), thereby allowing the partition ID tag oligonucleotide to be connected to the forward primer. In some embodiments, the connection sequence is the reverse complementary sequence of all or part (e.g., at least 6, 8, 10, 12, 14, 16 or more nucleotides) of the forward primer capture sequence or also has a sequence hybridized with the forward primer capture sequence. The variable partition ID tag sequence will be different between different partition ID tag oligonucleotides, thereby allowing the partition ID tag oligonucleotide to be distinguished from each other (thus distinguishing partitions). In some embodiments, the variable partition ID tag sequence is a sequence of about 6-about 20 nucleotides, for example, about 6-16, about 6-14, about 8-20, about 8-18, about 10-20, about 10-18 or about 12-20 nucleotides. The variable partition ID tag sequence can be random or specified, but will be different between the partition ID tag oligonucleotides in different partitions.

[0121] In some embodiments, multiple copies of the same partition ID tag oligonucleotide are provided in the partition. For example, this can be achieved when multiple copies of the partition ID tag oligonucleotide are connected to each other or to a common support delivered to the partition (optionally, the individual copies are subsequently released from each other within the partition).

[0122] In other embodiments (e.g., when the partition ID tag oligonucleotides are not connected to each other), multiple copies of the partition ID tag are introduced into each partition, but the copies in a specific partition do not have to have the same partition ID tag sequence. In these embodiments, the partition ID tag oligonucleotides have the same structure, but different partition ID tag sequences. If the length of the random partition ID tag sequence is long enough, the partition ID tag can be added as a reagent at a certain concentration, so that tens, hundreds or thousands of different tag sequences fill each partition, but the possibility of a specific tag sequence or a specific partition ID tag group filling two separate partitions will be close to zero. In these embodiments, the subsequent association of the forward primers from different beads with the same partition ID tag sequence (e.g., higher than the expected statistical level of random occurrence) will indicate that the two beads are present in the same partition. In the case where the unique partition ID tag groups are in different partitions, statistical analysis (e.g., histogram comparison) can be used to distinguish partition ID tags from the same or different partitions.

[0123] The reverse complementary sequence of the forward primer capture sequence can be the reverse complementary sequence of the entire capture sequence of the forward primer or can be the reverse complementary sequence of a partial capture sequence, as long as the reverse complementary sequence in the forward primer capture sequence and the partition ID tag oligonucleotide can be specifically hybridized in subsequent reactions. In other embodiments, the connection sequence is complementary to the bridging single-stranded nucleic acid sequence alone in the partition, so that when the capture sequence and the connection sequence are hybridized with the bridge sequence at the same time, the capture sequence and the connection sequence of the forward primer are allowed to be close to each other for connection. In some embodiments, the target reverse primer further comprises a universal (e.g., a "PCR handle") or other additional sequence to assist downstream operations or amplicon sequencing. For example, when using Illumina-based sequencing, the target reverse primer can have a 5'P5 or P7 sequence (optionally, with a forward primer having another of the two sequences).

[0124] The partition ID tag oligonucleotide can include a spacer sequence to determine the subsequent sequencing orientation. For example, in some embodiments, a constant sequence is included between the variable partition ID tag sequence and the connection sequence. In some embodiments, the 5′ of the variable partition ID tag sequence can also include a constant sequence. The constant sequence has a specific non-variable sequence and a set length, so that they can be identified in the nucleotide sequence, and the position of the adjacent variable partition ID tag sequence can therefore be indicated. The length of the constant sequence can be easily used for sequencing and unique sequence identification. In some embodiments, each constant sequence is between 10-150 nucleotides.

[0125] In some embodiments, the 5' end of the partition ID tag oligonucleotide includes a primer binding site, thereby allowing the amplicon containing the partition ID tag oligonucleotide to be amplified or otherwise manipulated by hybridizing with the primer binding site. In some embodiments, the partition ID tag oligonucleotide further includes a universal sequence or other additional sequence to assist in amplicon sequencing or downstream operations. For example, when using Illumina-based sequencing, the partition ID tag oligonucleotide can have a 5'P5 or P7 sequence (optionally, with a forward primer having the other of the two sequences).

[0126] The copy number of the partition ID tag oligonucleotide in a specific partition does not have to be very high. In fact, depending on the embodiment, it may be necessary to use as few copies as possible, because only a small amount of sequence readings of nucleic acids containing the partition ID tag sequence are required to evaluate the presence of multiple forward primers. Therefore, in some embodiments, each partition adds 1-50000 (e.g., 1-20, 5-50, 10-500, 100-20000) partition ID tag oligonucleotide copies on average. The number of copies is enough to fully confirm the bead occupancy in each partition, but it is not enough to retain most of the sequencing space of the test sample.

