WO2022256313A1

WO2022256313A1 - Validation of a unique molecular identifier associated with a nucleic acid sequence of interest

Info

Publication number: WO2022256313A1
Application number: PCT/US2022/031582
Authority: WO
Inventors: Wyatt James MCDONNELL; David Benjamin JAFFE; Katherine Pfeiffer; Ravi RAMENANI; Michael John Terry STUBBINGTON
Original assignee: 10X Genomics, Inc.
Priority date: 2021-06-01
Filing date: 2022-05-31
Publication date: 2022-12-08

Abstract

Provided herein are methods for validating a unique molecular identifier (UMI) of a barcoded nucleic acid molecule selected from a complex pool of barcoded nucleic acid molecules. The barcoded nucleic acid molecule may be a nucleic acid sequence of interest. As such, upon validating the unique molecular identifier, the barcoded nucleic acid molecule may be processed for further analysis, enrichment, and/or cloning. For example, the barcoded nucleic acid molecule may be enriched using primers that target the identification sequences including the validated unique molecular identifier. The barcoded nucleic acid molecule may be subsequently cloned, and the protein products can be analyzed.

Description

VALIDATION OF A UNIQUE MOLECULAR IDENTIFIER ASSOCIATED WITH A NUCLEIC ACID SEQUENCE OF INTEREST

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63/195,682, entitled “VALIDATION OF A UNIQUE MOLECULAR IDENTIFIER ASSOCIATED WITH A NUCLEIC ACID SEQUENCE OF INTEREST” and filed on lune 1, 2022, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates generally to the field of immunology, and particularly relates to methods, systems, and computer program products for validating the unique molecular identifier of a nucleic acid sequence of interest.

BACKGROUND

[0003] A sample may be processed for various purposes, such as identification of a type of moiety within the sample. The sample may be a biological sample. Biological samples may be processed, such as for detection of a disease (e.g., cancer) or identification of a particular species. There are various approaches for processing samples, such as polymerase chain reaction (PCR) and sequencing.

[0004] Biological samples may be processed within various reaction environments, such as partitions. Partitions may be wells or droplets. Droplets or wells may be employed to process biological samples in a manner that enables the biological samples to be partitioned and processed separately. For example, such droplets may be fluidically isolated from other droplets, enabling accurate control of respective environments in the droplets.

[0005] Biological samples in partitions may be subjected to various processes, such as chemical processes or physical processes. Samples in partitions may be subjected to heating or cooling, or chemical reactions, such as to yield species that may be qualitatively or quantitatively processed. Particular species yielded from the biological samples, e.g., nucleic acids, may be processed in selection and enrichment reactions to more efficiently recover nucleic acid sequences of interest. In order to avoid targeting incorrectly sequenced nucleic acid sequences, there is a need for validating the nucleic acid sequences selected for subsequent processes. SUMMARY

[0006] Provided herein are, inter alia, systems, methods, and articles of manufacture, including computer program products, for validating a unique molecular identifier to determine whether the read sequences associated with the unique molecular identifier identify the nucleic acid base occupying each position in an analyte comprising a nucleic acid sequence of interest with sufficient confidence. If the unique molecular identifier is validated, the corresponding nucleic acid sequence of interest may be selected for subsequent operations such as enrichment, cloning, analysis, and/or the like. For example, a nucleic acid sequence of interest may be enriched for further cloning and analysis using techniques that are low noise and/or high specificity. In some embodiments, methods herein can be useful for selection of a nucleic acid sequence of interest (for example, a candidate antibody) or antibody discovery applications.

[0007] In one aspect, there is provided a system that includes at least one processor and at least one memory. The at least one memory may include program code that provides operations when executed by the at least one processor. The operations may include: aligning, to a contig sequence, each read sequence of a plurality of read sequences associated with a unique molecular identifier, the aligning being based at least on a subsequence in each read sequence and a matching subsequence in the contig sequence; validating, based at least on the plurality of read sequences aligned to the contig sequence, a first position of a plurality of positions in a nucleic acid sequence of interest in the contig sequence, the first position being validated based on at least one of (i) a first base type occupying the first position in at least one of the plurality of read sequences matching a second base type occupying the first position in the contig sequence and (ii) a first quality score of the first base type exceeding a threshold value; and in response to validating the plurality of positions in the nucleic acid sequence of interest in the contig sequence, validating the unique molecular identifier. [0008] In another aspect, there is provided a system that includes at least one processor and at least one memory. The at least one memory may include program code that provides operations when executed by the at least one processor. The operations may include: aligning a plurality of read sequences associated with a unique molecular identifier to a contig sequence comprising a nucleic acid sequence of interest, wherein a read sequence of the plurality of read sequences is aligned to the contig sequence by aligning a subsequence of the read sequence to a matching subsequence in the nucleic acid sequence of interest; for a first position in the contig that corresponds to the nucleic acid sequence of interest, determining a quality score for each possible base type at the first position in the contig, based on quality scores of base types identified in positions of the plurality of reads that align to the first position in the contig; validating the first position in the contig based on at least one of (i) a first base type having a highest quality score matching a base type occupying the first position in the contig and (ii) the highest quality score exceeding a second highest quality score associated with a second base type occupying the first position in the contig by a threshold value; and validating the unique molecular identifier in response to validating each position in the contig that corresponds to the nucleic acid sequence of interest. [0009] In another aspect, there is provided a method for unique molecular identifier validation. The method may include: aligning, to a contig sequence, each read sequence of a plurality of read sequences associated with a unique molecular identifier, the aligning being based at least on a subsequence in each read sequence and a matching subsequence in the contig sequence; validating, based at least on the plurality of read sequences aligned to the contig sequence, a first position of a plurality of positions in a nucleic acid sequence of interest in the contig sequence, the first position being validated based on at least one of (i) a first base type occupying the first position in at least one of the plurality of read sequences matching a second base type occupying the first position in the contig sequence and (ii) a first quality score of the first base type exceeding a threshold value; and in response to validating the plurality of positions in the nucleic acid sequence of interest in the contig sequence, validating the unique molecular identifier.

[0010] In another aspect, there is provided a method for unique molecular identifier validation. The method may include: aligning a plurality of read sequences associated with a unique molecular identifier to a contig sequence comprising a nucleic acid sequence of interest, wherein a read sequence of the plurality of read sequences is aligned to the contig sequence by aligning a subsequence of the read sequence to a matching subsequence in the nucleic acid sequence of interest; for a first position in the contig that corresponds to the nucleic acid sequence of interest, determining a quality score for each possible base type at the first position in the contig, based on quality scores of base types identified in positions of the plurality of reads that align to the first position in the contig; validating the first position in the contig based on at least one of (i) a first base type having a highest quality score matching a base type occupying the first position in the contig and (ii) the highest quality score exceeding a second highest quality score associated with a second base type occupying the first position in the contig by a threshold value; and validating the unique molecular identifier in response to validating each position in the contig that corresponds to the nucleic acid sequence of interest.

[0011] In another aspect, there is provided a non-transitory computer readable medium storing instructions. The instructions may cause operations when executed by at least one data processor. The operations may include: aligning a plurality of read sequences associated with a unique molecular identifier to a contig sequence comprising a nucleic acid sequence of interest, wherein a read sequence of the plurality of read sequences is aligned to the contig sequence by aligning a subsequence of the read sequence to a matching subsequence in the nucleic acid sequence of interest; for a first position in the contig that corresponds to the nucleic acid sequence of interest, determining a quality score for each possible base type at the first position in the contig, based on quality scores of base types identified in positions of the plurality of reads that align to the first position in the contig; validating the first position in the contig based on at least one of (i) a first base type having a highest quality score matching a base type occupying the first position in the contig and (ii) the highest quality score exceeding a second highest quality score associated with a second base type occupying the first position in the contig by a threshold value; and validating the unique molecular identifier in response to validating each position in the contig that corresponds to the nucleic acid sequence of interest.

[0012] In another aspect, there is provided a method for enriching a nucleic acid sequence of interest associated with a valid unique molecular identifier. The method may include: aligning, to a contig sequence, each read sequence of a plurality of read sequences associated with a unique molecular identifier, the aligning being based at least on a subsequence in each read sequence and a matching subsequence in the contig sequence; validating, based at least on the plurality of read sequences aligned to the contig sequence, a first position of a plurality of positions in a nucleic acid sequence of interest in the contig sequence, the first position being validated based on at least one of (i) a first base type occupying the first position in at least one of the plurality of read sequences matching a second base type occupying the first position in the contig sequence and (ii) a first quality score of the first base type exceeding a threshold value; in response to validating the plurality of positions in the nucleic acid sequence of interest in the contig sequence, validating the unique molecular identifier; designing a primer configured to target the validated unique molecular identifier; and using the primer to enrich the nucleic acid sequence of interest associated with the unique molecular identifier.

[0013] In another aspect, there is provided a method for enriching a nucleic acid sequence of interest associated with a valid unique molecular identifier. The method may include: aligning a plurality of read sequences associated with a unique molecular identifier to a contig sequence comprising a nucleic acid sequence of interest, wherein a read sequence of the plurality of read sequences is aligned to the contig sequence by aligning a subsequence of the read sequence to a matching subsequence in the nucleic acid sequence of interest; for each position in the contig that corresponds to the nucleic acid sequence of interest, determining a quality score for each possible base type at the position in the contig, based on quality scores of base types identified in positions of the plurality of reads that align to the position in the contig; validating each position in the contig based on at least one of (i) a first base type having a highest quality score matching a base type occupying the position in the contig and (ii) the highest quality score exceeding a second highest quality score associated with a second base type occupying the position in the contig by a threshold value; validating the unique molecular identifier in response to validating each position in the contig that corresponds to the nucleic acid sequence of interest; designing a primer configured to target the validated unique molecular identifier; and using the primer to enrich the nucleic acid sequence of interest associated with the unique molecular identifier.

[0014] In another aspect, there is provided a non-transitory computer readable medium storing instructions. The instructions may cause operations when executed by at least one data processor. The operations may include: aligning, to a contig sequence, each read sequence of a plurality of read sequences associated with a unique molecular identifier, the aligning being based at least on a subsequence in each read sequence and a matching subsequence in the contig sequence; validating, based at least on the plurality of read sequences aligned to the contig sequence, a first position of a plurality of positions in a nucleic acid sequence of interest in the contig sequence, the first position being validated based on at least one of (i) a first base type occupying the first position in at least one of the plurality of read sequences matching a second base type occupying the first position in the contig sequence and (ii) a first quality score of the first base type exceeding a threshold value; and in response to validating the plurality of positions in the nucleic acid sequence of interest in the contig sequence, validating the unique molecular identifier . [0015] In some variations of the methods, systems, non-transitory computer readable media, and computer-implemented methods, one or more features disclosed herein including the following features can optionally be included in any feasible combination. The first base type may occupy the first position in a first read sequence and a second read sequence of the plurality of read sequences. The first quality score may be a value that is representative of a respective quality scores of the first base type in each of the first read sequence and the second read sequence.

[0016] In some variations, the value may include a sum, a mean, a medium, a mode, a maximum, or a minimum.

[0017] In some variations, the first base type may occupy the first position in at least a first read sequence of the plurality of read sequences. A third base type may occupy the first position in at least a second read sequence of the plurality of read sequences. The first quality score may be a highest quality score associated with the first position. A second quality score of the third base type may be a second highest quality score associated with the first position. The first position may be validated further based on the first quality score of the first base type exceeding the second quality score of the third base type by the threshold value.

[0018] In some variations, the first position may be invalid based at least on the first position not being covered by any one of the plurality of read sequences.

[0019] In some variations, the first base type and the second base type may include adenine (A), cytosine (C), guanine (G), or thymine (T).

[0020] In some variations, the first quality score may indicate an accuracy and/or a probability of error associated with a base call indicating the first base type.

[0021] In some variations, the first quality score may include a Phred quality score.

[0022] In some variations, the threshold value may be 15, 20, 25, or 30.

[0023] In some variations, the threshold value may be between 15 and 30.

[0024] In some variations, a second position of the plurality of positions in the nucleic acid sequence of interest in the contig may be validated based at least on the plurality of read sequences aligned to the contig. The second position may be validated based on at least one of (i) a third base type occupying the second position in at least one of the plurality of read sequences matching a fourth base type occupying the second position in the contig and (ii) a second quality score of the third base type exceeding the threshold value. [0025] In some variations, the subsequence and the matching subsequence may each be a continuous sequence of bases.

[0026] In some variations, the subsequence and the matching subsequence may be a longest sequence of matching bases between each read sequence and the contig sequence.

[0027] In some variations, the nucleic acid sequence of interest may include a variable (V) gene segment sequence and a joining (J) gene segment sequence.

[0028] In some variations, the first position of the plurality of positions in the nucleic acid sequence of interest may correspond to a start of the variable (V) gene segment sequence.

[0029] In some variations, a last position of the plurality of positions in the nucleic acid sequence of interest may correspond to an end of the joining (J) gene segment sequence.

[0030] In some variations, the nucleic acid sequence of interest may encode an antigen binding molecule or an antigen binding fragment of the antigen binding molecule.

[0031] In some variations, the antigen binding molecule or the antigen binding fragment of the antigen binding molecule may be a T cell receptor (TCR) or a fragment of the T cell receptor.

[0032] In some variations, the antigen binding molecule or the antigen-binding fragment of the antigen binding molecule may be a BCR, an antibody or an antigen binding fragment of the antibody or BCR.

[0033] In some variations, the validating may include examining one or more positions in the subsequence of each read sequence.

[0034] In some variations, the validating may include examining one or more additional positions in each read sequence between the subsequence and an indel-free alignment corresponding to a start of each read sequence and/or an end of each read sequence.

[0035] In some variations, the unique molecular identifier may be one of a plurality of unique molecular identifiers associated with a barcode sequence identifying a cell from which the nucleic acid sequence of interest is derived.

[0036] In some variations, the contig sequence may be a consensus sequence in which each position is occupied by a most frequently encountered nucleic acid base at a same position across a plurality of read sequences associated with the barcode.

[0037] In some variations, the unique molecular identifier, or a complement of the unique molecular identifier, may be comprised in a complementary deoxyribonucleic acid (cDNA) molecule comprising one or more sequences corresponding to an analyte. The unique molecular identifier may identify the analyte.

[0038] In some variations, the complementary deoxyribonucleic acid (cDNA) molecule may include a nucleic acid sequence of a heavy chain and/or a light chain of an antibody expressed by a cell.

[0039] In some variations, the cell may be a B cell or a T cell.

[0040] In some variations, the complementary deoxyribonucleic acid (cDNA) molecule may include a template switch oligonucleotide (TSO) sequence, a variable (V) gene segment sequence, a joining (I) gene segment sequence, a diversity (D) sequence, a constant (C) sequence, and a barcode sequence identifying a cell from which the complementary deoxyribonucleic acid (cDNA) molecule is derived.

[0041] In some variations, the barcode sequence may be a partition-specific barcode In some variations, the barcode sequence may be a partition-specific barcode that is unique to a partition containing a single one of the cell .

[0042] In some variations, an output corresponding to a result of validating the unique molecular identifier may be generated.

[0043] In some variations, a user interface displaying, at a client device, at least a portion of the output may be generated.

[0044] In some variations, at least a portion of the output may be sent, over a wired network and/or a wireless network, to the client device.

[0045] In some variations, a primer configured to target the validated unique molecular identifier may be designed.

[0046] In some variations, the primer may be configured to enrich the nucleic acid sequence of interest associated with the unique molecular identifier.

[0047] In some variations, the primer may enrich the nucleic acid sequence of interest through a complementary base pairing.

[0048] In some variations, the primer may be configured to enrich the nucleic acid sequence of interest during a nested polymerase chain reaction (PCR) amplification having a first amplification reaction and a second amplification reaction. [0049] In some variations, the first amplification reaction may include using an outer F (forward) primer and an outer R (reverse) primer configured to enrich the nucleic acid sequence of interest associated with the unique molecular identifier and/or a barcode sequence of a cell from which the nucleic acid sequence of interest is derived.

[0050] In some variations, the outer F primer may be a sequence complementary to the validated unique molecular identifier and/or the barcode sequence. The outer R primer may be a sequence complementary to (i) a complement of one or more of the plurality of read sequences associated with the validated unique molecular identifier, (ii) a portion of the nucleic acid sequence of interest that encodes at least a part of a B cell receptor (BCR) constant sequence, or (iii) a portion of the nucleic acid sequence of interest that encodes a junction (J) region and/or isotype region of the B cell receptor.

[0051] In some variations, the second amplification reaction may include using an inner F (forward) primer and an inner R (reverse) primer to further enrich a product of the first amplification reaction.

[0052] In some variations, the inner F (forward) primer may be complementary to (i) a variable (V) gene segment sequence of the nucleic acid sequence of interest or (ii) nucleotides of at least a portion of the leader sequence and/or encoding framework region (FWR)l of the B cell receptor, or fragment thereof. The inner R (reverse) primer may be a sequence complementary to (iii) a constant (C) gene segment sequence and a joining (J) gene segment sequence of the nucleic acid sequence of interest or complement thereof, or (iv) at least a portion of the nucleic acid sequence of interest that encodes a complementarity region (CDR)3, a FWR4, a J region, a D region, and/or a V region, or a junction between any one or more thereof, of the BCR or fragment thereof (or a complement thereof).

[0053] In some variations, the outer F primer may be a sequence complementary to the validated unique molecular identifier and/or the barcode sequence. The outer R primer may be a sequence complementary to (i) a complement of one or more of the plurality of read sequences associated with the validated unique molecular identifier, (ii) a portion of the nucleic acid sequence of interest that encodes at least a part of a T cell receptor (TCR) constant sequence, or (iii) a portion of the nucleic acid sequence of interest that encodes a junction (J) region and/or isotype region of the T cell receptor. [0054] In some variations, the inner F (forward) primer may be complementary to (i) a variable (V) gene segment sequence of the nucleic acid sequence of interest or (ii) nucleotides of at least a portion of the leader sequence and/or encoding framework region (FWR)l of the T cell receptor, or fragment thereof. The inner R (reverse) primer may be a sequence complementary to (iii) a constant (C) gene segment sequence and a joining (J) gene segment sequence of the nucleic acid sequence of interest or complement thereof, or (iv) at least a portion of the nucleic acid sequence of interest that encodes a complementarity region (CDR)3, a FWR4, a J region, a D region, and/or a V region, or a junction between any one or more thereof, of the TCR or fragment thereof (or a complement thereof) [0055] In some variations, the inner F (forward) primer may be further complementary to at least a portion of a template switch oligonucleotide (TSO) sequence of the nucleic acid sequence of interest.

[0056] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0057] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0059] FIG. 1 shows an example of a microfluidic channel structure for partitioning individual biological particles.

[0060] FIG. 2 shows an example of a microfluidic channel structure for delivering barcode carrying beads to droplets.

[0061] FIG. 3 shows an example of a microfluidic channel structure for co-partitioning biological particles and reagents.

[0062] FIG. 4 shows an example of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets.

[0063] FIG. 5 shows an example of a microfluidic channel structure for increased droplet generation throughput.

[0064] FIG. 6 shows another example of a microfluidic channel structure for increased droplet generation throughput.

[0065] FIG. 7A shows a cross-section view of another example of a microfluidic channel structure with a geometric feature for controlled partitioning. FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

[0066] FIG. 8 illustrates an example of a barcode carrying bead.

[0067] FIG. 9 illustrates a workflow for the enrichment of a nucleic acid sequence of interest.

[0068] FIG. 10 illustrates a nested PCR scheme for amplification of a nucleic acid sequence of interest.

[0069] FIG. 11 shows exemplary labelling agents comprising reporter oligonucleotides attached thereto.

[0070] FIG. 12A shows a workflow for the analysis of one or more analytes.

[0071] FIG. 12B-C show processing of nucleic acid molecules derived from a cell to append a barcode sequence.

[0072] FIG. 13A-C show a workflow for the analysis of multiple analytes using labelling agents.

[0001] FIG. 14A depicts a schematic diagram illustrating an example of validating a unique molecular identifier, in accordance with some example embodiments. [0002] FIG. 14B depicts a system diagram illustrating an example of an analysis system, in accordance with some example embodiments.

[0003] FIG. 14C depicts a flowchart illustrating an example of a process for validating a unique molecular identifier (UMI), in accordance with some example embodiments.

[0004] FIG. 14D depicts a table illustrating a proportion of invalid unique molecular identifiers associated different read coverage and read lengths, in accordance with some example embodiments. [0005] FIG. 14E depicts another table illustrating a proportion of invalid unique molecular identifiers associated with different read coverage, in accordance with some example embodiments. [0006] FIG. 14F depicts a block diagram illustrating an example of a computing system, in accordance with some example embodiments.

[0007] FIG. 14G depicts examples of invalid unique molecular identifiers, in accordance with some example embodiments.

[0008] FIG. 14H depicts an example of an output associated with unique molecular validation, in accordance with some example embodiments.

[0073] FIG. 141 depicts another example of an output associated with unique molecular validation, in accordance with some example embodiments.

[0074] FIG. 15 shows exemplary labelling agents comprising reporter oligonucleotides attached [0075] FIG. 16 illustrates an example of primer design configured to yield a clonable sequence from a nucleic acid sequence of interest using enrichment methods provided herein.

[0076] FIG. 17 provides a pictorial outline for a method of enriching a nucleic acid sequence of interest.

[0077] FIG. 18 provides a pictorial outline of a nucleic acid sequence that is compatible with a vector, including incorporation of the nucleic acid sequence into the vector.

[0078] FIG. 19 provides a pictorial outline of an exemplary probe and scheme for capture-based enrichment of nucleic acid sequences of interest.

[0079] FIG. 20 shows products of a nested (FIG. 20A) versus one-step (FIG. 20B) PCR amplification reaction to enrich for a target nucleic acid sequence of interest, e.g., encoding a fragment of a BCR.

[0080] FIG. 21 shows BioA results indicating that nested PCR cleanly amplifies a target product of interest, e.g., nucleic acid sequence encoding a fragment of a BCR, for three out of four cell clones from a pooled barcoded cDNA library. (FIG. 21B-D; clone B, C, and D). A nested PCR amplification targeting a fourth cell clone yielded multiple products (FIG. 21A; clone A).

[0081] FIG. 22 shows sequencing results of the enrichment products following nested amplification for a nucleic acid sequence of interest from a pooled barcoded cDNA library, e.g., a target nucleic acid sequence encoding a fragment of a BCR produced from Clone A (an expanded clonotype with multiple subclonotypes), when the forward outer primer lacked sufficient specificity. [0082] FIG. 23 shows sequencing results of the enrichment products following nested amplification for a nucleic acid sequence of interest from a pooled barcoded cDNA library, e.g., a target nucleic acid sequence encoding a fragment of a BCR produced from Clone C (a single cell clone with many valid UMIs), when the forward outer primer lacked sufficient specificity.

[0083] FIG. 24 shows sequencing results of the enrichment products following nested amplification for a nucleic acid sequence of interest from a pooled barcoded cDNA library, e.g., a target nucleic acid sequence encoding a fragment of a BCR produced from Clone B (an expanded clonotype with a single unique subclone), when the forward outer primer bound with sufficient specificity to the cell barcode and UMI.

[0084] FIG. 25 illustrates another example of a barcode carrying bead.

DETAILED DESCRIPTION

[0085] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0086] Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0087] The terms “a,” “an,” and “the,” as used herein, generally refers to singular and plural references unless the context clearly dictates otherwise. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”. [0088] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0089] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0090] The term “barcode,” as used herein, generally refers to a label, or identifier, that conveys or is capable of conveying information about an analyte. A barcode can be part of an analyte. A barcode can be independent of an analyte. A barcode can be a tag attached to an analyte (e.g., nucleic acid molecule) or a combination of the tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)). A barcode may be unique. Barcodes can have a variety of different formats. For example, barcodes can include: polynucleotide barcodes; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences.

A barcode can be attached to an analyte in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and/or after sequencing of the sample. Barcodes can allow for identification and/or quantification of individual sequencing-reads.

[0091] The term “real time,” as used herein, can refer to a response time of less than about 1 second, a tenth of a second, a hundredth of a second, a millisecond, or less. The response time may be greater than 1 second. In some instances, real time can refer to simultaneous or substantially simultaneous processing, detection or identification.

[0092] The term “subject,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g., bird), or other organism, such as a plant. For example, the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and/or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient. A subject can be a microorganism or microbe (e.g., bacteria, fungi, archaea, viruses).

[0093] The term “genome,” as used herein, generally refers to genomic information from a subject, which may be, for example, at least a portion or an entirety of a subject’s hereditary information. A genome can be encoded either in DNA or in RNA. A genome can comprise coding regions (e.g., that code for proteins) as well as non-coding regions. A genome can include the sequence of all chromosomes together in an organism. For example, the human genome ordinarily has a total of 46 chromosomes. The sequence of all of these together may constitute a human genome.

[0094] The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be used synonymously. An adaptor or tag can be coupled to a polynucleotide sequence to be “tagged” by any approach, including ligation, hybridization, or other approaches.

[0095] The term “sequencing,” as used herein, generally refers to methods and technologies for determining the sequence of nucleotide bases in one or more polynucleotides. The polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA). Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by Illumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or Life Technologies (Ion Torrent®). Alternatively or in addition, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification. Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human), as generated by the systems from a sample provided by the subject. In some examples, such systems provide sequencing reads (also “reads” herein). A read may include a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule that has been sequenced. In some situations, systems and methods provided herein may be used with proteomic information.

[0096] The term “bead,” as used herein, generally refers to a particle. The bead may be a solid or semi-solid particle. The bead may be a gel bead. The gel bead may include a polymer matrix (e.g., matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Polymers in the polymer matrix may be randomly arranged, such as in random copolymers, and/or have ordered structures, such as in block copolymers. Cross-linking can be via covalent, ionic, or inductive, interactions, or physical entanglement. The bead may be a macromolecule. The bead may be formed of nucleic acid molecules bound together. The bead may be formed via covalent or non- covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA). The bead may be formed of a polymeric material. The bead may be magnetic or non-magnetic. The bead may be rigid. The bead may be flexible and/or compressible. The bead may be disruptable or dissolvable. The bead may be a solid particle (e.g., a metal-based particle including but not limited to iron oxide, gold or silver) covered with a coating comprising one or more polymers. Such coating may be disruptable or dissolvable. [0097] The term “sample,” as used herein, generally refers to a biological sample of a subject. The biological sample may comprise any number of macromolecules, for example, cellular macromolecules. The sample may be a cell sample. The sample may be a cell line or cell culture sample. The sample can include one or more cells. The sample can include one or more microbes. The biological sample may be a nucleic acid sample or protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may be a cell-free or cell free sample. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.

[0098] The term “biological particle,” as used herein, generally refers to a discrete biological system derived from a biological sample. The biological particle may be a macromolecule. The biological particle may be a small molecule. The biological particle may be a virus. The biological particle may be a cell or derivative of a cell. The biological particle may be an organelle. The biological particle may be a rare cell from a population of cells. The biological particle may be any type of cell, including without limitation prokaryotic cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or other animal cell type, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from single cell or multicellular organisms. The biological particle may be a constituent of a cell. The biological particle may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The biological particle may be or may include a matrix (e.g., a gel or polymer matrix) comprising a cell or one or more constituents from a cell (e.g., cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof, from the cell. The biological particle may be obtained from a tissue of a subject. The biological particle may be a hardened cell. Such hardened cell may or may not include a cell wall or cell membrane. The biological particle may include one or more constituents of a cell, but may not include other constituents of the cell.

An example of such constituents is a nucleus or an organelle. A cell may be a live cell. The live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix, or cultured when comprising a gel or polymer matrix.

[0099] The term “macromolecular constituent,” as used herein, generally refers to a macromolecule contained within or from a biological particle. The macromolecular constituent may comprise a nucleic acid. In some cases, the biological particle may be a macromolecule. The macromolecular constituent may comprise DNA. The macromolecular constituent may comprise RNA. The RNA may be coding or non-coding. The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), for example. The RNA may be a transcript. The RNA may be small RNA that are less than 200 nucleic acid bases in length, or large RNA that are greater than 200 nucleic acid bases in length. Small RNAs may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). The RNA may be double-stranded RNA or single- stranded RNA. The RNA may be circular RNA. The macromolecular constituent may comprise a protein. The macromolecular constituent may comprise a peptide. The macromolecular constituent may comprise a polypeptide.

[00100] The term “molecular tag,” as used herein, generally refers to a molecule capable of binding to a macromolecular constituent. The molecular tag may bind to the macromolecular constituent with high affinity. The molecular tag may bind to the macromolecular constituent with high specificity. The molecular tag may comprise a nucleotide sequence. The molecular tag may comprise a nucleic acid sequence. The nucleic acid sequence may be at least a portion or an entirety of the molecular tag. The molecular tag may be a nucleic acid molecule or may be part of a nucleic acid molecule. The molecular tag may be an oligonucleotide or a polypeptide. The molecular tag may comprise a DNA aptamer. The molecular tag may be or comprise a primer. The molecular tag may be, or comprise, a protein. The molecular tag may comprise a polypeptide. The molecular tag may be a barcode.

[00101] The term “partition,” as used herein, generally, refers to a space or volume that may be suitable to contain one or more species or conduct one or more reactions. A partition may be a physical compartment, such as a droplet or well. The partition may isolate space or volume from another space or volume. The droplet may be a first phase (e g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet may be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition may comprise one or more other (inner) partitions. In some cases, a partition may be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and/or remote physical compartments.

[00102] Provided herein are methods for the enriching a nucleic acid sequence of interest from a plurality of nucleic acid molecules, such as a library of nucleic acid molecules. The methods provided herein can be used, for example, to enrich a nucleic acid sequence of interest so that it may be further cloned or analyzed. Methods herein can provide a low noise, high specificity, or both. In some embodiments, methods herein can be useful for selection of a nucleic acid sequence of interest (for example, a candidate antibody) or antibody discovery applications.

[00103] A general workflow of methods provided herein is provided in FIG. 9. Briefly, (1) a library can be generated (e.g., a barcoded library of sequences of a single cell immune repertoire of a subject); (2) sequences of interest can be identified (e.g., a V(D)J sequence, such as paired TCR (e.g., TRA/TRB), BCR, or antibody (e.g., heavy/light chain) sequences), for example by sequencing;

(3) the sequence(s) of interest can be enriched from the library (e.g., by using 1 or 2 rounds of PCR);

(4) cloning (and optionally expressing) the enriched sequence(s) of interest into an appropriate expression vector; and (5) optionally analyzing the protein (e.g., antibody) encoded by the sequence of interest. [00104] An analyte, such as an analyte comprising a nucleic acid sequence of interest, may include or be processed to include, an identification sequence and one or more read sequences. In some embodiments, the analyte is abarcoded analyte, e.g., a barcoded nucleic acid molecule, e.g., a member of a barcoded nucleic acid library generated according to any one of the methods described herein. In the example shown in FIG. 10, a barcoded nucleic acid molecule (e.g., a barcoded complementary deoxyribonucleic acid (cDNA) molecule) may include a barcode sequence, a unique molecular identifier sequence (UMI), and the nucleic acid sequence of interest or a portion thereof (e.g., a nucleic acid sequence encoding a variable (V) gene segment sequence, a diversity (D) sequence, a joining (J) gene segment sequence, a constant (C) gene segment sequence). The identification sequence may include a barcode sequence that uniquely identifies a biological particle (e.g., a cell, cell bead, or nucleus of a cell) from which the nucleic acid sequence of interest is derived. In the case of partitions containing a single cell, a single cell bead, or a single nucleus, the barcode sequence may be a partition-specific barcode sequence. Furthermore, the identification sequence may include a unique molecular identifier (UMI) that is unique to the nucleic acid sequence of interest. In some embodiments, the identification sequence includes the barcode sequence and the UMI sequence. As shown in FIG. 10, the barcoded nucleic acid molecule may further comprise any one or more of: a first read sequence, a template switch oligonucleotide (TSO), and a second read sequence.

[00105] As described herein, following the generation of barcoded nucleic acid molecules subsequent operations can be performed. Such subsequent operations can include subsequent amplification (e.g., via polymerase chain reaction (PCR)). These operations may occur in bulk (e.g. , outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations. In some cases, such subsequent amplification may produce a plurality of amplicons comprising, e.g., the same barcode sequence and the same unique molecular identifier (UMI) sequence, indicating that such amplicons were derived from a single originating template, e.g., a barcoded nucleic acid molecule comprising the barcode sequence and the UMI sequence.