[0127] The partition ID tag oligonucleotide can be added to the partition as a free oligonucleotide, or attached to a solid support (different or the same solid support as the one to which the forward primer is attached). Figure 2 Various aspects are described. In some embodiments, the partition ID tag oligonucleotide is delivered to the partition as a free oligonucleotide, i.e., not attached to a solid support. In some embodiments, the partition ID tag oligonucleotide can be single-stranded (see, e.g., Figure 2 ) or part thereof (see, e.g., Figure 7 ) or fully double stranded. An exemplary partially double stranded option includes providing two partition ID tag oligonucleotides that hybridize at a partition ID tag sequence, wherein one partition ID tag oligonucleotide has the reverse complement sequence of the partition ID tag sequence of the other partition ID tag oligonucleotide or a portion thereof. Figure 2 and 7 -9 describe examples of this embodiment. In these embodiments, the partially double-stranded molecule has a 3' overhang that contains the capture sequence or its reverse complement, resulting in the capture sequence being able to hybridize or otherwise interact with the capture sequence of the forward primer. This is advantageous, for example, in an assay where there are no multiple rounds of capture and extension. The double-stranded format allows each strand (forward and reverse complement of the tag sequence) to bind to a different forward primer in the same capture / extension step.

[0128] In some embodiments, the single-stranded or double-stranded partition ID tag oligonucleotide is attached to a solid support when delivered to the partition. See e.g. Figure 10-12 . For example, in some embodiments, the partition ID tag oligonucleotide can be connected to a bead. In some embodiments, the partition ID tag oligonucleotide is connected to a solid support (e.g., different beads) different from the solid support to which the forward primer is connected. Alternatively, the partition ID tag oligonucleotide can be delivered to a solid support (e.g., beads) that is connected to the same solid support as the forward primer. In these embodiments, it may be desirable to include more forward primers than the partition ID tag oligonucleotide on the solid support, for example, the number of partition ID tag oligonucleotides is limited to the value shown in the required number of partition ID tag oligonucleotides for each partition. In either case, the number of beads delivered by each partition can be adjusted so that a large number (e.g., at least a large portion) of partitions include a bead.

[0129] In some embodiments, once introduced into the partition, the partition ID tag oligonucleotide is cut from the solid support (e.g., beads), so that the free partition ID tag oligonucleotide is more capable of interacting as described herein. Therefore, in some embodiments, the partition ID tag oligonucleotide is connected to the solid support by a cleavable joint. In some cases, the tag reverse primer is connected to the solid support (e.g., beads) by a disulfide bond (e.g., by a sulfide of a solid support and covalently attached to the 5' or 3' end of the oligonucleotide, or a disulfide bond between the sulfides of the nucleic acid). In such cases, the oligonucleotide can be cut off by contacting the solid support with a reducing agent, such as a thiol or phosphine reagent, including but not limited to β-mercaptoethanol, dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP). In some embodiments, the cleavable joint is a restriction enzyme site cut by a restriction enzyme (e.g., an endonuclease, such as a type II endonuclease or a type IIS endonuclease). For example, in some embodiments, the cleavable linker includes a type II restriction enzyme binding site (e.g., HhaI, HindIII, NotI, BbvCI, EcoRI, BglI) or a type IIS restriction enzyme binding site (e.g., FokI, AlwI, BspMI, MnII, BbvI, BccI, MboI). In some embodiments, the cleavable linker includes a uridine incorporation site in the partial nucleotide sequence. The uridine incorporation site can be cleaved, for example, using a uracil glycosylase (e.g., uridine N-glycosylase or uridine DNA glycosylase). In some embodiments, the cleavable linker includes a photocleavable nucleotide. Photocleavable nucleosides include, for example, photocleavable fluorescent nucleotides and photocleavable biotinylated nucleotides. See, for example, Li et al., PNAS, 2003, 100: 414-419; Luo et al., Methods Enzymol. 2014. 549: 115-131.

[0130] In some embodiments, the partition ID tag oligonucleotide and the forward and reverse primers are connected to each other through a cleavable linker (e.g., as described above) and are also connected to a solid support (e.g., a bead). In this embodiment, the cleavage agent separates the forward primer and the partition ID tag oligonucleotide from each other and from the solid support in the partition.

[0131] In other embodiments, when delivered to a partition, one or more partition ID tag oligonucleotides can be connected (optionally, through a cleavable linker, as described above) to another molecule. As a non-limiting example, one or more partition ID tag oligonucleotides can be connected to a protein (including but not limited to an antibody), biotin, polyethylene glycol (PEG), a gel (including but not limited to agarose or acrylamide gel) or other molecules. In some embodiments, the molecule to which the partition ID tag oligonucleotide is attached is an affinity reagent, i.e., a reagent having an affinity for other target molecules. Non-limiting examples of affinity reagents include, for example, antibodies and biotin.

[0132] In some embodiments, the 3' end of the partition ID tag oligonucleotide cannot be extended by a polymerase. For example, in some embodiments, the 3' end of the partition ID tag oligonucleotide is a dideoxynucleotide (e.g., ddC). Extension of the closed partition ID tag eliminates or reduces the possibility that the forward primer that initially captured it will subsequently preferentially capture the partition ID tag.