[00106] It may be desirable to be able to select barcoded nucleic acid molecules from a complex pool of barcoded nucleic acid molecules (or amplicons or derivatives thereof) based on an identification sequence described herein, e.g., a unique molecular identifier sequence (UMI), if the selected barcoded nucleic acid molecule comprises a nucleic acid sequence of interest. Such selected barcoded nucleic acid molecules may be processed for further analysis, enrichment, and/or cloning according to one or more methods described herein. In some cases, during the processes for generating barcoded analytes or subsequent operations, some of the analytes from the biological particle associated with a given UMI may be damaged. In such cases, selection of barcoded nucleic acid molecules for further analysis, enrichment, and/or cloning based on UMI sequence may run a significant risk of selecting molecules that do not comprise the full nucleic acid sequence of interest but instead comprise a truncated, damaged, or otherwise altered version of the sequence of interest. Therefore, provided herein are methods for validating a UMI of a barcoded nucleic acid molecule as comprising the full nucleic acid sequence of interest.

[00107] A single unique molecular identifier may be associated with multiple read sequences (also “reads” herein), each of which being a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule (e.g., a complementary deoxyribonucleic acid (cDNA) molecule and/or the like) that has been sequenced. Moreover, a single barcode may be associated with read sequences having different unique molecular identifiers (UMIs). The read sequences associated with a single barcode, which may correspond to a biological particle (e.g., a cell, cell bead, nucleus of a cell, and/or the like) from which the nucleic acid sequence of interest is derived, may therefore undergo an assembly process to identify one or more continuous sequences of nucleic acid bases called contig sequences (also “contigs” herein). A contig sequence may be a consensus sequence in which each position is occupied by the most frequently encountered nucleic acid base at the same position across the read sequences associated with the barcode. Moreover, a contig sequence may include at least a portion of the nucleic acid sequence of interest or may comprise the nucleic acid sequence of interest. The nucleic acid sequence of interest can be any sequence of interest identified from a sequencing library, e.g., a sequencing library comprising a plurality of barcoded nucleic acid molecules disclosed herein. A nucleic acid sequence of interest can be, for example, a V-J sequence between a start of a variable (V) gene segment sequence and an end of a joining (J) gene segment sequence, an antigen binding molecule, an antigen binding fragment of an antigen binding molecule, and/or the like. The resulting assembly may be a scaffold of contig sequences separated by one or more gaps. [00108] The read sequences associated with a unique molecular identifier (UMI) may be validated in order to ensure that a nucleic acid base for each position within an analyte, such as a nucleic acid sequence of interest (e.g., a complementary deoxyribonucleic acid (cDNA) molecule and/or the like), is identified with sufficient confidence. A valid unique molecular identifier may be targeted for subsequent enrichment, cloning, and/or analysis, for example, by designing a corresponding primer targeting the unique molecular identifier via complementary base pairing. Conversely, a unique molecular identifier (UMI) may be invalid when the read sequences associated with the unique molecular identifier fail to identify each nucleic acid base in the analyte with sufficient confidence. Some nucleic acid bases may be sequenced incorrectly due to damage to the underlying nucleic acid sequence while other errors may arise during the sequencing process itself. As shown in FIGS. 14D-E, the proportion of invalid unique molecular identifiers may be lower at higher read coverage (e.g., quantity of reads per cell (RPC)) and/or longer read lengths (e.g., quantity of base pairs sequenced). Nevertheless, ensuring the validity of each unique molecular identifier (UMI) may be imperative in order to avoid targeting invalid unique molecular identifiers for subsequent enrichment, cloning, and/or analysis.

[00109] In some example embodiments, the read sequences associated with a unique molecular identifier (UMI) may be validated based at least on a corresponding contig sequence which, as noted, may be a consensus sequence in which each position is occupied by the most frequently encountered nucleic acid base at the same position across the read sequences associated with a barcode (e.g., a partition-specific barcode). FIG. 14A depicts a schematic diagram illustrating an example of validating a unique molecular identifier, in accordance with some example embodiments. In the example shown in FIG. 14A, a unique molecular identifier (UMI) may be associated with multiple read sequences including, for example, a first read sequence 140a, a second read sequence 140b, a third read sequence 140c, a fourth read sequence 140d, and/or the like. Moreover, as shown in FIG. 14A, the first read sequence 140a, the second read sequence 140b, the third read sequence 140c, and the fourth read sequence 140d may be aligned to a corresponding contig sequence 145. [00110] Each of the first read sequence 140a, the second read sequence 140b, the third read sequence 140c, and the fourth read sequence 140d may be aligned to the contig sequence 145 based at least on a continuous subsequence of nucleic acid bases in which the base types present in the read sequence match the base types present in the contig sequence 145. FIG. 14A shows an example of this alignment being performed based on a longest continuous subsequence of matching bases between each read sequence and the contig sequence 145. For example, the first read sequence 140a is aligned to the contig sequence 145 based on the longest matching subsequence TTCG while the second read sequence 140b is aligned to the contig sequence 145 based on the longest matching subsequence CAGATGA. It should be appreciated that the alignment between a read sequence and the contig sequence 145 may be performed based on other criteria such as a continuous sequence of more than a threshold quantity of matching bases.

[00111] In some example embodiments, the validation of the unique molecular identifier (UMI) may be performed based on a quality score (Q-score) assigned to the identification of the nucleic acid base (also “base call” herein) at each position in the read sequences associated with the unique molecular identifier. The validation may be performed for positions in the read sequence beyond the matching subsequence. As such, the positions that are examined in each read sequence may extend to an indel-free alignment corresponding to a start of the read sequence and/or an end of the read sequence. For example, as shown in FIG. 14A, the nucleic acid base G in a first position of the first read sequence 140a is assigned a first quality score of 10 while the nucleic acid base T in a second position of the first read sequence 140a is assigned a second quality score of 30. A quality score may be assigned to the base call at a position within a read sequence, for example, by a sequencing platform during a sequencing run to indicate an accuracy of the base call and/or a probability of an error in the base call. Different sequencing platforms may apply a different quality score.

Moreover, quality scores may be recalibrated to reflect changes in a corresponding sequencing platform such as updates to hardware, software, chemistry, and/or the like.

[00112] One example of a quality score is a Phred quality score Q , which is logarithmically related to a probability P of an error being present in a base call (e.g., Q = -10 log₁₀ P ). Table 1 below depicts the Phred quality score scale, which ranges from a score of 10 indicative of a 90% accurate base call to a score of 60 indicative of a 99.9999% accurate base call.

[00113] Table 1

[00114] In some example embodiments, the validation of a unique molecular identifier (UMI) may include validating each position in a target sequence 150 within the contig sequence 145 based on the quality score assigned to the base call at the corresponding position each read sequence covering the position. The target sequence 150 may be a nucleic acid sequence of interest such as, for example, a V-J sequence between a start of a variable (V) gene segment sequence and an end of a joining (J) sequence. A position within the target sequence 150 may be validated when a type of nucleic acid base (also “base type” herein) occupying the position is identified by at least one read sequence with sufficient confidence. For example, one or more first read sequences may identify a first type of nucleic acid base as occupying a position with the target sequence 150. In some cases, one or more second read sequences may identify a second type of nucleic acid base as occupying the position within the target sequence 150 while one or more third read sequences may identify a third type of nucleic acid base as occupying the same position within the target sequence 150.

[00115] The base calls for each type of nucleic acid base may be associated with a quality score, such as a Phred quality score and/or the like. A position in the target sequence 150 may be validated when the position is covered by at least one read sequence providing one or more base calls that identify the nucleic acid base occupying the position with sufficient confidence. When one or more read sequences provide a same base call for a position in the target sequence 150, the position may be validated when the base call matches the type of nucleic acid base occupying the position in the contig sequence 145 and the total quality score associated with the base call exceeds a threshold value. Alternatively, when different read sequences provide different base calls for the same position, validation may be performed based on a first type of nucleic acid base having a highest total quality score and a second type of nucleic acid base having a second highest total quality score. For instance, the position may be validated when the type of nucleic acid base having the highest total quality score matches the type of nucleic acid base occupying the position in the contig sequence 145. Furthermore, the position may be validated when the highest total quality score exceeds the second highest total quality score by a threshold value.

[00116] To further illustrate, a first position 155a within the target sequence 150 may be covered by the first read sequence 140a, the second read sequence 140b, the third read sequence 140c, and the fourth read sequence 140d. In the example shown in FIG. 14A, the third read sequence 140c and the fourth read sequence 140d may provide a different base call than each of the first read sequence 140a and the second read sequence 140b. The first position 155a may be validated based at least on the nucleic acid base adenine (A), which has the highest total quality score of 60, matching the type of nucleic acid base occupying the first position 155a in the target sequence 150 of the contig sequence 145. In addition, the first position 155a may be validated based at least on the highest total quality score of 60 being more than a threshold greater than the second highest total quality score of 40 associated with nucleic acid base guanine (G). Contrastingly, the first position 155a may fail to validate if the type of nucleic acid base with the highest total quality score does not match the type of nucleic acid base occupying the first position 155a in the target sequence 150 of the contig sequence 145 and/or if the highest total quality score is not more than the threshold greater than the second highest total quality score.

[00117] In some example embodiments, the total quality score for a nucleic acid base occupying the same position in multiple read sequences may be a sum of the individual quality scores assigned to the nucleic acid base in each read sequence. For instance, in the example shown in FIG. 14A, the total quality score for the nucleic acid base adenine (A) occupying the first position 155a may correspond to a sum of the respective quality scores assigned to the nucleic acid base adenine (A) occupying the first position 155a in the third read sequence 140c and the fourth read sequence 140d. Nevertheless, it should be appreciated that the total quality score for a nucleic acid base occupying the same position in multiple read sequences may be another summary value that is representative of the individual quality scores assigned to the nucleic acid base in each read sequence. Examples of other summary values may include a mean, a medium, a mode, a maximum, and a minimum of the individual quality scores assigned to the same nucleic acid base in each read sequence.

[00118] A second position 155b in the target sequence 150 is an example of a position covered by at least one read sequence providing a same base call, such as the second read sequence 140b in the example shown in FIG. 14A. The second position 155b may be validated based at least on the nucleic acid base thymine (T) at the second position 155b in the second read sequence 140b matching the type of nucleic acid base occupying the second position 155b in the contig sequence 145. Furthermore, the second position 155b may be validated if the total quality score associated with the base calls across the read sequences covering the second position 155b exceeds a threshold value. It should be appreciated that a position in the target sequence 150 that is not covered any read sequences associated with the unique molecular identifier (UMI), such as a third position 155c, may fail to validate.

[00119] In some example embodiments, a unique molecular identifier may be validated if every position within the target sequence 150 of the contig sequence 145 is validated. As noted, a position in the target sequence 150 may be validated if the position is covered by at least one read sequence associated with the unique molecular identifier and the at least one read sequence provides one or more base calls that identify the nucleic acid base occupying the position with sufficient confidence.. A validated unique molecular identifier (UMI) may be selected for subsequent enrichment, cloning, and/or analysis. For example, a primer may be designed to target a validated unique molecular identifier via a complementary base pairing. The primer may be configured to enrich the target sequence 150 which, as noted, may be a nucleic acid sequence of interest such as, for example, a V-J sequence between a start of a variable (V) gene segment sequence and an end of a joining (J) sequence. For instance, the target sequence 150 may be enriched using various techniques disclosed herein including a nested polymerase chain reaction (PCR) amplification strategy having a first amplification reaction and a second amplification reaction.

[00120] The present disclosure also provides computer systems configured to implement the various methods disclosed herein including, for example, methods for validating a unique molecular identifier (UMI). For example, the computer systems disclosed herein may be configured to implement methods for validating each position in a nucleic acid sequence of interest, such as a V-J sequence between a start of a variable (V) gene segment sequence and an end of a joining (J) sequence, covered by one or more read sequences associated with the unique molecular identifier. These methods may include validating each position in the nucleic acid sequence of interest based on the quality scores of the base calls at the corresponding positions in the read sequences associated with the unique molecular identifier. For instance, a position in the nucleic acid sequence of interest may be validated if the position is covered by at least one read sequence providing one or more base calls that identify the nucleic acid base occupying the position with sufficient confidence. Moreover, these methods may include validating the unique molecular identifier (UMI) upon validating each position within the nucleic acid sequence of interest. [00121] FIG. 14B depicts a system diagram illustrating an example of an analysis system 1500, in accordance with some example embodiments. Referring to FIG. 14B, the analysis system 1500 may include a validation engine 1502, a sequencing platform 1504, and a client device 1506. As shown in FIG. 14B, the validation engine 1502, the sequencing platform 1504, and the client device 1506 may be communicatively coupled via a network 1505. The network 1505 may be a wired network and/or a wireless network including, for example, a local area network (LAN), a virtual local area network (VLAN), a wide area network (WAN), a public land mobile network (PLMN), the Internet, and/or the like. The client device 1506 may be a processor-based device including, for example, a smartphone, a tablet computer, a laptop computer, a desktop computer, a workstation, a wearable apparatus, an Intemet-of-Things (IoT) appliance, and/or the like.

[00122] Referring again to FIG. 14B, in some example embodiments, the validation engine 1502 may receive, from the sequencing platform 1504, one or more read sequences associated with a unique molecular identifier (UMI). For example, the validation engine 1502 may receive, from the sequencing platform 1504, the first read sequence 140a, the second read sequence 140b, the third read sequence 140c, and the fourth read sequence 140d. Furthermore, the validation engine 1502 may validate the unique molecular identifier based at least on whether the read sequences associated with the unique molecular identifier provide one or more base calls that identify the nucleic acid base occupying each position within the target sequence 150 with sufficient confidence. For example, one or more read sequences may provide a same base call for a position in the target sequence 150, in which case the position may be validated when the base call matches the type of nucleic acid base occupying the position in the contig sequence 145 and the total quality score associated with the base calls exceeds a threshold value. Alternatively, when the read sequences provide different base calls for the same position within the target sequence 150, the position may be validated if the base calls having the highest total quality score matches the type of nucleic acid base occupying the position in the contig sequence 145 and the highest total quality score exceeds the second highest total quality score by a threshold value.

[00123] FIG. 14C depicts a flowchart illustrating an example of a process 1450 for validating a unique molecular identifier, in accordance with some example embodiments. Referring to FIGS. 14A-C, the process 1450 may be performed by the validation engine 1502, for example, to validate a unique molecular identifier (UMI) having the first read sequence 140a, the second read sequence 140b, the third read sequence 140c, and the fourth read sequence 140d.

[00124] At 1452, the validation engine 1502 may align, to a contig sequence, one or more read sequences associated with a unique molecular identifier. In some example embodiments, each read sequence associated with a unique molecular identifier (UMI) may be aligned to a contig sequence based on a matching subsequence of nucleic acid bases which, as noted, may be a continuous subsequence of nucleic acid bases in which the base types present in the read sequence match the base types present in the contig sequence. In some cases, the read sequences associated with the unique molecular identifier may be aligned to the contig sequence based on the longest matching subsequence between each read sequence and the contig sequence. For instance, in the example shown in FIG. 14A, the first read sequence 140a is aligned to the contig sequence 145 based on the matching subsequence TTCG while the second read sequence 140b is aligned to the contig sequence 145 based on the matching subsequence CAGATGA.

[00125] At 1454, the validation engine 1502 may validate, based at least on the aligned read sequences, each position within a nucleic acid sequence of interest in the contig sequence. In some example embodiments, the validation engine 1502 may validate the unique molecular identifier (UMI) based on a quality score (Q-score) assigned to the base calls provided by the read sequences at each position within the nucleic acid sequence of interest. The validation engine 1502 may examine positions in each read sequence beyond the matching subsequence present in each read sequence. For example, the positions examined in each read sequence may extend to an indel-free alignment corresponding to a start of the read sequence and/or an end of the read sequence.

[00126] A position in the target sequence 150 shown in FIG. 14A may be validated if the first read sequence 140a, the second read sequence 140b, the third read sequence 140c, and the fourth read sequence 140d provides one or more base calls for the position that identify the nucleic acid base occupying the position with sufficient confidence. As noted, when one or more read sequences provide a same base call for a position in the target sequence 150, that position may be validated when the base call matches the type of nucleic acid base occupying the position in the contig sequence 145 and the total quality score associated with the base calls exceeds a threshold value. Alternatively, when the read sequences provide different base calls for the same position within the target sequence 150, the position may be validated if the base calls having the highest total quality score matches the type of nucleic acid base occupying the position in the contig sequence 145 and the highest total quality score exceeds the second highest total quality score by a threshold value. In some example embodiments, the threshold value may be selected from a range between 15 and 30 such as, for example, 15, 20, 25, 30, and/or the like.