[0133] After forming a partition comprising forward and reverse primers and partition ID tag oligonucleotides, a molecular method can be performed to detect the target nucleic acid in the partition. Therefore, in various embodiments, the sample nucleic acid is also in the partition. Exemplary molecular methods can include any molecular method for detecting nucleic acid, including but not limited to template-based primer extension (e.g., polymerase chain reaction) or a method for specific hybridization with target nucleic acid by forward primer capture sequence detection. In some embodiments, the method includes connection. When the partition is complete or after the partition contents have been merged (therefore the method is "in bulk"), any of these molecular methods can be performed. In any one of the options, in the partition, the ID tag oligonucleotide is allowed to hybridize with the forward primer to form a hybridization product. The hybridization product can then be extended or otherwise amplified in the partition or in batches. As described above, in addition to ligation (and optionally, amplification) of the forward primer to the target nucleic acid (if any), the reaction also ligates available copies of the partition ID tag oligonucleotide to some copies of the forward primer in the partition, thereby forming a polynucleotide comprising a forward primer in the partition having the partition ID tag, wherein the forward primer includes a forward primer barcode sequence.

[0134] In some embodiments, the extension and / or amplification reaction comprises the use of a polymerase, e.g., a DNA polymerase. The DNA polymerase that can be used for the methods described herein can be any polymerase that can replicate a DNA molecule. In some embodiments, the DNA polymerase is a thermostable polymerase. Thermostable polymerases are isolated from multiple thermophilic bacteria, such as Thermus aquaticus (Taq), Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo), Bacillus sterothermophilus (Bst), Sulfolobus acidocaldarius (Sac), Sulfolobus solfataricus (Sso), Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), and Methanobacterium thermoautotrophicum (Mth), as well as other species. DNA polymerases are known in the art and are commercially available. In some embodiments, the DNA polymerase is Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT TM 、DEEPVENT TM , or an active mutant, variant, or derivative thereof. In some embodiments, the polymerase is Taq DNA polymerase. In some embodiments, the DNA polymerase is a high-fidelity DNA polymerase (e.g., iProof TM High-fidelity DNA polymerase, High-fidelity DNA polymerase, High-fidelity DNA polymerase, Taq high-fidelity DNA polymerase, In some embodiments, the DNA polymerase is a fast-start polymerase (e.g., FastStart TM Taq DNA polymerase or FastStart TM High-fidelity DNA polymerases). In some embodiments, the polymerase is a strand-displacing polymerase (e.g., phi29 or Bst DNA polymerase, large fragment).

[0135] In some embodiments, the connection of the forward primer to the partition ID tag oligonucleotide includes connecting the forward primer to the partition ID tag oligonucleotide. In some embodiments, the partition comprises a bridging single-stranded nucleic acid. The bridging single-stranded nucleic acid can play a role in making the forward primer and the partition ID tag oligonucleotide approach each other to connect to each other. For example, the bridging single-stranded nucleic acid can include the reverse complementary sequence of the 3' sequence of the target nucleic acid and the partition ID tag oligonucleotide binding sequence (for example, 5' binding sequence) from 3' to 5', so that the hybridization of the forward primer and the partition ID tag oligonucleotide makes the 3' end of the forward primer adjacent to the 5' end of the partition ID tag oligonucleotide, and wherein the 3' end of the forward primer is connected to the 5' end of the partition ID tag oligonucleotide. In some embodiments, the length of the bridging single-stranded nucleic acid is 10-100 nucleotides. In some embodiments, the ligation reaction includes using a ligase, for example, a DNA ligase. Exemplary ligases for methods described herein include but are not limited to T4DNA ligase and T4RNA ligase. Nucleic acid ligation methods have been described in the art, see, for example, Li et al., Anal Biochem, 2006, 349: 242-246; and Kuhn et al., FEBS J., 2005, 212: 5991-6000.

[0136] After the molecules in the partitions are reacted (e.g., amplification or additional ligation of forward primers and partition ID tag oligonucleotides in the partitions, and optional amplification or other manipulation of target nucleic acids in the partitions), the contents of the partitions are released prior to downstream applications, for example, to pool multiple partitions for downstream applications such as sequencing reactions. Disruption of the partitions can be achieved by any of a number of methods, including, but not limited to, electrical methods and introduction of destabilizing fluids. See, e.g., Zeng et al., Anal Chem 2011, 83: 2083-2089. Methods for disrupting partitions are also described, for example, in US2013 / 0189700, the contents of which are incorporated herein by reference.

[0137] In some embodiments, the partitions are disrupted by mixing the partitions (e.g., droplets) with a destabilizing fluid. In some embodiments, the destabilizing fluid is chloroform. In some embodiments, the destabilizing fluid comprises a perfluorinated ethanol. In some embodiments, the destabilizing fluid comprises a fluorinated oil, such as a perfluorocarbon oil.