[00127] At 1456, the validation engine 1502 may validate the unique molecular identifier based at least on a result of validating each position within the nucleic acid sequence of interest in the contig sequence. For example, the validation engine 1502 may validate the unique molecular identifier (UMI) associated with the first read sequence 140a, the second read sequence 140b, the third read sequence 140c, and the fourth read sequence 140d if the validation engine 1502 is able to validate each position within the target sequence 150 of the contig sequence 145. In some cases, instead of validating every position within the target sequence 150, the unique molecular identifier may be validated when the validation engine 1502 is able to validate more than a threshold quantity of positions within the target sequence 150. This threshold quantity of positions may vary depending on the type of the target sequence 150. For instance, for some types of the target sequence 150 , the threshold quantity of positions for validating a unique molecular identifier may be 95% of the positions within the target sequence 150. Alternatively, for other types of the target sequence 150, such as the light chain or the heavy chain of an antibody, the threshold quantity of positions that must be validated in order to validate a corresponding unique molecular identifier may be 99% of the positions within the target sequence 150.

[00128] At 1458, the validation engine 1502 may generate an output corresponding to a result of validating the unique molecular identifier. In some example embodiments, the validation engine 1502 may generate, based at least on the result of validating the unique molecular identifier, an output for display by a user interface 1545 at the client device 1506. For example, if the validation engine 1502 is able to successfully validate the unique molecular identifier (UMI), the validation engine 1502 may generate an output indicating a successful validation of the unique molecular identifier. Alternatively and/or additionally, the output may include one or more validated unique molecular identifiers. The validated unique molecular identifiers may be recommended for subsequent enrichment, cloning, and/or analysis.

[00129] In some example embodiments, the output generated by validation engine 1502 may include a report of (i) the total number of unique molecular identifiers associated with at least a portion of a target nucleic sequence present in a contig sequence and (ii) the number of validated unique molecular identifiers for the target nucleic acid sequence present in the contig sequence. To further illustrate, FIGS. 14H-I depict examples of outputs associated with unique molecular identifier validation, in accordance with some example embodiments. The contig sequences shown FIGS. 14H-I may correspond to one or more nucleic acid sequences of interest (or portions thereof), which may encode an antigen binding molecule, such as a B cell receptor (BCR), or an antigen binding fragment of the antigen binding molecule. As shown in FIGS. 14H-I, the output of the validation engine 1502 may indicate a total quantity of unique molecular identifiers as well as a total quantity of validated unique molecular identifiers for each nucleic acid sequence of interest and the corresponding contig sequence.

[00130] As noted, the nucleic acid sequences of interest shown in FIGS. 14H-I may encode an antigen binding molecule, such as a B cell receptor (BCR), or an antigen binding fragment of the antigen binding molecule. For example, each barcode corresponding to a biological particle (e.g., a cell, a cell bead, a cell nucleus, and/or the like) may be associated with a light chain (e.g., chain 1) and a heavy chain (e.g., chain 2) of a biological particle, which are B cell receptors (BCRs) in the examples shown in FIGS. 14H-I. The contig sequence associated with each chain may correspond to a nucleic acid sequence of interest (or target nucleic acid sequence). Moreover, as shown in FIGS. 14H-I, the nucleic acid sequence of interest in each chain (e.g., chain 1 and chain 2) may be covered by a total quantity u of unique molecular identifiers and an rival quantity of validated unique molecular identifiers. Nucleic acid sequences having valid unique molecular identifier may be selected for subsequent operations such as amplification (e.g., via polymerase chain reaction (PCR)).

[00131] FIG. 14F depicts a block diagram illustrating an example of a computer system 1401, in accordance with some example embodiments. Referring to FIGS. 14A-F, the computer system 1401 may be configured to implement one or more of the validation engine 1502, the sequencing platform 1504, and the client device 1506. The computer system 1401 may be programmed or otherwise configured to (i) design a nucleic acid primer as described herein, control an amplification reaction as provided herein, execute cloning and/or expression of a nucleic acid sequence of interest and/or protein product of a nucleic acid sequence of interest provided herein, or analyze a protein product of a nucleic acid sequence of interest provided herein. The computer system 1401 can regulate various aspects of the present disclosure, such as, for example, amount of primer, buffer, nucleic acid, or other reagent added to an amplification reaction, thermocycling of an amplification reaction, conditions for introducing an enriched nucleic acid sequence of interest to a vector, conditions for expressing a protein product of a nucleic acid sequence of interest, and/or providing reagents and/or adjusting conditions for an experiment for analysis of a protein product of a nucleic acid sequence of interest. The computer system 1401 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[00132] The computer system 1401 includes a central processing unit (also “processor” and “computer processor” herein) 1405, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1401 also includes memory or memory location 1410 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1415 (e.g., hard disk), communication interface 1420 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1425, such as cache, other memory, data storage and/or electronic display adapters. The memory 1410, storage unit 1415, interface 1420 and peripheral devices 1425 are in communication with the central processing unit 1405 through a communication bus (solid lines), such as a motherboard. The storage unit 1415 can be a data storage unit (or data repository) for storing data. The computer system 1401 can be operatively coupled to a computer network (“network”) 1430 with the aid of the communication interface 1420. The network 1430 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1430 in some cases is a telecommunication and/or data network. The network 1430 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1430, in some cases with the aid of the computer system 1401, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1401 to behave as a client or a server.

[00133] The central processing unit 1405 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1410. The instructions can be directed to the central processing unit 1405, which can subsequently program or otherwise configure the central processing unit 1405 to implement methods of the present disclosure. Examples of operations performed by the central processing unit 1405 can include fetch, decode, execute, and writeback.

[00134] The central processing unit 1405 can be part of a circuit, such as an integrated circuit.

One or more other components of the system 1401 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[00135] The storage unit 1415 can store files, such as drivers, libraries and saved programs. The storage unit 1415 can store user data, e.g., user preferences and user programs. The computer system 1401 in some cases can include one or more additional data storage units that are external to the computer system 1401, such as located on a remote server that is in communication with the computer system 1401 through an intranet or the Internet.

[00136] The computer system 1401 can communicate with one or more remote computer systems through the network 1430. For instance, the computer system 1401 can communicate with a remote computer system of a user (e.g., operator). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1401 via the network 1430.

[00137] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1401, such as, for example, on the memory 1410 or electronic storage unit 1415. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the central processing unit 1405. In some cases, the code can be retrieved from the storage unit 1415 and stored on the memory 1410 for ready access by the central processing unit 1405. In some situations, the electronic storage unit 1415 can be precluded, and machine-executable instructions are stored on memory 1410.

[00138] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.

[00139] Aspects of the systems and methods provided herein, such as the computer system 1401, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., readonly memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[00140] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM,

DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[00141] The computer system 1401 can include or be in communication with an electronic display 1435 that comprises a user interface (UI) 1440 for providing, for example, enrichment yield, results of analysis of a protein product of a nucleic acid sequence of interest, etc. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.

[00142] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1405 The algorithm can, for example, validate a unique molecular identifier (UMI), control enrichment of a nucleic acid sequence of interest, control cloning of a nucleic acid sequence of interest, and/or assess or analyze a protein product of a nucleic acid sequence of interest. [00143] Devices, systems, compositions and methods of the present disclosure may be used for various applications, such as, for example, processing a single analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein) from a single biological particle (e.g., cell, cell bead, or nucleus of a cell). For example, a biological particle (e.g., a cell or cell bead, or nucleus of a cell) is partitioned in a partition (e.g., droplet), and multiple analytes from the biological particle are processed for subsequent processing. The multiple analytes may be from the single biological particle. This may enable, for example, simultaneous proteomic, transcriptomic and genomic analysis of the biological particle.

[00144] Libraries of nucleic acid molecules that have nucleic acid sequences, e.g., nucleic acid sequences encoding proteins, such as paired T cell receptors (TCRs), B cell receptors (BCRs), and antibodies or antigen binding fragments thereof, can be employed to provide an enriched nucleic acid sequence of interest of the nucleic acid sequences, e.g., encoding an amino acid sequence of interest (e.g., a specific T cell receptor, B cell receptor, or antibody or antigen binding fragment thereof). The library can be generated, for example, by isolating and/or amplifying RNA encoding the amino acid sequence of interest, or using DNA, e.g., genomic DNA. The RNA library can be reverse transcribed to yield a cDNA library, and identification sequences (e.g., barcode sequence or unique molecular identification sequences) can be appended to members of the library and can be used to identify members of the library.

[00145] In some instances, a barcoded nucleic acid library comprising immune molecules (e.g., from single cells) is generated as described herein. For example, in some embodiments, RNA molecules are processed as generally described in FIGS. 12B-C. Referring to FIG. 12B, in some instances, nucleic acid molecules derived from a cell (such as RNA molecules) are processed to append a cell (e.g., partition) specific barcode sequence 1222 to these molecules or derivatives thereof (e.g., cDNA molecules). For example, referring to FIG. 12B, in some embodiments, primer 1250 comprises a sequence complementary to a sequence of RNA molecule 1260 from a cell (such as an RNA encoding for an immune molecule, such as a light or heavy chain antibody sequence). In some instances, primer 1250 comprises one or more adapter sequences 1251 that are not complementary to RNA molecule 1260. In some instances, primer 1250 comprises a poly-T sequence. In some instances, primer 1250 comprises a sequence complementary to a target sequence in an RNA molecule. In some instances, primer 1250 comprises a sequence complementary to a region of an immune molecule, such as the constant region of an RNA encoding a TCR, BCR, or antibody molecule. Primer 1250 is hybridized to RNA molecule 1260 and cDNA molecule 1270 is generated in a reverse transcription reaction. In some instances, the reverse transcriptase enzyme is selected such that several non-templated bases 1280 (e.g., a poly-C sequence) are appended to the cDNA. Nucleic acid barcode molecule 1290 comprises a sequence 1224 complementary to the non- templated bases, and the reverse transcriptase performs a template switching reaction onto nucleic acid barcode molecule 1290 to generate a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and a sequence of cDNA 1270 (or a portion thereof). In another example, referring to FIG. 12C, in some embodiments, nucleic acid barcode molecule 1290 comprises sequence 1223 complementary to a sequence of RNA molecule 1260 from a cell. In some instances, sequence 1223 comprises a sequence specific for an RNA molecule. In some instances, sequence 1223 comprises a poly-T sequence. In some instances, sequence 1223 comprises a sequence specific for an RNA molecule. In some instances, sequence 1223 comprises a sequence complementary to a region of an immune molecule, such as the constant region of an RNA encoding a TCR, BCR, or antibody molecule. Sequence 1223 is hybridized to RNA molecule 1260 and a cDNA molecule 1270 is generated in a reverse transcription reaction generating a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and a sequence of cDNA 1270 (or a portion thereof). Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g., to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g., U.S. Pat. Pub. 20180105808, which is hereby incorporated by reference in its entirety. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform. In some instances, one or more labelling agents capable of binding to or otherwise coupling to one or more cell features may be used to characterize cells and/or cell features as described herein (e.g., to characterize immune receptor or antigen specificity of immune molecules).

[00146] Molecules of the library can have the structure, from 5’ to 3’, of identification sequence to coding sequence. For example, molecules of the library can have the structure, from 5’ to 3’, of: (1) barcode sequence; (2) unique molecular identifier sequence; (3) template switch oligonucleotide sequence (4) immune molecule variable sequence (e.g., V(D)J sequence, as provided herein); and (5) immune receptor constant sequence. In some embodiments, one or more adapter sequences (such as sequencing platform specific sequences, such as a sequencing primer or primer binding sequence, e.g., an Illumina R1 or R2) can be located either 5’ or 3’ to the sequence of a molecule of a library, or both.

[00147] In some instances, a barcoded gene expression library is generated (e.g., from single cells as described herein) from a plurality of cells comprising an immune molecule, such as a TCR, BCR, or antibody. The barcoded library can then be sequenced and analyzed to identify paired immune molecule sequences from single cells (e.g., comprising a common barcode sequence), such as paired TCRs (e.g., TRA TRB), paired BCRs (light/heavy chain sequences), and paired antibody sequences (light/heavy chain sequences). Immune molecules of interest (e.g., paired light/heavy chain antibodies) can then be directly enriched (e.g., amplified) from the barcoded library for subsequent processing and analysis in, e.g., an expression vector. In some instances, primers are designed to amplify paired immune molecules, e.g., light and heavy chain antibody sequences, from the library for cloning into one or more suitable expression vectors.

[00148] Enrichment of a nucleic acid sequence of interest from, e.g., a barcoded gene expression library, can allow expedited isolation of the nucleic acid, and the expression and/or analysis for the amino acid sequence which it encodes. For example, enrichment (e.g., using one or more PCR reactions) of sequences of interest (e.g., a V(D)J sequence, such as paired TCR (e.g., TRA/TRB), BCR, or antibody (e.g., heavy/light chain) sequences) and direct cloning of those enriched sequences (such as a light and heavy chain sequence of an antibody) into an appropriate expression vector can be utilized to avoid costly and time consuming methodologies (such as gene synthesis) employed to generate an expression vector configured to express immune molecules (e.g., antibodies) of interest. A nucleic acid sequence of interest can be enriched by amplifying the nucleic acid sequence of interest based on an identification sequence (e.g., barcode and/or UMI) associated with the nucleic acid sequence of interest, for example, by using a scheme such as is illustrated in FIG. 9 In some embodiments, the nucleic acid sequence of interest is enriched by amplifying the nucleic acid sequence of interest based at least in part on a validated identification sequence (e.g., a UMI that has been validated by a method or system disclosed herein). In some embodiments, a nested amplification approach can be employed to further enrich the nucleic acid sequence of interest. [00149] As part of an enrichment protocol, a nucleic acid primer can be designed that anneals to one or more identification sequences in a molecule that harbors a nucleic acid sequence of interest, e.g., a barcode sequence or unique molecule identifier. Another primer can be designed to anneal to a sequence downstream of the identification sequence, and can be configured such that the nucleic acid sequence can be amplified using the primers, e.g., by polymerase chain reaction. A second round of amplification can be performed using a different set of primers to further enrich the nucleic acid sequence of interest.

[00150] After enriching a nucleic acid sequence of interest, it can be cloned into a vector and subsequently expressed in an expression system. Such cloning and expression can yield protein for analysis. For example, a candidate T cell receptor, B cell receptor, or antibody or antigen binding fragment thereof can be expressed in an expression system where such a nucleic acid sequence of interest is cloned. Such a protein can be a therapeutic candidate, a gene of interest, a protein variant of interest, or another protein to be analyzed. In some instances, primers are designed to amplify paired immune molecule sequences from single cells (e.g., comprising a common barcode sequence), such as paired TCRs (e.g., TRA/TRB), paired BCRs (light/heavy chain sequences), and paired antibody sequences (light/heavy chain sequences). These amplified, paired immune molecule sequences (e.g., paired light and heavy chain antibody sequences) can then be optionally processed for subsequent cloning into an expression vectors for expression of functional immune molecules (e.g., a plasmid configured to co-express paired immune molecule subunits, such as an antibody heavy and light chain).

[00151] Methods provided herein can comprise providing a plurality of nucleic acid molecules. Nucleic acid molecules described herein can comprise ribonucleic acids (e.g., RNA, such as RNA molecules provided herein) or deoxyribonucleic acids (e.g., DNA or cDNA). A nucleic acid molecule can comprise G, A, T, U, C, or bases that are capable of base pairing reliably with a complementary nucleotide. 7-deaza-adenine, 7-deaza-guanine, adenine, guanine, cytosine, thymine, uracil, 2-deaza-2-thio-guanosine, 2-thio-7-deaza-guanosine, 2-thio- adenine, 2-thio- 7-deaza- adenine, isoguanine, 7-deaza-guanine, 5,6-dihydrouridine, 5,6- dihydrothymine, xanthine, 7-deaza- xanthine, hypoxanthine, 7-deaza-xanthine, 2,6 diamino-7- deaza purine, 5- methyl-cytosine, 5- propynyl-uridine, 5-propynyl-cytidine, 2-thio-thymine or 2-thio-uridine are examples of such bases, although many others are known A nucleic acid molecule can comprise an LNA, a PNA, a UNA, or an morpholino oligomer, for example. A nucleic acid molecule used herein may contain natural or non- natural nucleotides or linkages.

[00152] A nucleic acid molecule can comprise an identification sequence. An identification sequence can identify, for example, the nucleic acid molecule, the source of the nucleic acid sample, or another property of the nucleic acid sample. For example, the nucleic acid molecule can comprise one or more of: an adapter sequence, a primer or primer binding sequence, a sequencing primer or sequencing primer binding sequence (such as an R1 or partial R1 sequence), a unique molecular identifier (UMI), a polynucleotide sequence (such as a poly-A or poly-C sequence), or a sequence configured to bind to the flow cell of a sequencer (such as a P5 or P7, or partial sequences thereof).

It is to be understood that the nucleic acid molecule can further comprise a cell barcode sequence, e.g., a partition-specific barcode.

[00153] In some embodiments, a nucleic acid molecule of a plurality of nucleic acid molecules can comprise two or more of a barcode, a unique molecular identification sequence, and a template switch oligonucleotide sequence. For example, a nucleic acid molecule can comprise a barcode and a unique molecular identification sequence, a barcode and a template switch oligonucleotide sequence, or a unique molecular identification sequence and a template switch oligonucleotide sequence. [00154] An example of such a nucleic acid molecule is included in the top panel of FIG. 10. [00155] In some embodiments, a nucleic acid sequence of interest can be engineered to comprise a restriction site (e.g., using PCR primers comprising restriction sites). In some embodiments, a restriction site can be utilized for cloning after enrichment of the nucleic acid sequence of interest. [00156] A nucleic acid molecule of a plurality of nucleic acid molecules can comprise a nucleic acid sequence that can code for an amino acid sequence. In some embodiments, the amino acid sequence can be of a T cell receptor or a B cell receptor. In some embodiments, the amino acid sequence can be of an antibody or antigen binding fragment thereof.

[00157] In some embodiments, a nucleic acid molecule of a plurality of nucleic acid molecules can comprise a nucleic acid sequence of interest, such as a nucleic acid sequence described herein. [00158] As used herein, the term “antibody” can refer to an immunoglobulin (Ig), polypeptide, or a protein (e.g., BCR) having a binding domain which is, or is homologous to, an antigen-binding domain. The term can further include “antigen-binding fragments” and other interchangeable terms for similar binding fragments as described herein Native antibodies and native immunoglobulins (Igs)can be heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains. Antibodies can further refer to camelid antibodies. In some cases, camelid antibodies are not tetrameric. Each light chain can be linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages can vary among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain can have regularly spaced intrachain disulfide bridges. Each heavy chain can have at one end a variable domain (“VH”) followed by a number of constant domains (“CH”). Each light chain can have a variable domain at one end (“VL”) and a constant domain (“CL”) at its other end; the constant domain of the light chain can be aligned with the first constant domain of the heavy chain, and the light-chain variable domain can be aligned with the variable domain of the heavy chain. Particular amino acid residues can form an interface between the light- and heavy-chain variable domains.

[00159] In some instances, an antibody or an antigen-binding fragment thereof comprises an isolated antibody or antigen-binding fragment thereof, a purified antibody or antigen-binding fragment thereof, a recombinant antibody or antigen-binding fragment thereof, a modified antibody or antigen-binding fragment thereof, or a synthetic antibody or antigen-binding fragment thereof. [00160] Antibodies and antigen-binding fragments herein can be partly or wholly synthetically produced. An antibody or antigen-binding fragment can be a polypeptide or protein having a binding domain which can be, or can be homologous to, an antigen binding domain. In some instances, an antibody or an antigen-binding fragment thereof can be produced in an appropriate in vivo animal model and then isolated and/or purified.

[00161] Depending on the amino acid sequence of the constant domain of its heavy chains, immunoglobulins (Igs) can be assigned to different classes. Major classes of immunoglobulins can include: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. An Ig or portion thereof can, in some cases, be a human Ig. In some instances, a C_//3 domain can be from an immunoglobulin. In some cases, a chain or a part of an antibody or antigen binding fragment thereof, a modified antibody or antigen-binding fragment thereof, or a binding agent can be from an Ig. In such cases, an Ig can be IgG, an IgA, an IgD, an IgE, or an IgM. In cases where the Ig is an IgG, it can be a subtype of IgG, wherein subtypes of IgG can include IgGl, an IgG2a, an IgG2b, an IgG3, and an IgG4. In some cases, a C_#3 domain can be from an immunoglobulin selected from the group consisting of an IgG, an IgA, an IgD, an IgE, and an IgM.

[00162] The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (“K” or “K”) or lambda (“l”), based on the amino acid sequences of their constant domains.

[00163] A “variable region” of an antibody can refer to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain can consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain can be held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. CDRs can be determined by methods such as: (1) an approach based on cross-species sequence variability (/.<?., Rabat etal ., Sequences of Proteins of Immunological Interest, (5th ed ., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Iazikani el al. (1997) ./. Molec. Biol. 273:927-948)). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches.

[00164] With respect to antibodies, the term “variable domain” can refer to the variable domains of antibodies that are used in the binding and specificity of each particular antibody for its particular antigen. In some cases, the variability is not evenly distributed throughout the variable domains of antibodies. In some cases, it is concentrated in three segments called hypervariable regions (also known as CDRs) in both the light chain and the heavy chain variable domains. More highly conserved portions of variable domains can be called the “framework regions” or “FRs.” The variable domains of unmodified heavy and light chains can contain four FRs (FR1, FR2, FR3, and FR4), largely adopting a b-sheet configuration interspersed with three CDRs which can form loops connecting and, in some cases, part of the b-sheet structure. The CDRs in each chain can be held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Rabat et al ., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669).

[00165] “Antibodies” useful in the present disclosure can encompass monoclonal antibodies, polyclonal antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, heteroconjugate antibodies, humanized antibodies, human antibodies, deimmunized antibodies, mutants thereof, fusions thereof, immunoconjugates thereof, antigen-binding fragments thereof, and/or any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. In some embodiments, an antibody can be a murine antibody.

[00166] An antibody can be a human antibody. As used herein, a “human antibody” can be an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or that has been made using any suitable technique for making human antibodies. Human antibodies can include antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al. , 1996, Nature Biotechnology , 14:309-314; Sheets etal ., 1998, PNAS USA, 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks etal., 1991, J Mol. Biol., 222:581). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro). See , e.g., Cole et al ., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147 (l):86-95; and U.S. Pat. No. 5,750,373.

[00167] Any of the antibodies herein can be bispecific. Bispecific antibodies can be antibodies that have binding specificities for at least two different antigens and can be prepared using the antibodies disclosed herein. Exemplary methods for making bispecific antibodies are described (see, e.g, Suresh el al, 1986, Methods in Knzymology 121:210). The recombinant production of bispecific antibodies can be based on the coexpression of two immunoglobulin heavy chain-light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, 1983, Nature, 305, 537-539). Bispecific antibodies can be composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure, with an immunoglobulin light chain in only one half of the bispecific molecule, can facilitate separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This approach is described, for example, in PCT Publication No. WO 94/04690.

[00168] Functional fragments of any of the antibodies herein are also contemplated. The terms “antigen-binding portion of an antibody,” “antigen-binding fragment,” “antigen-binding domain,” “antibody fragment,” or a “functional fragment of an antibody” can refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Representative antigenbinding fragments include a Fab, a Fab', a F(ab')i, a Fv, a scFv, a dsFv, a variable heavy domain, a variable light domain, a variable NAR domain, bi-specific scFv, a bi-specific Fab2, a tri-specific Fab3, an AVIMER®, a minibody, a diabody, a maxibody, a camelid, a VHH, a minibody, an intrabody, fusion proteins comprising an antibody portion (e.g., a domain antibody), and a single chain binding polypeptide.

[00169] “F(ab')2” and “Fab¹” moieties can be produced by treating an Ig with a protease such as pepsin and papain, and include antibody fragments generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two heavy chains. For example, papain can cleave IgG upstream of the disulfide bonds existing between the hinge regions in each of the two heavy chains to generate two homologous antibody fragments in which an light chain composed of VL and CL (light chain constant region), and a heavy chain fragment composed of VH and CH₇I (gΐ) region in the constant region of the heavy chain) are connected at their C terminal regions through a disulfide bond. Each of these two homologous antibody fragments can be called Fab'. Pepsin can also cleave IgG downstream of the disulfide bonds existing between the hinge regions in each of the two heavy chains to generate an antibody fragment slightly larger than the fragment in which the two above-mentioned Fab' are connected at the hinge region. This antibody fragment can be called F(ab')2.

[00170] The Fab fragment can also contain the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments can differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteine(s) from the antibody hinge region. Fab'-SH can be a Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments can be produced, for example, as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments can also be employed.

[00171] A “Fv” as used herein can refer to an antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region can consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent or covalent association (disulfide linked Fvs have been described, see, e.g.. Reiter el al. (1996) Nature Biotechnology 14:1239-1245). In this configuration that the three CDRs of each variable domain can interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, a combination of one or more of the CDRs can from each of the VH and VL chains confer antigen-binding specificity to the antibody. For example, the CDRH3 and CDRL3 can be sufficient to confer antigen-binding specificity to an antibody when transferred to VH and VL chains of a recipient antibody or antigen-binding fragment thereof and this combination of CDRs can be tested for binding, specificity, affinity, etc. using, for example, techniques described herein. In some cases, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although likely at a lower specificity or affinity than when combined with a second variable domain. Furthermore, although the two domains of a Fv fragment (VL and VH) can be coded for by separate genes, they can be joined using recombinant methods, for example by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); Bird etal. (1988) Science 242:423-426; Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn etal. (1998) Nat. Biotechnol. 16:778). Such scFvs can be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to an Fc region cDNA or genomic sequences in order to generate expression vectors encoding complete Ig ( e.g ., IgG) molecules or other isotypes. VH and VL can also be used in the generation of Fab, Fv, or other fragments of Igs using either protein chemistry or recombinant DNA technology.

[00172] “Single-chain Fv” or “sFv” antibody fragments can comprise the VH and VL domains of an antibody, wherein these domains can be present in a single polypeptide chain. In some embodiments, the Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[00173] Also contemplated herein is the use of an AVIMER® as an antigen or antigen binding moiety. The term “AVIMER®” can refer to a class of therapeutic proteins of human origin, which can be unrelated to antibodies and antibody fragments, and can be composed of several modular and reusable binding domains, referred to as A-domains (also referred to as class A module, complement type repeat, or LDL-receptor class A domain). They can be developed from human extracellular receptor domains by in vitro exon shuffling and phage display (Silverman et al, 2005, Nat. Biotechnol. 23:1493-1494; Silverman et al., 2006, Nat. Biotechnol. 24:220). The resulting proteins can contain multiple independent binding domains that can exhibit improved affinity and/or specificity compared with single-epitope binding proteins. Each of the known 217 human A- domains can comprise ~35 amino acids (~4 kDa); and these domains can be separated by linkers that can average five amino acids in length. Native A-domains fold quickly and efficiently to a uniform, stable stmcture mediated primarily by calcium binding and disulfide formation. A conserved scaffold motif of only 12 amino acids can be required for this common structure. The end result can be a single protein chain containing multiple domains, each of which represents a separate function. Each domain of the proteins can bind independently, and the energetic contributions of each domain can be additive.

[00174] Antigen-binding polypeptides can also include heavy chain dimers such as, for example, antibodies from camelids and sharks. Camelid and shark antibodies can comprise a homodimeric pair of two chains of V-like and C-like domains (neither has a light chain). Since the VH region of a heavy chain dimer IgG in a camelid does may not have to make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts a light chain can be changed to hydrophilic amino acid residues in a camelid. VH domains of heavy-chain dimer IgGs can be called VHH domains. Shark Ig-NARs can comprise a homodimer of one variable domain (termed a V-NAR domain) and five C-like constant domains (C-NAR domains). In camelids, the diversity of antibody repertoire can be determined by the CDRs 1, 2, and 3 in the VH or VHH regions. The CDR3 in the camel VHH region can be characterized by its relatively long length, averaging 16 amino acids (Muyldermans et ah, 1994, Protein Engineering 7(9): 1129). This can be in contrast to CDR3 regions of antibodies of many other species. For example, the CDR3 of mouse VH can have an average of 9 amino acids. Libraries of camelid-derived antibody variable regions, which can maintain the in vivo diversity of the variable regions of a camelid, can be made by, for example, the methods disclosed in U.S. Patent Application Ser. No. 20050037421.

[00175] As used herein, a “maxibody” can refer to a bivalent scFv covalently attached to the Fc region of an immunoglobulin, see , e.g., Fredericks etal. , Protein Engineering, Design & Selection, 17:95-106 (2004) and Powers etal, Journal of Immunological Methods, 251:123-135 (2001). [00176] As used herein, a “dsFv” can be a Fv fragment obtained, for example, by introducing a Cys residue into a suitable site in each of a heavy chain variable region and a light chain variable region, and then stabilizing the heavy chain variable region and the light chain variable region by a disulfide bond. The site in each chain, into which the Cys residue can be introduced, can be determined based on a conformation predicted by molecular modeling. In the present disclosure, for example, a conformation can be predicted from the amino acid sequences of the heavy chain variable region and light chain variable region of the above-described antibody, and DNA encoding each of the heavy chain variable region and the light chain variable region, into which a mutation has been introduced based on such prediction, can be then constructed. The DNA construct can be incorporated then into a suitable vector and prepared from a transformant obtained by transformation with the aforementioned vector.

[00177] Single chain variable region fragments (“scFv”) of antibodies are described herein. Single chain variable region fragments may be made by linking light and/or heavy chain variable regions by using a short linking peptide. Bird et al. (1988) Science 242:423-426. The single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect, or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resultant scFv can be isolated using any suitable protein purification techniques.

[00178] Diabodies can be single chain antibodies. Diabodies can be bivalent, bispecific antibodies in which VH and VL domains can be expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger, P., etal ., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); and Poljak, R.

T, etal., Structure, 2:1121-1123 (1994)).

[00179] As used herein, a “minibody” can refer to a scFv fused to CH3 via a peptide linker (hingeless) or via an IgG hinge has been described in Olafsen, et al, Protein Eng Des Sel. April 2004; 17(4):315-23.

[00180] As used herein, an “intrabody” can refer to a single chain antibody which can demonstrate intracellular expression and can manipulate intracellular protein function (Biocca, et al, EMBO J. 9:101-108, 1990; Colby etal, Proc Natl Acad. Sci. USA. 101:17616-21, 2004).

Intrabodies, which can comprise cell signal sequences which can retain the antibody construct in intracellular regions, may be produced, for example, as described in Mhashilkar et al, ( EMBO .!., 14:1542-51, 1995) and Wheeler et al. ( FASEBJ . 17:1733-5. 2003). Transbodies can be cell- permeable antibodies in which a protein transduction domains (PTD) can be fused with single chain variable fragment (scFv) antibodies as in, for example, Heng et al. (Med Hypotheses. 64: 1105-8, 2005). [00181] An antibody or antigen binding fragment can bind an epitope. An epitope can be a portion of an antigen or other macromolecule capable of forming a binding interaction with the variable region binding pocket of an antigen binding molecule such as an antibody or antigen binding fragment thereof. Such binding interactions can be manifested as an intermolecular contact with one or more amino acid residues of one or more CDRs. Antigen binding can involve, for example, a CDR3, a CDR3 pair or, in some cases, interactions of up to all six CDRs of the VH and VL chains. An epitope can be a linear peptide sequence (/.<?., “continuous”) or can be composed of noncontiguous amino acid sequences (i.e., “conformational” or “discontinuous”). An antigen binding molecule such as an antibody or antigen binding fragment thereof can recognize one or more amino acid sequences; therefore, an epitope can define more than one distinct amino acid sequence. Epitopes recognized by antigen binding molecules such as antibodies or antigen binding fragments thereof can be determined by peptide mapping or sequence analysis techniques. In some embodiments, binding interactions can be manifested as intermolecular contacts between an epitope on an antigen and one or more amino acid residues of a CDR. Epitopes recognized by antigen binding molecules such as antibodies or antigen binding fragments thereof can be determined, for example, by peptide mapping or sequence analysis techniques. Binding interactions can manifest as intermolecular contacts between an epitope on an antigen and one or more amino acid residues of a complementarity determining region (CDR).

[00182] An epitope can be determined, for example, using one or more epitope mapping techniques. Epitope mapping can comprise experimentally identifying the epitope on an antigen. Epitope mapping can be performed by any acceptable method, for example X-ray co crystallography, cryogenic electron microscopy, array-based oligo-peptide scanning, site-directed mutagenesis mapping, high-throughput shotgun mutagenesis epitope mapping, hydrogen-deuterium exchange, cross-linking coupled mass spectrometry, yeast display, phage display, proteolysis, or a combination thereof.