[0138] In some embodiments, the method further comprises purifying the target nucleic acid (e.g., a target nucleic acid associated with a particle described herein) released by the partition, e.g., to separate the target nucleic acid from other partition components. In some embodiments, the purification step comprises using a column-based purification method (Zymo Select-a-Size Clean & Concentrator) or a solid phase reversible immobilization (SPRI) paramagnetic bead reagent. SPRI paramagnetic bead reagents are commercially available, e.g., in the Agencourt AMPure XP PCR purification system (Beckman-Coulter, Berria, California).

[0139] In some embodiments, nucleic acids from partitions described herein are analyzed by sequencing or genotyping methods. In some embodiments, nucleic acids are analyzed by sequencing, for example, high-throughput sequencing. In some embodiments, nucleic acids comprising a forward primer sequence (comprising a forward primer barcode) and a partition ID barcode oligonucleotide (comprising a tag sequence) are sequenced. When multiple forward primer barcodes are associated with the same tag sequence, it can be assumed that these multiple forward primers are evaluated to be in the same partition. When deconvoluting sequencing information from a partition, the user can use this information to improve the sequencing results. For example, the user can exclude data from the partitions in which there is more than one forward primer from the sequencing data of the remaining partitions. Alternatively, the user can only use information from a unique forward primer in the partition in which there are multiple forward primers. Alternatively, the sequencing information of all forward primers from a partition can be compiled and processed, as if from a single partition.

[0140] Methods for high-throughput sequencing and genotyping are known in the art. For example, such sequencing techniques include, but are not limited to, pyrosequencing, sequencing by ligation, single-molecule sequencing, sequencing by synthesis (SBS), massive simultaneous cloning, massive simultaneous single-molecule SBS, massive simultaneous single-molecule real-time, massive simultaneous single-molecule nanopore technology, etc. Morozova and Marra provide a review of some such techniques, see Genomics, 92: 255 (2008), which is hereby incorporated by reference in its entirety.

[0141] Exemplary DNA sequencing techniques include fluorescence-based sequencing techniques (see, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, New York, which is hereby incorporated by reference in its entirety). In some embodiments, automated sequencing techniques as understood in the art are used. In some embodiments, the present technology provides for simultaneous sequencing of partitioned amplicons (PCT Application No. WO 2006 / 0841,32, which is hereby incorporated by reference in its entirety). In some embodiments, DNA sequencing is accomplished by simultaneous oligonucleotide extension (see, e.g., U.S. Pat. Nos. 5,750,341 and 6,306,597, both of which are hereby incorporated by reference in their entirety). Additional examples of sequencing technologies include: Church polyclonal 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; incorporated herein by reference in their entirety), 454 picoliter pyrosequencing technology (Margulies et al., 2005 Nature 558, 114). 437, 376-380; U.S. Publication No. 2005 / 0130173; hereby incorporated by reference in its entirety), Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Pat. Nos. 6,787,308 and 6,833,246; hereby incorporated by reference in their entirety), Lynx massive simultaneous excellent sequencing technology (Brenner et al., (2000). Nat. Biotechnol. 18: 630-634; U.S. Pat. Nos. 5,695,934, 5,714,330; hereby incorporated by reference in their entirety) and Adessi PCR cloning technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO 2000 / 018957; hereby incorporated by reference in its entirety).

[0142] Generally, high-throughput sequencing has the common feature of large-scale synchronization, and the purpose of high-throughput strategies is to make the cost lower than that of earlier sequencing methods (see, e.g., Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; both are hereby incorporated by reference in their entirety). Such methods can be roughly divided into two categories: those that generally use template amplification and those that do not. Methods that require amplification include pyrophosphate sequencing commercialized by Roche with the 454 technology platform (e.g., GS 20 and GS FLX), the Solexa platform sold by Illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform sold by Applied Biosystems. Non-amplification methods are also called single-molecule sequencing, and examples include the HeliScope platform sold by Helicos BioSciences, platforms sold by VisiGen, Oxford Nanopore Technologies, Life Technologies / Ion Torrent, and Pacific Biosciences.

[0143] In pyrosequencing (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbial., 7:287-296; U.S. Pat. Nos. 6,210,891 and 6,258,568; each of which is incorporated herein by reference in its entirety), template DNA is fragmented, end-repaired, adapters are ligated, and single template molecules are captured for in situ clonal amplification using beads loaded with oligonucleotides complementary to the adapters. Each bead loaded with a single template type is partitioned into water-in-oil microbubbles, and the template is clonally amplified using a technique known as emulsion PCR. After amplification, the emulsion is broken and the beads are placed in each well of a picoliter microplate, which serves as a flow cell in the sequencing reaction. In the presence of a sequencing enzyme and a luminescent reporter such as luciferase, an ordered iterative introduction of each of the four dNTP reagents occurs in the flow cell. When the appropriate dNTP is added to the 3′ end of the sequencing primer, the generated ATP causes a light pulse in the well, which is recorded by a CCD camera. Read lengths greater than or equal to 400 bases can be achieved, and 10 6 sequence reads, yielding up to 500 million base pairs (Mb) of sequence.

[0144] In the Solexa / Illumina platform (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbial., 7:287-296; U.S. Pat. Nos. 6,833,246, 7,115,400 and 6,969,488; each of which is incorporated herein by reference in its entirety), sequencing data is generated in a shorter read format. In this method, single-stranded fragmented DNA is end-repaired to produce 5′-phosphorylated blunt ends, followed by Klenow-mediated addition of a single A base to the 3′ ends of these fragments. The addition of the A facilitates the addition of a T-ended adapter oligonucleotide, which will be used to capture the template-adapter molecule on the surface of a flow cell into which an oligonucleotide anchor is inserted. The anchor is used as a PCR primer, but due to the length of the template and its proximity to other adjacent anchor oligonucleotides, PCR extension results in the molecule "arching over" hybridized adjacent anchor oligonucleotides to form a bridge structure on the surface of the flow cell. These DNA circles are denatured and cleaved. The positive strand is then sequenced by a reversible dye terminator. The sequence of the incorporated nucleotides is determined by detecting fluorescence after incorporation, and each fluorophore and blocker are removed before the next round of dNTP addition. Sequence read lengths range from 36 nucleotides to more than 50 nucleotides, with an overall output of more than 1 billion nucleotide pairs analyzed per run.

[0145] The sequencing of nucleic acid molecules using SOLiD technology (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbial., 7: 287-296; U.S. Pat. Nos. 5,912,148; and 6,130,073; each of which is incorporated herein by reference in its entirety) also includes fragmenting the template, connecting oligonucleotide adapters, connecting beads, and emulsion PCR clonal amplification. Thereafter, the beads carrying the template are immobilized on the derivatized surface of the glass flow chamber, and a primer complementary to the adapter oligonucleotide is annealed. However, the primer is not used for 3′ extension, but is used to provide a 5′ phosphate group for connection to the interrogation probe, which contains two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels. In the SOLiD system, there are 16 possible combinations of the two bases at the 3′ end of each probe in the interrogation probe and one of four fluorescent labels at the 5′ end. Fluorescent colors, and thus each probe identified, correspond to a specified color-space coding scheme. Multiple rounds (usually 7) of probe annealing, ligation, and fluorescence detection are followed by denaturation, and then a second round of sequencing is performed using a primer that is one base offset relative to the initial primer. In this way, the template sequence can be reconstructed computationally, and the template base is queried twice for higher accuracy. The average sequence read length is 35 nucleotides, and the overall output is more than 4 billion bases per sequencing run.

[0146] In some embodiments, nanopore sequencing is used (see, e.g., Astier et al., J. Am. Chem. Soc. 2006 Feb 8;128(5)1705-10, incorporated herein by reference). The principle of nanopore sequencing involves what happens when a nanopore is immersed in a conducting fluid and a voltage (volts) is applied across the nanopore. Under these conditions, a small current can be observed to flow through the nanopore due to ionic conduction, and the amount of current is extremely sensitive to the size of the nanopore. As each base of the nucleic acid passes through the nanopore, it causes a change in the amplitude of the current passing through the nanopore, which is different for each of the four bases, allowing the sequence of the DNA molecule to be determined.

[0147] In some embodiments, the HeliScope (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbial, 7:287-296; U.S. Pat. Nos. 7,169,560, 7,282,337, 7,482,120, 7,501,245, 6,818,395, 6,911,345, and 7,501,245; each of which is incorporated herein by reference in its entirety) by Helicos BioSciences Corporation is used. Template DNA is fragmented and polyadenylated at the 3′ end, and the last adenosine carries a fluorescein label. The denatured polyadenylated template fragments are attached to poly(dT) oligonucleotides on the surface of the flow cell. The initial physical position of the captured template is recorded by a CCD camera, and the label is then cut off and washed away. Sequencing is achieved by adding polymerase and serial addition of fluorescently labeled dNTP reagents. Incorporation events generate fluorescent signals corresponding to the dNTPs, and a CCD camera captures the signals before each round of dNTP addition. Sequence reads are 25-50 nucleotides in length, with an overall output of over 1 billion nucleotide pairs analyzed per run.