[00183] An antibody or antigen binding fragment thereof can comprise a V(D)J sequence. The variable region of each immunoglobulin heavy or light chain can be encoded by a plurality of subgenes. These subgenes can comprise variable (V), diversity (D) and joining (J) segments, and can be combined to yield a V(D)J sequence. A heavy chain can comprise V, D and/or J segments, and a light chain can comprise V and/or J segments. Multiple copies of the V, D and J gene segments exist, and can be tandemly arranged in the genomes of mammals. In the bone marrow, each developing B cell can assemble an immunoglobulin variable region, for example by randomly selecting and combining one V, one D and one J gene segment (or one V and one J segment in the light chain). As there can be multiple copies of each type of gene segment, and different combinations of gene segments can be used to generate each immunoglobulin variable region, this process can generate a large number of antibodies with different paratopes, and in some embodiments, different antigen specificities. The rearrangement of several subgenes (e.g., in the V2 family) for lambda light chain immunoglobulin can be coupled with the activation of microRNA miR-650, which can further influence the biology of B-cells.

[00184] A plurality of nucleic acid molecules can comprise a library of nucleic acid molecules.

As a non-limiting example, a library of nucleic acid molecules can comprise complementary deoxyribonucleic acid (cDNA molecules). In some cases, a library of nucleic acid molecules can comprise a library of cDNA molecules. In some embodiments, a library of nucleic acid molecules can comprise a library of variants of a nucleic acid molecule. Variants of a nucleic acid molecule can comprise variants of a nucleic acid molecule that codes for an amino acid sequence, such as an amino acid sequence of an antibody or antigen binding fragment thereof. In some embodiments, variants of a nucleic acid molecule can comprise a nucleic acid sequence coding for an amino acid sequence of a T cell receptor or a B cell receptor.

[00185] For example, variants of an antibody or antigen binding fragment thereof can comprise variants in a variable region. In some embodiments, variants in a variable region can comprise a variant in a V sequence, a variant in a D sequence, a variant in a J sequence, or a combination thereof. Variants of an antibody or antigen binding fragment thereof can have different specificity (e.g., having specificity for different antigens) or different affinity (e.g., having different affinity for a same antigen).

[00186] A nucleic acid molecule of a plurality of nucleic acid molecules can comprise a plurality of nucleic acid molecules that can comprise a nucleic acid sequence coding for a V amino acid sequence, a D amino acid sequence, a J amino acid sequence, or a combination thereof. In some embodiments, a nucleic acid molecule of a plurality of nucleic acid molecules can comprise a nucleic acid sequence coding for a V(D)J amino acid sequence. In some embodiments, a nucleic acid sequence of interest can comprise a nucleic acid sequence coding for a V(D)J amino acid sequence. In some embodiments, different nucleic acid sequences of a plurality of nucleic acid molecules can comprise nucleic acid sequences coding for different V(D)J amino acid sequences. Different V(D)J amino acid sequences can comprise different V sequences, different D sequences, different J sequences, or a combination thereof.

[00187] A plurality of nucleic acid molecules can correspond to a plurality of cell surface proteins from a plurality of cells. In some cases, the plurality of cell surface proteins from a plurality of cells can be different. In some cases, different cell surface proteins can be variations of a cell surface protein. Examples of cell surface proteins can include T cell receptors (e.g., of T cells), B cell receptors (e.g., of B cells), or antibodies or antigen binding fragments thereof. Cell surface proteins can be naturally occurring or synthetic. In some cases, a cell surface protein can be a modified natural protein.

[00188] In some embodiments, providing the plurality of nucleic acid molecules can comprise generating the plurality of nucleic acid molecules. The plurality of nucleic acid molecules generated can comprise a plurality of identification sequences that identify said plurality of nucleic acid molecules.

[00189] Generation of a library of nucleic acid molecules can be accomplished for example by collecting nucleic acid molecules and appending identification sequences (e.g., as described herein to include barcodes, unique molecular identifiers, and/or template switch oligonucleotides) to the nucleic acid molecules. For example, nucleic acid molecules coding for T cell receptors, B cell receptors, or antigens or antibody fragments thereof can be isolated from samples (e.g., cells) and labeled with an identification sequence using methods provided herein. A library can be generated, for example, as described in U.S. Patent No.: 10,550,429, which is incorporated herein in its entirety. Once a library is created, a member of the library can be created, one of the library can be enriched using a primer complementary to at least a portion of the identification sequence. In some embodiments, the enriched member of the library (i.e., nucleic acid sequence of interest) can be cloned and expressed, and in some cases further analysis can be performed on the amino acid product of the nucleic acid sequence of interest (e.g., T cell receptor, B cell receptor, or antibody or antigen binding fragment thereof).

[00190] A nucleic acid sequence of interest can be enriched from a library of nucleic acid molecules. A library of nucleic acid molecules may be a cDNA library generated from a single cell, e.g., B cell, from the immune repertoire of a subject. Such a library may be generated, for example, by isolating and/or amplifying RNA, and reverse transcribing the RNA library to yield a cDNA library. The library may be a barcoded gene expression library generated from a cell, e.g., B cell, partitioned with a barcoded bead. The cell, following lysis or permeabilization, may have its RNA reverse transcribed, and during reverse transcription, have identification sequences (e.g., barcode sequence or unique molecular identification sequences) appended thereto (e.g., generating a whole transcriptome barcoded gene expression library). See, e.g., FIG. 12B or FIG. 13C. Depending on sequences included in the identification sequence, the library may be a sequencing library. Methods for enriching a nucleic acid sequence of interest can comprise an amplification reaction. Examples of amplification reactions can include linear amplification, polymerase chain reaction (PCR), and nested PCR. In some embodiments, a different amplification reaction can be employed.

[00191] PCR can comprise denaturation, annealing, and extension steps. Denaturation can comprise exposing the nucleic acid to a temperature capable of melting the nucleic acid. In some cases, denaturation can occur between 94 °C and 98 °C. In some cases, denaturation can occur at 94 °C, 95 °C, 96 °C, 97 °C, or 98 °C. Denaturation can last for at least 15 seconds, at least 30 seconds, at least 45 seconds, at least 60 seconds, at least 75 seconds, at least 90 seconds, at least 105 seconds, at least 120 seconds, at least 135 seconds, at least 150 seconds, at least 165 seconds, or at least 180 seconds. Annealing can comprise exposing the melted nucleic acid to a temperature which can allow the binding of a primer to the nucleic acid. In some cases, annealing can occur between 50 °C and 75 °C. In some cases, annealing can occur between 55 °C and 70 °C. In some instances, annealing can occur at 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, or 70 °C. Annealing can last for at least 15 seconds, at least 30 seconds, at least 45 seconds, at least 60 seconds, at least 75 seconds, at least 90 seconds, at least 105 seconds, at least 120 seconds, at least 135 seconds, at least 150 seconds, at least 165 seconds, or at least 180 seconds. Extension can comprise exposing the nucleic acid to a temperature at which extension can occur, thereby amplifying the nucleic acid, for example by a polymerase present in the partition with the nucleic acid. Extension can occur between 65 °C and 75 °C. In some cases, extension can occur at 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, 73 °C, 74 °C, or 75 °C. The steps of denaturation, annealing, and extension can be repeated for a number of cycles. In some cases, PCR cycling can proceed for at least 1 cycle. In some cases, PCR cycling can proceed for at least 5, 10, 15, 20, 25, 30, 35, or 40 cycles. In some cases, PCR cycling can proceed for between 1 cycle and 40 cycles, between 1 cycle and 35 cycles, between 1 cycle and 30 cycles, between 1 cycle and 25 cycles, between 1 cycle and 20 cycles, between 1 cycle and 15 cycles, between 1 cycle and 10 cycles, between 1 cycle and 5 cycles, between 5 cycles and 40 cycles, between 5 cycles and 35 cycles, between 5 cycles and 30 cycles, between 5 cycles and 25 cycles, between 5 cycles and 20 cycles, between 5 cycles and 15 cycles, between 5 cycles and 10 cycles, between 10 cycles and 40 cycles, between 10 cycles and 35 cycles, between 10 cycles and 30 cycles, between 10 cycles and 25 cycles, between 10 cycles and 20 cycles, between 10 cycles and 15 cycles, between 15 cycles and 40 cycles, between 15 cycles and 35 cycles, between 15 cycles and 30 cycles, between 15 cycles and 25 cycles, between 15 cycles and 20 cycles, between 20 cycles and 40 cycles, between 20 cycles and 35 cycles, between 20 cycles and 30 cycles, between 20 cycles and 25 cycles, between 25 cycles and 40 cycles, between 25 cycles and 35 cycles, between 25 cycles and 30 cycles, between 30 cycles and 40 cycles, between 30 cycles and 35 cycles, or between 35 cycles and 40 cycles.

[00192] Methods for enriching a nucleic acid sequence of interest, such as an amplification reaction, can comprise contacting the nucleic acid sequence of interest with PCR reaction, e.g., reagents for a PCR reaction. In some cases, reagents for a PCR reaction can comprise a polymerase, one or more sets primers, and a dNTP mixture. In some cases, e.g., in an amplification reaction, a polymerase can be a DNA polymerase, an RNA polymerase, or a reverse transcriptase. In some cases, a set of primers can comprise at least 2 primers which can be complementary to a region of a nucleic acid of interest, such that the region of the nucleic acid of interest can be amplified via PCR using the primer pair. In some cases, a partition can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sets of two primers. In some cases, a partition can comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sets of two primers. In some cases, a partition can comprise one or more probes. A probe can be a DNA binding dye, a hydrolysis probe, a molecular beacon, a dual hybridization probe, an eclipse probe, or an ampliflouor probe. In some cases, a probe can be a SYBR green probe, a Taqman probe, a Scorpions PCR primer, a LUX PCR primer, or a QZyme PCR primer. In some cases, a probe can comprise a label, which can be colored, opaque, radiopaque, fluorescent, radioactive, or otherwise detectable. In some cases, a partition can comprise additional reagents, which can comprise magnesium, salt, glycerol, buffer, dye, or other reagents. A first set of partitions and a second set of partitions can be obtained. In some cases, these partitions can each comprise a nucleic acid molecule, e.g., a target nucleic acid molecule, which can be amplified and detected. A set of partitions can comprise a plurality of partitions. In some cases, a set of partitions can comprise at least 1, at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000, or at least 10,000,000 partitions. In some cases, a set of partitions can comprise a set of droplets, e.g., an aqueous droplet in an emulsion. For example, a first set of partitions can comprise a first set of droplets, and a second set of partitions can comprise a second set of droplets.

[00193] Methods for enriching a nucleic acid sequence of interest, such as an amplification reaction, can comprise contacting the nucleic acid sequence of interest with a nucleic acid primer. In some cases, a nucleic acid primer can be an oligonucleotide suitable for a PCR reaction (e.g., a PCR primer).

[00194] A nucleic acid primer can comprise an oligonucleotide. An oligonucleotide can be a molecule which can be a chain of nucleotides. Oligonucleotides described herein can comprise ribonucleic acids. Oligonucleotides described herein can comprise deoxyribonucleic acids. In some cases, oligonucleotides can be of any sequence, including a user-specified sequence.

[00195] In some embodiments, an oligonucleotide can comprise G, A, T, U, C, or bases that are capable of base pairing reliably with a complementary nucleotide. 7-deaza-adenine, 7-deaza- guanine, adenine, guanine, cytosine, thymine, uracil, 2-deaza-2-thio-guanosine, 2-thio-7-deaza- guanosine, 2-thio- adenine, 2-thio- 7-deaza-adenine, isoguanine, 7-deaza-guanine, 5,6- dihydrouridine, 5,6- dihydrothymine, xanthine, 7-deaza-xanthine, hypoxanthine, 7-deaza-xanthine, 2,6 diamino-7- deaza purine, 5- methyl-cytosine, 5-propynyl-uridine, 5-propynyl-cytidine, 2-thio- thymine or 2-thio-uridine are examples of such bases, although many others are known. An oligonucleotide can comprise an LNA, a PNA, a UNA, or an morpholino oligomer, for example. The oligonucleotides used herein may contain natural or non- natural nucleotides or linkages.

[00196] An oligonucleotide can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides long. In some cases, an oligonucleotide can be between 10-30, between 10-50, between 10-70, between 10-100, between 20-50, between 20-70, between 20-100, between 30-50, between 30-70, between 30-100, between 40-70, between 40-100, between 50-70, between 50-100, between 60-70, between 60-80, between 60-90, or between 60-100 nucleotides in length. In some cases, an oligonucleotide can be no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, or no more than 100 nucleotides long.

[00197] In some cases, an oligonucleotide can be wholly single stranded. In some cases, an oligonucleotide can be partially double stranded. A partially double stranded region can be at the 3 ’ end of the oligonucleotide, at the 5’ end of the oligonucleotide, or between the 5’ end and 3’ end of the oligonucleotide. In some cases, there may be more than one double stranded region.

[00198] Methods can comprise using a nucleic acid primer complementary to a portion of the nucleic acid sequence of interest (e.g., the identification sequence or a portion thereof). In some embodiments, a nucleic acid primer complementary to at least a portion of the identification sequence of a nucleic acid sequence of interest can be complimentary to at least a portion of a barcode sequence, at least a portion of a template switch oligonucleotide sequence, at least a portion of a unique molecular identifier sequence, or a combination thereof. In some embodiments, a nucleic acid primer can be complementary to a barcode sequence and a read sequence of the nucleic acid sequence of interest, or a portion thereof. In some embodiments, a nucleic acid primer can be complementary to a barcode sequence and a unique molecular identifier sequence, or a portion thereof. This can be done to amplify the nucleic acid sequence of interest, for example using an amplification reaction provided herein (e.g., PCR or nested PCR).

[00199] In some embodiments, a nucleic acid primer can further comprise a nucleic acid sequence that can be complementary to at least a portion of a coding sequence of the nucleic acid sequence of interest. In some embodiments, the nucleic acid primer can comprise a nucleic acid sequence that can be complementary to a variable region of the nucleic acid sequence of interest, such as the variable region of a T cell receptor, the variable region of a B cell receptor, or the variable region of an antigen or antibody binding fragment thereof. As an example, the nucleic acid primer can comprise a nucleic acid sequence that is complementary to a V(D)J sequence or a portion thereof, a V sequence of the V(D)J sequence or a portion thereof, a D sequence of the V(D)J sequence or a portion thereof, or a J sequence of the V(D)J sequence or a portion thereof. In some embodiments, a nucleic acid primer can be complementary to a portion of the variable region of the nucleic acid sequence of interest that is different than that of a different nucleic acid molecule.

[00200] In some embodiments, a nucleic acid primer can further comprise a non-binding handle. A non-binding handle can be a nucleic acid sequence on the nucleic acid primer that is not complementary to a segment of the nucleic acid sequence of interest. In some embodiments, a nonbinding handle may not bind any nucleic acid sequence in the plurality of nucleic acid molecules. In some embodiments, a non-binding handle can be utilized for cloning into recipient vector or to enable pairing of specific heavy/light (or TRA/TRB) sequences using overlap extension or a similar method after enrichment of the nucleic acid molecule.

[00201] In some embodiments, methods provided herein can further comprise using another nucleic acid primer to amplify said nucleic acid sequence of interest, wherein said another nucleic acid primer is different from said nucleic acid primer. For example, a method can comprise using a first nucleic acid primer and a second nucleic acid primer (e.g., a forward primer and a reverse primer for a PCR reaction).

[00202] In some embodiments, the another nucleic acid primer can comprise a non-binding handle. Such a non-binding handle can be a nucleic acid sequence on the nucleic acid primer that is not complementary to a segment of the nucleic acid sequence of interest. In some embodiments, a non-binding handle may not bind any nucleic acid sequence in the plurality of nucleic acid molecules. In some embodiments, a non-binding handle can be utilized for cloning into recipient vector or to enable pairing of specific heavy/light (or TRA/TRB) sequences using overlap extension or a similar method after enrichment of the nucleic acid molecule.

[00203] In some methods, the nucleic acid primer and another (e.g., a second) nucleic acid primer (e.g., a forward primer and a reverse primer) can be configured to anneal to sequences flanking at least a portion of said nucleic acid sequence of interest. For example, a nucleic acid primer can be configured to anneal to a sequence upstream of the nucleic acid sequence of interest, and a second nucleic acid primer can be configured to anneal to a complement of a sequence downstream of the nucleic acid sequence of interest. In some embodiments, two such nucleic acid primers can be configured to yield a copy of the nucleic acid sequence of interest after an amplification reaction such as PCR. [00204] In some embodiments, a second nucleic acid primer can comprise a nucleic acid sequence complementary to a binding sequence on the nucleic acid sequence of interest, or a complement thereof. Such a binding sequence can be on a coding section of the nucleic acid sequence of interest, or upstream or downstream of the coding section of the nucleic acid sequence of interest. In some embodiments, the second nucleic acid primer can be complementary to at least a portion of a nucleic acid sequence coding for a constant region of an amino acid sequence coded for by the nucleic acid sequence of interest, such as a T cell receptor, a B cell receptor, or an antibody or antigen binding fragment thereof, or a complement of such a nucleic acid sequence.