[0148] Ion torrent technology is a DNA sequencing method based on the detection of hydrogen ions released by DNA polymerization (see, e.g., Science 327(5970):1190(2010); U.S. Patent Application Nos. 2009 / 0026082; 2009 / 0127589; 2010 / 0301398; 2010 / 0197507; 2010 / 0188073 and 2010 / 0137143; all of which are incorporated herein by reference in their entirety for all purposes). The microwell contains the template DNA strand to be sequenced. Below the microwell layer is an ultrasensitive ISFET ion sensor. All layers are contained in a CMOS semiconductor chip, which is similar to those used in the electronics industry. Hydrogen ions are released when dNTPs are incorporated into the growing complementary strand, triggering the ultrasensitive ion sensor. If homopolymeric repeating series are present in the template series, multiple dNTP molecules will be incorporated in a single cycle. This results in the release of a corresponding amount of hydrogen and a proportionally higher electronic signal. This technology is different from other sequencing technologies in that it does not use modified nucleotides and optical elements. The single-base accuracy of the ion flow sequencer is about 99.6% for every 50 bases read, and each run produces about 100Mb. The read length is 100 base pairs. The accuracy of 5-repeat homopolymeric repeat sequences is about 98%. The advantages of ion semiconductor sequencing are fast sequencing speed and low initial and running costs.

[0149] Another exemplary nucleic acid sequencing method applicable to the methods described herein is a sequencing method developed by Stratos Genomics and used with Xpandomer. The sequencing method generally includes providing a daughter chain produced by template-directed synthesis. The daughter chain generally includes a plurality of subunits coupled according to a continuous nucleotide sequence corresponding to all or part of the target nucleic acid, each subunit containing a tether, at least one probe or nucleobase residue and at least one selectively cleavable bond. The selectively cleavable bond is cleaved to obtain an Xpandomer, and its length is greater than the length of the plurality of subunits of the daughter chain. Xpandomer generally includes a tether and a reporter element, and the reporter element is used to resolve the genetic information of the continuous nucleotide sequence corresponding to all or part of the target nucleic acid in the sequence. The reporter element of the Xpandomer is then measured. Supplementary details of the Xpandomer-based method are documented in the literature, such as U.S. Patent Publication No. 2009 / 0035777, which is incorporated herein by reference in its entirety.

[0150] Other single-molecule sequencing methods include real-time sequencing by synthesis using the VisiGen platform (Voelkerding et al., Clinical Chem., 55:641-58, 2009; U.S. Patent No. 7,329,492, U.S. Patent Application Serial Nos. 11 / 671,956 and 11 / 781,166; each of which is incorporated herein by reference in its entirety), in which an immobilized primed DNA template is chain extended using a fluorescein-modified polymerase and a fluorescein acceptor molecule, resulting in detectable fluorescence resonance energy transfer (FRET) upon nucleotide addition.

[0151] Another real-time single-molecule sequencing system developed by Pacific Biosciences (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7:287-296; U.S. Patent Nos. 7,170,050, 7,302,146, 7,313,308 and 7,476,503; each of which is incorporated herein by reference in its entirety) utilizes a 50-100 nm diameter single-molecule sequencing system containing approximately 20 zeolitic cells (10 -21 L) Reaction wells of reaction volume. Sequencing reactions are performed using immobilized template, modified Φ29 DNA polymerase, and high local concentration of fluorescein-labeled dNTPs. High local concentration and continuous reaction conditions allow the use of laser excitation, optical waveguides, and CCD cameras to capture incorporation events in real time through fluorescence signal detection.

[0152] In some embodiments, the single-molecule real-time (SMRT) DNA sequencing method uses a zero-mode waveguide (ZMW) developed by Pacific Biosciences or a similar method. With this technology, DNA sequencing is performed on SMRT chips, each of which contains thousands of zero-order waveguides (ZMW). ZMWs are holes with diameters of a few tenths of a nanometer, made in a 100nm metal film that is placed on a silica substrate. Each ZMW becomes a probe that provides a detection volume of only 20 zeolitres (10 -21 L). With this volume, the activity of a single molecule can be detected against a background of thousands of labeled nucleotides. ZMWs are sequenced by synthesis, providing a window for observing DNA polymerase. Within each ZMW chamber, a single DNA polymerase molecule is bound to the bottom surface and thus permanently retained within the detection volume. The phospholinked nucleotides, each labeled with a fluorophore of a different color, are then introduced into the reaction solution at high concentrations that increase enzyme speed, accuracy, and processivity. Due to the small size of the ZMWs, even at these high concentrations, the detection volume is occupied by the nucleotides for a very small proportion of the time. In addition, due to the short diffusion distance of the transported nucleotides, the stopover in the detection volume is very fast, lasting only a few microseconds. The result is a very low background.

[0153] Methods and systems for such real-time sequencing that can be adapted for use with the present invention are described in the literature, for example, in U.S. Patent Nos. 7,405,281, 7,315,019, 7,313,308, 7,302,146, and 7,170,050; U.S. Patent Publication Nos. 2008 / 0212960, 2008 / 0206764, 2008 / 0199932, 2008 / 0199874, 2008 / 0176769, 2008 / 0176316, 2008 / 0176241, 2008 / 0165346, 2008 / 0160531, 2008 / 0157005, 2008 / 0153100, 2008 / 0153095, 2008 / 0152281, 2008 / 0152280, 2008 / 0145278, 2008 / 0128627, 2 008 / 0108082, 2008 / 0095488, 2008 / 0080059, 2008 / 0050747, 2008 / 0032301, 2008 / 0030628, 2008 / 0009007, 2007 / 0238679, 2007 / 0231804, 2007 / 0206187, 2007 / 0196846, 2007 / 018875 0, 2007 / 0161017, 2007 / 0141598, 2007 / 0134128, 2007 / 0128133, 2007 / 0077564, 2007 / 0072196 and 2007 / 0036511, and Korlach et al. (2008) "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures," PNAS 105(4): 1176-81, all of which are hereby incorporated by reference in their entirety.

[0154] In another aspect, a partitioned library is provided that includes a plurality of partitions (e.g., at least about 100; 200; 300; 500; 750; 1000; 2500; 5000; 7500; 10,000; 15,000; 20,000; 30,000, 40,000, 50,000, 70,000, 100,000, 150,000, 200,000, 300,000, 400,000, 500,000, 700,000, 1,000,000 or more partitions). In some embodiments, at least some of the partitions include (1) at least one forward primer, as described herein, optionally attached to beads or other solid supports, and (2) a partition ID tag oligonucleotide, and optionally, (3) a target reverse primer (which may also be added once the partitions have been assembled in bulk, or optionally, not included at all). In some embodiments, a partition comprises 0, 1 or more than one particle / partition. In some embodiments, a partition has an average of about one (e.g., 0.5-1.5) particle / partition. In some embodiments, a partition has an average of about two (e.g., 1.5-2.5) particles / partition. In some embodiments, a partition has an average of about three (e.g., 2.5-3.5) particles / partition. In some embodiments, a partition ID tag oligonucleotide is also connected to a solid support, which can be a solid support identical or different from the solid support connected to the forward primer. In some embodiments, a partition comprises an amplicon, which comprises a forward primer and a partition ID tag oligonucleotide or its complementary sequence.

[0155] In some embodiments, at least some of the partitions in the partitioned library (e.g., most, substantially all, or all of the partitions of the partitioned library) also include a sample (e.g., one or more target nucleic acids, or one or more cells). In some embodiments, the sample comprising the target nucleic acid comprises DNA, RNA, or a combination or hybrid thereof. In some embodiments, the sample is a sample comprising cells, such as a single cell sample. In some embodiments, the sample is a sample as described elsewhere herein.

[0156] In some embodiments, the partition further comprises other reagents or components for polymerization, amplification, reverse transcription, or primer extension as described herein (e.g., polymerase, salts, nucleotides, buffers, stabilizers, primers, detectable agents, or nuclease-free water).

[0157] In another aspect, a kit for generating a plurality of partitions described herein is provided. In some embodiments, the kit comprises: (a) a plurality of beads comprising a solid support surface, the solid support surface having a plurality of forward primers coupled thereto, wherein the forward primer comprises a barcode sequence, and wherein at least a majority of the plurality of oligonucleotide primers coupled to the solid support surface comprises the same barcode sequence; and (b) a partition ID tag oligonucleotide. In some embodiments, the kit further comprises (c) a target reverse primer. In some embodiments, the partition ID tag oligonucleotide is also connected to a solid support, which may be a solid support identical or different from the solid support connected to the forward primer.

[0158] In some embodiments, the kit further comprises one or more reagents for polymerization, amplification, reverse transcription, or primer extension as described herein (e.g., a polymerase, salts, nucleotides, buffers, stabilizers, primers, detectable agents, or nuclease-free water).

[0159] In some embodiments, the kits further comprise instructions for performing the methods described herein.

[0160] Example

[0161] Fig.13 The table provided in this article provides Figure 6 The data of the published experiments. 8 The target DNA is a single cell nuclear DNA that is labeled. The partition ID tags are delivered to the partitions at a level of 0, 10, 100 or 1000 unique partition ID tags / partition. The barcode is cut by its corresponding bead, so that it captures the target molecule and the partition ID tag, and the molecules of the barcode / target and barcode / partition ID tag combination are amplified for sequencing.

[0162] Fig.13 The table in represents bioinformatics data from sequencing samples with different partition ID tag concentrations. As expected, as the partition ID tag concentration increases, the relative number of sequencing reads for barcode / partition ID tag molecules relative to barcode / target molecules increases (column 2).

[0163] "Good" barcodes in the sequenced samples were determined using known methods, and the % of "good" barcodes that were associated with at least one partition ID tag were bioinformatically determined (column 3).