[00205] The second nucleic acid primer can be at least partially complementary to a variable region of a nucleic acid sequence of interest (e.g., a V(D)J sequence, a V sequence, a D sequence, or a J sequence). For example, the second nucleic acid primer can be further complementary to at least a portion of a nucleic acid sequence coding for a J region of an antibody or antigen binding fragment thereof.

[00206] Methods can further comprise a second enrichment step, such as a second amplification reaction. A second amplification reaction can comprise linear amplification, PCR, or another amplification scheme. In some embodiments, a second PCR reaction can be implemented to enrich or further enrich a nucleic acid sequence of the plurality of nucleic acid molecules. As an example, a nested PCR scheme can be utilized to provide enrichment of a nucleic acid sequence of interest. [00207] A second round of amplification can comprise contacting the nucleic acid sequence with a third primer and a fourth primer. A third primer or a fourth primer can comprise an oligonucleotide as provided herein. A third primer and a fourth primer can be configured to specifically enrich the nucleic acid sequence. In some embodiments, the third primer can be different than the first primer, or the fourth primer can be different than the second primer.

[00208] The third primer can be complementary to at least a portion of the identification sequence. In some embodiments, the third primer can be complementary to a portion of a barcode of the identification sequence. In some cases, the third primer can be complementary to a 5’ end of a barcode of the identification sequence.

[00209] In some embodiments, a third primer can be complementary to a portion of the identification sequence upstream of a barcode of the identification sequence. For example, a third primer can be complementary to at least a portion of a read sequence of a nucleic acid molecule. In some embodiments, a third primer can be complementary to at least a portion of a variable sequence, such as a nucleic acid sequence coding for a V(D)J sequence.

[00210] A fourth primer can be complementary to the complement of another segment of the nucleic acid molecule, such that the nucleic acid sequence of interest can be flanked by the third primer and the fourth primer. In some embodiments, the fourth primer can be complementary to a nucleic acid sequence downstream of a coding sequence of the nucleic acid sequence of interest. In some embodiments, the fourth primer can be complementary to at least a portion of the complement of a constant region of the nucleic acid sequence of interest.

[00211] In some methods, others of the plurality of nucleic acid molecules can be not amplified. For example, nucleic acid molecules that do not comprise the nucleic acid sequence of interest can be not amplified. In some methods, others of the plurality of nucleic acid molecules can be amplified by less than a threshold amount. For example, a nucleic acid sequence of interest can be amplified by more than 100 times, more than 1000 times, more than 10,000 times, more than 100,000 times, more than 1,000,000 times, or more than 10,000,000 times more than others of the plurality of nucleic acid molecules.

[00212] Methods provided herein can further comprise determining an enrichment level of the nucleic acid sequence of interest. Enrichment can be determined, for example, by fluorescence, gel electrophoresis, sequencing, or another acceptable method for determining enrichment.

[00213] A nucleic acid sequence of interest can be enriched by a factor of at least 1000 at least 10,000, at least 100,000, at least 1,000,000, or at least 10,000,000. In some embodiments, a nucleic acid sequence of interest can be enriched by a factor sufficient for cloning the nucleic acid sequence of interest. In some embodiments, a nucleic acid sequence of interest can be further enriched by a second amplification step by a factor of at least 1000, at least 10,000, at least 100,000, at least 1,000,000, or at least 10,000,000.

[00214] Also provided herein are methods comprising enriching a nucleic acid sequence of interest based on at least a portion of a constant region of said nucleic acid sequence of interest. This enrichment can yield an enriched nucleic acid sequence of interest. In some embodiments, the method can further comprise modification of the enriched nucleic acid sequence yielding a modified enriched nucleic acid sequence. A pictorial outline of such a method is provided in FIG. 17. The modified enriched nucleic acid sequence can be compatible with a vector. For example, the modified enriched nucleic acid sequence can have a structure (e.g., a nucleic acid sequence) that can be directly incorporated into a vector. A vector can be a vector suitable for cloning or expression of the modified enriched nucleic acid sequence or other use of the modified enriched nucleic acid sequence. A vector can be any vector described herein. A pictorial outline of a nucleic acid sequence that is compatible with a vector (including incorporation of the nucleic acid sequence into the vector) is provided in FIG. 18.

[00215] A nucleic acid sequence of interest can be a nucleic acid sequence described herein. For example, a nucleic acid sequence of interest can code for at least a portion of a cell surface protein of a cell, such as a T cell receptor (or fragment thereof) or a B cell receptor (or fragment thereof). A nucleic acid sequence of interest can comprise a constant region. In some embodiments, the nucleic acid sequence of interest can comprise a sequence encoding a V(D)J sequence or a portion thereof, such as a V sequence (or portion thereof), a D sequence (or portion thereof), or a J sequence (or portion thereof), as described herein. In some embodiments, the constant region of a nucleic acid sequence of interest can comprise a sequence encoding a V(D)J sequence or a portion thereof, such as a V sequence (or portion thereof), a D sequence (or portion thereof), or a J sequence (or portion thereof), as described herein. In some embodiments, a nucleic acid sequence of interest can comprise a barcode (e.g., as provided herein), a UMI (e.g., as provided herein), or a 5’ untranslated region (5’ UTR) of a gene of interest (e.g., a TCR gene or BCR gene). In some embodiments, the nucleic acid sequence of interest can comprise complementary deoxyribonucleic acid (cDNA) of an RNA transcript of interest (e.g., a TCR or BCR transcript).

[00216] Enriching can be performed using a first nucleic acid primer. A first nucleic acid primer can be complementary to a region of the nucleic acid sequence of interest. For example, the first nucleic acid primer can be complementary at least to a barcode or portion thereof on said nucleic acid sequence of interest. In some embodiments, the first nucleic acid primer can be complementary to a UMI sequence or a portion thereof on said nucleic acid sequence of interest. In some embodiments, the first nucleic acid primer can be complementary at least to a 5’ untranslated region (5’ UTR) or a portion thereof on said nucleic acid sequence of interest. In some embodiments, the first nucleic acid primer can be a framework leader (FWR1) primer.

[00217] Enriching can be performed using a second nucleic acid primer. In some embodiments, the second nucleic acid primer can be used with the first nucleic acid primer to enrich the nucleic acid sequence of interest. In some embodiments, the second nucleic acid primer can be complementary at least to a constant region or portion thereof on said nucleic acid sequence of interest. In some embodiments, the second nucleic acid primer can be complementary at least to a V(D)J sequence or portion thereof on said nucleic acid sequence of interest. In some embodiments, the second nucleic acid primer can be complementary at least to a J sequence or portion thereof on said nucleic acid sequence of interest. In some embodiments, the second nucleic acid primer can be complementary at least to a nucleic acid sequence of a junction region or portion thereof on said nucleic acid sequence of interest.

[00218] Enriching can be performed using hybridization capture. In some embodiments, the hybridization capture can be based on hybridization of a nucleic acid probe to a sequence on said nucleic acid sequence of interest such as a constant sequence or a junction sequence. For example, a probe can hybridize to a portion of a junction sequence such as a V(D)J sequence or a portion thereof, such as a V sequence or a portion thereof, a D sequence or a portion thereof, or a J sequence or a portion thereof. In some embodiments, a probe can hybridize to a V sequence and a D sequence (or a portion thereof) or a D sequence and a J sequence (or a portion thereof). The probe may comprise a functional group (such as a biotin molecule) to enable purification of the hybridized target nucleic acid molecule (e.g., using streptavidin conjugated beads, such as magnetic beads).

See, e.g., FIG. 19.

[00219] A nucleic acid primer used for enriching can be selected based on Rapid Amplification of cDNA Ends (RACE) sequencing. RACE sequencing can be a technique used to obtain a sequence (e.g., 5’ RACE) of a nucleic acid (e.g., an RNA transcript), such as a nucleic acid (e.g., an RNA transcript) found within a cell. RACE sequencing can result in the production of a cDNA copy of a sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies (see RT-PCR). The amplified cDNA copies can be sequenced and can map to a unique genomic region. In some embodiments, the RACE-products can be sequenced by next generation sequencing technologies.

[00220] In some embodiments, a method can further comprise cloning a modified enriched nucleic acid into a vector, such as a vector the modified enriched nucleic acid sequence is compatible with. Cloning can be performed using any acceptable method, including methods provided herein (e.g., in the cloning section). [00221] Nucleic acid primers should not be interpreted to be specific to a particular nucleic acid strand. For example, in some embodiments, a first nucleic acid molecule can be complementary to a complement of an identification sequence as described herein. In some embodiments, a second nucleic acid molecule can be complementary to a binding sequence as designed herein.

[00222] In yet further embodiments of enriching for a nucleic acid sequence of interest, in which the nucleic acid sequence of interest is a BCR or TCR, or fragment thereof, the enrichment may be performed via first and second amplification reactions. The first reaction may be performed with first and second primers in which: (i) the first primer has a sequence complementary to at least a portion of the barcode sequence and/or the UMI sequence, and (ii) the second primer has a sequence complementary to a complement of at least a portion of the nucleic acid sequence of interest that encodes a junction (J) region and/or isotype region of the BCR or TCR, or fragment thereof. The second reaction may be performed with third and fourth primers in which: (i) the third primer includes a sequence complementary to nucleotides of at least a portion of the leader sequence and/or encoding framework region (FWR)l of the BCR or TCR, or fragment thereof, and (ii) the fourth primer includes a sequence complementary to a complement of at least a portion of the nucleic acid sequence of interest that encodes a complementarity region (CDR)3, FWR4, a J region, a D region, and/or a V region, or a junction between any one or more thereof, of the BCR or TCR, or fragment thereof.

[00223] In certain embodiments of the method, in the first amplification reaction, the first primer may include a sequence complementary to at least a portion of the barcode sequence and the UMI sequence. In certain other embodiments, the first primer may include a sequence complementary to the barcode sequence and the UMI sequence. In some embodiments, the second primer may include a sequence complementary to the complement of the nucleic acid sequence of interest that encodes at least a portion of the J region of the BCR or TCR, or fragment thereof. In other embodiments, the second primer may include a sequence complementary to the complement of the nucleic acid sequence of interest that encodes at least a portion of the isotype region of the BCR or TCR, or fragment thereof. In yet other embodiments, the second primer may include a sequence complementary to the complement of the nucleic acid sequence of interest that encodes at least a portion of the J region and the isotype region of the BCR or TCR, or fragment thereof. In certain embodiments of the method, in the first amplification reaction, the first primer may include a sequence complementary to at least a portion of the barcode sequence and the UMI sequence and the second primer may include a sequence complementary to the complement of the nucleic acid sequence of interest that encodes at least a portion of the J region and the isotype region of the BCR or TCR,or fragment thereof.

[00224] In certain embodiments of the method, in the second amplification reaction, the third primer may include a sequence complementary to nucleotides of at least a portion of the leader sequence or encoding FWR1 of the BCR or TCR, or fragment thereof. In other certain embodiments of the methods, the third primer may include a sequence complementary to nucleotides encoding at least a portion of the FWR1 of the BCR or FCR, or fragment thereof. In other embodiments of the method, the fourth primer may include a sequence complementary to the complement of at least a portion of the nucleic acid sequence of interest that encodes the CDR3, and junction extending into the J region of the BCR or TCR or fragment thereof. In yet other embodiments of the methods, the fourth primer may include a sequence complementary to the complement of at least a portion of the nucleic acid sequence of interest that encodes the D and J regions, or a junction between the D and I regions of the BCR or TCR or fragment thereof. In yet still other embodiments of the methods, the fourth primer may include a sequence complementary to the complement of at least a portion of the nucleic acid sequence of interest that encodes the V and J regions, or a junction between the V and I regions, of the BCR or TCR, or fragment thereof. In still other embodiments of the methods, the fourth primer may include a sequence complementary to the complement of at least a portion of the nucleic acid sequence of interest that encodes the V and D regions, or a junction between the V and D regions, of the BCR or TCR, or fragment thereof. In yet a further embodiment of the methods, the fourth primer may include a sequence complementary to the complement of at least a portion of the nucleic acid sequence of interest that encodes the V, D and J regions of the BCR or TCR, or fragment thereof. In certain embodiments of the method, in the second amplification reaction, the third primer may include a sequence complementary to nucleotides of at least a portion of the leader sequence of or encoding FWR1 the BCR or TCR, or fragment thereof and the fourth primer may include sequence complementary to the complement of at least a portion of the nucleic acid sequence of interest that encodes the CDR3, and junction extending into the J region of the BCR or TCR, or fragment thereof. [00225] In an embodiment of the method, the first amplification reaction may employ a first primer that includes a sequence complementary to at least a portion of the barcode sequence and the UMI sequence and a second primer that includes a sequence complementary to the complement of the nucleic acid sequence of interest that encodes at least a portion of the J region and the isotype region of the BCR or TCR, or fragment thereof. The first amplification may be followed by a second amplification reaction that may employ a third primer that includes a sequence complementary to nucleotides of at least a portion of the leader sequence of or that encodes FWR1 of the BCR or TCR, or fragment thereof and the fourth primer may include a sequence complementary to the complement of at least a portion of the nucleic acid sequence of interest that encodes the CDR3, and junction extending into the J region of the BCR or TCR, or fragment thereof. Modification of an enriched nucleic acid sequence of interest

[00226] Further modification of a nucleic acid sequence of interest can be performed, for example after the nucleic acid sequence of interest has been enriched. In some embodiments, modification of a nucleic acid sequence of interest can be performed in preparation for analysis of the nucleic acid sequence of interest, to analyze the nucleic acid sequence of interest, or to prepare the nucleic acid sequence of interest for cloning.

[00227] Methods can further comprise performing fragmentation of a nucleic acid sequence of interest. Nucleic acid fragmentation (e.g. footprinting), such as by OH radicals, can be a tool to probe nucleic acid structure and nucleic acid-protein interactions. Such methods can provide structural information with single base pair resolution. Footprinting can refer to assays in which either the binding of a ligand to a specific sequence of bases or the conformation of the nucleic acid inhibits nicking of the phosphodiester backbone of nucleic acid polymer by a reagent. Intimate interactions between proteins and nucleic acids can be widely examined by footprinting methods. A prerequisite of such assays can be the ability to produce and detect high-quality nucleic acid fragmentation around the protein-protected areas. Nucleic acid fragmentation can be achieved by using a variety of enzymatic and chemical reagents. This can be highly related to the development of chemical hydroxyl radical footprinting using Fenton chemistry and peroxonitrous acid. Hydroxyl radicals can engender breaks of the phosphodiester backbone in a non-specific sequence manner and, hence, can be utilized for footprinting assays. Using hydroxyl radical methods over enzymatic footprinting can be advantageous because it can provide great sensitivity to nucleic acid structures, such as sequence-dependent curvature and RNA folding.

[00228] Methods can further comprise A-tailing of a nucleic acid sequence of interest. A- tailing can comprise an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule. A-tailing can be performed to prepare a T-vector for use in TA cloning or to A-tail a PCR product produced by a high-fidelity polymerase (e.g., other than Taq) for use in TA cloning. TA cloning can be a rapid method of cloning PCR products that can utilize stabilization of the single-base extension (adenosine) produced by Taq polymerase by the complementary T (thymidine) of the T-vector prior to ligation and transformation. This technique may not utilize restriction enzymes and PCR products can be used directly without modification. Additionally, in some embodiments PCR primers do not need to be designed with restriction sites, making the process less complicated. In some embodiments, A-tailing can be non-directional, meaning the insert can go into the vector in both orientations.

[00229] Methods can further comprise performing a sample index polymerase chain reaction (SI- PCR) on a nucleic acid sequence of interest. SI-PCR can utilize different pairs of index primers on a nucleic acid molecule. In some cases, index primers can beadded to individual samples in a second thermocycling step, for example after initial amplification of the target region. This can allow mixing of many samples together (e.g., up to 96) and simultaneous sequencing of the samples. Following sequencing, for example on an Illumina MiSeq, software can be able to identify these indexes on each sequence read, in some cases allowing separation of the reads for each different nucleic acid molecule.

[00230] Methods can further comprise V(D)J enrichment of a nucleic acid sequence of interest. This can be accomplished, for example, using PCR or another amplification method to amplify a V(D)J sequence or a fragment thereof from the enriched nucleic acid sequence of interest.