[0164] Two barcodes (X and Y) are determined to "overlap" or potentially occupy the same partition if they share 2 or more common partition ID tags, and if the number of sequencing reads for the partition ID tags shared between X and Y is ≥ 10 of the smaller of the following two values: 1) the number of total sequencing reads where the partition ID tag is associated with X, or 2) the number of total sequencing reads where the partition ID tag is associated with Y. Column 4 shows the % of barcodes in each sample that were determined not to overlap in this way and therefore to occupy a partition alone. In the systems tested, the number of partition ID tags required for each partition to begin determining which barcodes share a partition with other barcodes was greater than 100, but likely less than 1000, as shown by the inability to find a large number of overlapping barcodes at a maximum of 100 partition ID tags per partition.

[0165] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art should understand various modifications or changes made therefrom, and they are included in the subject matter and interests of this application and the scope of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. Multiple partitions, where The partitions include: one or more forward primers having a barcode and a capture sequence that is complementary to: a 3' sequence of the target nucleic acid, or a reverse complement thereof, wherein different partitions contain different forward primers having different barcode sequences, and A partition ID tag oligonucleotide comprising a variable partition ID tag sequence and a complementary sequence to the capture sequence.

2. The plurality of partitions of claim 1, wherein: At least some of the partitions contain the target nucleic acid.

3. The plurality of partitions of claim 1, wherein: The partition further comprises a target reverse primer having a 3' sequence identical to the 5' sequence of the target nucleic acid, or a reverse complement thereof.

4. The plurality of partitions of claim 1, wherein: The forward primer is attached to beads.

5. The plurality of partitions of claim 4, wherein: Multiple partition ID tag oligonucleotides are linked to each other.

6. The plurality of partitions of claim 5, wherein: The plurality of partition ID tag oligonucleotides are linked to each other via beads.

7. The plurality of partitions of claim 5, wherein: The forward primer and the partition ID tag oligonucleotide are linked to each other via a cleavable linker, and the method further comprises cleaving the cleavable linker prior to the amplifying.

8. The plurality of partitions of claim 5, wherein: The beads contain at least 10 (eg, 100, 1000, 10000) times more forward primers than partition ID tag oligonucleotides attached to the beads.

9. A plurality of partitions as claimed in any one of claims 4 to 8, wherein: Primers are cleaved by the beads prior to the amplification.

10. The plurality of partitions of claim 1, wherein: The partition ID tag oligonucleotide is single stranded.

11. The plurality of partitions of claim 1, wherein: The variable partition ID tag sequence is double-stranded.

12. The plurality of partitions of claim 11, wherein: The 3′ end of each strand of the double-stranded variable partition ID tag sequence is connected to the complementary sequence of the capture sequence.

13. A method for detecting the presence or absence of multiple barcodes in a partition, the method comprising: A partition is formed, the partition comprising: a forward primer comprising a barcode and a capture sequence, wherein the capture sequence is complementary to the 3' sequence of the target nucleic acid, wherein different partitions comprise different forward primers having different barcode sequences, a partition ID tag oligonucleotide comprising a variable partition ID tag sequence and a reverse complement of the capture sequence; wherein the forward primer and the partitioning tag oligonucleotide are attached to the same solid support, and wherein the solid support is a bead; in the partition, hybridizing at least one forward primer to the partition ID tag oligonucleotide to form a hybridization product; extending the hybridization product with a polymerase to form an extension product; The extension products are amplified to form amplicons, and the amplicons are sequenced, wherein if different forward primers form amplicons with the same variable partition ID tag sequence, the different forward primers are considered to be from the same partition.

14. The method of claim 13, wherein: At least some of the partitions contain the target nucleic acid.

15. The method of claim 13, wherein: In the partition formation step, the forward primer and partition tag oligonucleotide are attached to the same bead when delivered to the partition.

16. The method of claim 13, wherein: Multiple copies of the partition ID tag oligonucleotides are linked to each other.

17. The method of claim 16, wherein: The multiple copies are connected to each other via beads.

18. The method of claim 16, wherein: During partition formation, the forward primer and the partition ID tag oligonucleotide are linked to each other via a cleavable linker, and the method further comprises cleaving the cleavable linker prior to the amplifying.

19. The method of claim 16, wherein: The beads contain at least 10 times the number of forward primers as compared to the partition ID tag oligonucleotides attached to the beads.

20. The method of claim 13, wherein: Primers are cleaved by the beads prior to the amplification.

21. The method of claim 13, wherein: The partition ID tag oligonucleotide is single stranded.

22. The method of claim 13, wherein: The variable partition ID tag sequence is double-stranded.

23. The method of any one of claims 13 to 22, wherein: The partition ID tag oligonucleotide has a blocked 3' end such that a polymerase cannot extend the blocked 3' end during amplification.

24. The method of any one of claims 13 to 23, wherein: The partitions are droplets.

25. The method of claim 13, wherein: The partition ID tag oligonucleotide comprises a double-stranded variable partition ID tag oligonucleotide and a single-stranded 3' end comprising the reverse complement sequence of the capture sequence.

26. The method of claim 14, wherein: The target nucleic acid within the partition is from a single cell.

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