[00231] Modification of a nucleic acid sequence of interest or enriched nucleic acid sequence of interest can comprise addition of Gibson ends to said amplified nucleic acid sequences. Addition of Gibson ends (e.g., Gibson Assembly) can allow cloning or joining of two nucleic acid sequences without restriction sites. In some cases, addition of Gibson ends can allow joining of any two fragments regardless of sequence. Gibson assembly can be performed in a manner to leave no scar between joined nucleic acid sequence. Gibson assembly can be used to combine a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of fragments. Gibson assembly can be performed, for example, as described in Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO.

Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6(5):343-345. doi:10.1038/nmeth.l318 which is incorporated by reference herein in its entirety.

[00232] Gibson assembly can simultaneously combine a plurality of DNA fragments, e.g., based on sequence identity. The DNA fragments contain an about 20-40 base pair overlap with adjacent DNA fragments. These DNA fragments can be mixed with one or more enzymes (e.g., a cocktail of 3 enzymes), along with other buffer components. In some embodiments, the enzymes can include an exonuclease, a DNA polymerase, and a DNA ligase.

[00233] During Gibson assembly, modification of a nucleic acid sequence of interest or enriched nucleic acid sequence of interest can comprise combining a second nucleic acid of interest with the nucleic acid of interest or enriched nucleic acid of interest. In some embodiments, the second nucleic acid sequence of interest can be enriched. Such combining can comprise, for example, using one or more overlap extension primers to link the nucleic acid sequence of interest or enriched nucleic acid sequence of interest to the second nucleic acid sequence of interest. In some cases, such case can comprise using a nucleic acid linker to join the second nucleic acid sequence of interest to the nucleic acid sequence of interest or the enriched nucleic acid sequence of interest.

[00234] During Gibson assembly, a second nucleic acid sequence of interest can be a nucleic acid sequence described herein. For example, a second nucleic acid sequence of interest can code for at least a portion of a cell surface protein of a cell, such as a T cell receptor (or fragment thereof) or a B cell receptor (or fragment thereof). In some cases, an enriched nucleic acid sequence of interest can comprise one chain of a T cell receptor (or fragment thereof) or first chain of a B cell receptor (or fragment thereof), while a second nucleic acid sequence of interest can comprise a second chain of a T cell receptor (or fragment thereof) or a B cell receptor (or fragment thereof). A second nucleic acid sequence of interest can comprise a constant region. In some embodiments, the second nucleic acid sequence of interest can comprise a sequence coding for a V(D)J sequence or a portion thereof, such as a V sequence (or portion thereof), a D sequence (or portion thereof), or a J sequence (or portion thereof), as described herein. In some embodiments, the constant region of a second nucleic acid sequence of interest can comprise a sequence coding for a V(D)J sequence or a portion thereof, such as a V sequence (or portion thereof), a D sequence (or portion thereof), or a J sequence (or portion thereof), as described herein. In some embodiments, a second nucleic acid sequence of interest can comprise a barcode (e.g., as provided herein), a UMI (e.g., as provided herein), or a 5’ untranslated region (5’ UTR). In some embodiments, the second nucleic acid sequence of interest can comprise complementary deoxyribonucleic acid (cDNA).

[00235] During Gibson assembly, the exonuclease can chew back DNA from the 5' end, and in some cases does not inhibit polymerase activity, thus allowing the reaction to occur in one single process. The resulting single-stranded regions on adjacent DNA fragments can anneal. The DNA polymerase can incorporate nucleotides to fill in any gaps. The DNA ligase can covalently join the DNA of adjacent segments, thereby removing any nicks in the DNA. Either linear or closed circular molecules can be assembled. In some embodiments, PCR can be utilized to perform the Gibson assembly.

[00236] Existing antibody cloning methods can be time-consuming or difficult, and can require considerable automation and expensive plate-based reagents in order to succeed. Human labor can be used instead, but this can become impractical when cloning thousands of antibodies or TCRs, which can become a common procedure, for example during pandemic antibody discovery efforts and during antibody discovery campaigns for pharmaceutical research. Methods for enriching nucleic acid sequences (including those coding for antibodies, TCRs, or fragments thereof) and methods for cloning those sequences provided herein can leverage cDNA and amplified sequences to efficiently recover targets of interest from one or more single cell libraries. By way of example, employing primer and probes as provided herein, it is possible to enrich specifically for particular BCR/antibody or TCR nucleic acid sequences of interest from complex, pooled, cDNA libraries. Primers and probes that target sequences encoding a BCR/antibody or TCR target of interest at, for instance, a junction leading to or including J region sequences, are demonstrated to selectively enrich for specific BCRs/antibodies or TCRs, including from a library of pooled donor samples comprising B cells expressing numerous BCRs/antibodies of different sequences (or T cells expressing TCRs), e.g., from a sequencing library prepared from pooled donor samples comprising thousands (e.g., 5,000-10,000) of BCR sequences (or TCR sequences). Accordingly, using the approaches described herein, including, e.g., the UMI validation approaches described herein, users can sequence thousands to hundreds of thousands of antibodies and target select antibodies for recovery and cloning with high accuracy. This can be particularly powerful when combined with other components provided herein (e.g., barcoding) to screen, e g. for antigen specificity or other multiomic data.

[00237] Methods provided herein can further comprise cloning a nucleic acid sequence of interest into a vector. A vector can be a nucleic acid (e.g., DNA) molecule used as a vehicle to artificially carry foreign genetic material into a cell, where it can be replicated and/or expressed. Examples of vectors can include a viral vector, a plasmid, a bacteriophage, a cosmid, or an artificial chromosome. [00238] In some embodiments, a vector can be modified by the addition of genetic material coding for a protein. For example, a vector can comprise a nucleic acid sequence that can be combined with the nucleic acid sequence of interest. For example, a vector can comprise a nucleic acid sequence that can be combined with the nucleic acid sequence of interest to yield a nucleic acid sequence for a protein of interest, such as an antibody or antigen binding fragment thereof, T cell receptor, or B cell receptor. For example, a vector can comprise at least a portion of a constant region of a T cell receptor, a B cell receptor, or an antibody or antigen binding fragment thereof [00239] In some embodiments, a vector can comprise a promoter. A promoter can be a sequence of DNA to which one or more proteins can bind that can initiate transcription of a single RNA from the DNA downstream of it. This RNA may encode a protein, or can have a function in and of itself, such as tRNA, mRNA, or rRNA. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand). Promoters can be about 100-1000 base pairs long. Examples of promoters can include bacterial promoters or eukaryotic promoters. [00240] In some embodiments, cloning can comprise a vector restriction digest (e.g., cutting of the nucleic acid sequence of the vector at a restriction site, or site recognized by a restriction enzyme). A restriction digest of a vector can comprise digesting the vector at a restriction site. A restriction site can be a DNA sequence on the vector that can contain a specific sequence of nucleotides (e.g., 4-8 bases long) that can be recognized by a restriction enzyme. In some embodiments, a restriction site can be a palindromic sequence. In some embodiments, a restriction enzyme (e.g., a restriction enzyme that can recognize the restriction site) can cut the sequence between two nucleic acids within the restriction site or nearby the restriction site. An example of a restriction site can be, for example, a fspl restriction site that can be recognized by the fspl restriction enzyme. Non-limiting examples of restriction sites that can be employed are provided in Table 1.

Table 1: Restriction enzymes and their recognition sequences

[00241] A cloning vector can have features that can allow a gene to be conveniently inserted into the vector or removed from it. Examples can include a multiple cloning site (MCS) or polylinker, which can contain unique restriction site(s). The restriction site(s) in the MCS can be first cleaved by restriction enzymes, then a PCR-amplified target gene, e.g. nucleic acid sequence of interest, also digested with the same enzymes is ligated into the vectors using DNA ligase. It can be inserted into the vector in a specific direction if so desired. The restriction sites may be further used for sub cloning into another vector if necessary. [00242] Other cloning vectors may employ topoisomerase instead of ligase, and cloning can be performed more rapidly without the need for restriction digest of the vector or insert. In this TOPO cloning method, a linearized vector can be activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process. Another method of cloning without the use of DNA digest and ligase can be by DNA recombination, for example as used in the Gateway cloning system. The gene, once cloned into the cloning vector, may be conveniently introduced into a variety of expression vectors by recombination.

[00243] A vector can comprise a reporter gene. A reporter gene can be used in some cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified. Such features can include the lacZa fragment for a complementation in blue-white selection, and/or marker gene or reporter genes in frame with and flanking the MCS to facilitate the production of fusion proteins. Examples of fusion partners that may be used for screening can include the green fluorescent protein (GFP) and luciferase.

[00244] In some embodiments, cloning can comprise combining two or more nucleic acid sequences. For example, two or more nucleic acid sequences can be joined to yield a coding sequence for an amino acid sequence of interest (e.g., a T cell receptor, a B cell receptor, or an antibody or antigen binding fragment thereof). Two or more nucleic acid sequences can comprise a nucleic acid sequence of a heavy chain of an antibody or antigen binding fragment and a nucleic acid sequence of a light chain. Two or more nucleic acid sequences can comprise a nucleic acid sequence of an alpha chain of a T cell receptor and a nucleic acid sequence of a beta chain of a T cell receptor. In such a method, a full antibody or antigen binding fragment thereof, B cell receptor, T cell receptor or other amino acid can be cloned in a single vector and expressed as a single nucleic acid sequence or amino acid sequence.

[00245] After cloning, the nucleic acid sequence of interest or the amino acid product of the nucleic acid sequence of interest can be expressed. Expression can be performed in any acceptable expression system, including a bacterial expression system, a yeast expression system, an insect cell expression system, a viral expression system, or a mammalian cell expression system. In some embodiments, expression can be in a live animal. [00246] The protein product of the nucleic acid sequence of interest can be analyzed. For example, the affinity, specificity, enzymatic activity, solubility, stability, or other property of the protein product can be analyzed. Examples of assays can include ELISA, western blot, enzymatic assay, dot blot, Bradford protein assay, neutralization assay, immunoassay, or another assay.

[00247] In an aspect, the systems and methods described herein provide for the compartmentalization, depositing, or partitioning of one or more particles (e.g., biological particles, macromolecular constituents of biological particles, beads, reagents, etc.) into discrete compartments or partitions (referred to interchangeably herein as partitions), where each partition maintains separation of its own contents from the contents of other partitions. The partition can be a droplet in an emulsion. The partition can be a well. A partition may comprise one or more other partitions. [00248] A partition may include one or more particles. A partition may include one or more types of particles. For example, a partition of the present disclosure may comprise one or more biological particles and/or macromolecular constituents thereof. A partition may comprise one or more gel beads. A partition may comprise one or more cell beads. A partition may include a single gel bead, a single cell bead, or both a single cell bead and single gel bead. A partition may include one or more reagents. Alternatively, a partition may be unoccupied. For example, a partition may not comprise a bead. A cell bead can be a biological particle and/or one or more of its macromolecular constituents encased inside of a gel or polymer matrix, such as via polymerization of a droplet containing the biological particle and precursors capable of being polymerized or gelled. Unique identifiers, such as barcodes, may be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a microcapsule (e.g., bead), as described elsewhere herein. Microfluidic channel networks (e.g., on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms may also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.

[00249] The partitions can be flowable within fluid streams. The partitions may comprise, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core. In some cases, the partitions may comprise a porous matrix that is capable of entraining and/or retaining materials within its matrix. The partitions can be droplets of a first phase within a second phase, wherein the first and second phases are immiscible. For example, the partitions can be droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase). In another example, the partitions can be droplets of a non-aqueous fluid within an aqueous phase. In some examples, the partitions may be provided in a water-in-oil emulsion or oil-in-water emulsion. A variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295, which is entirely incorporated herein by reference for all purposes. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in, for example, U.S. Patent Application Publication No. 2010/0105112, which is entirely incorporated herein by reference for all purposes.

[00250] In the case of droplets in an emulsion, allocating individual particles to discrete partitions may in one non-limiting example be accomplished by introducing a flowing stream of particles in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams. Fluid properties (e.g., fluid flow rates, fluid viscosities, etc.), particle properties (e.g., volume fraction, particle size, particle concentration, etc.), microfluidic architectures (e.g., channel geometry, etc.), and other parameters may be adjusted to control the occupancy of the resulting partitions (e.g., number of biological particles per partition, number of beads per partition, etc ). For example, partition occupancy can be controlled by providing the aqueous stream at a certain concentration and/or flow rate of particles. To generate single biological particle partitions, the relative flow rates of the immiscible fluids can be selected such that, on average, the partitions may contain less than one biological particle per partition in order to ensure that those partitions that are occupied are primarily singly occupied. In some cases, partitions among a plurality of partitions may contain at most one biological particle (e.g., bead, DNA, cell or cellular material). In some embodiments, the various parameters (e.g., fluid properties, particle properties, microfluidic architectures, etc.) may be selected or adjusted such that a majority of partitions are occupied, for example, allowing for only a small percentage of unoccupied partitions. The flows and channel architectures can be controlled as to ensure a given number of singly occupied partitions, less than a certain level of unoccupied partitions and/or less than a certain level of multiply occupied partitions. [00251] FIG. 1 shows an example of a microfluidic channel structure 100 for partitioning individual biological particles. The channel structure 100 can include channel segments 102, 104, 106 and 108 communicating at a channel junction 110. In operation, a first aqueous fluid 112 that includes suspended biological particles (or cells) 114 may be transported along channel segment 102 into junction 110, while a second fluid 116 that is immiscible with the aqueous fluid 112 is delivered to the junction 110 from each of channel segments 104 and 106 to create discrete droplets 118, 120 of the first aqueous fluid 112 flowing into channel segment 108, and flowing away from junction 110. The channel segment 108 may be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested. A discrete droplet generated may include an individual biological particle 114 (such as droplets 118). A discrete droplet generated may include more than one individual biological particle 114 (not shown in FIG. 1). A discrete droplet may contain no biological particle 114 (such as droplet 120). Each discrete partition may maintain separation of its own contents (e.g., individual biological particle 114) from the contents of other partitions.

[00252] The second fluid 116 can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 118, 120. Examples of particularly useful partitioning fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010/0105112, which is entirely incorporated herein by reference for all purposes.

[00253] As will be appreciated, the channel segments described herein may be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structure 100 may have other geometries. For example, a microfluidic channel structure can have more than one channel junction. For example, a microfluidic channel structure can have 2, 3, 4, or 5 channel segments each carrying particles (e.g., biological particles, cell beads, and/or gel beads) that meet at a channel junction. Fluid may be directed to flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can comprise compressors (e.g., providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid may also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.

[00254] The generated droplets may comprise two subsets of droplets: (1) occupied droplets 118, containing one or more biological particles 114, and (2) unoccupied droplets 120, not containing any biological particles 114. Occupied droplets 118 may comprise singly occupied droplets (having one biological particle) and multiply occupied droplets (having more than one biological particle). As described elsewhere herein, in some cases, the majority of occupied partitions can include no more than one biological particle per occupied partition and some of the generated partitions can be unoccupied (of any biological particle). In some cases, though, some of the occupied partitions may include more than one biological particle.

[00255] In some cases, it may be desirable to minimize the creation of excessive numbers of empty partitions, such as to reduce costs and/or increase efficiency. While this minimization may be achieved by providing a sufficient number of biological particles (e.g., biological particles 114) at the partitioning junction 110, such as to ensure that at least one biological particle is encapsulated in a partition, the Poissonian distribution may expectedly increase the number of partitions that include multiple biological particles. As such, where singly occupied partitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions can be unoccupied.

[00256] In some cases, the flow of one or more of the biological particles (e.g., in channel segment 102), or other fluids directed into the partitioning junction (e.g., in channel segments 104, 106) can be controlled such that, in many cases, no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied. These flows can be controlled so as to present a non-Poissonian distribution of single-occupied partitions while providing lower levels of unoccupied partitions. The above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in many cases, the use of the systems and methods described herein can create resulting partitions that have multiple occupancy rates of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases, less than about 5%, while having unoccupied partitions of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less.

[00257] As will be appreciated, the above-described occupancy rates are also applicable to partitions that include both biological particles and additional reagents, including, but not limited to, microcapsules or beads (e.g., gel beads) carrying barcoded nucleic acid molecules.

[00258] FIG. 2 shows an example of a microfluidic channel structure 200 for delivering barcode carrying beads to droplets. The channel structure 200 can include channel segments 201, 202, 204, 206 and 208 communicating at a channel junction 210. In operation, the channel segment 201 may transport an aqueous fluid 212 that includes a plurality of beads 214 (e.g., with nucleic acid molecules, oligonucleotides, molecular tags) along the channel segment 201 into junction 210. The plurality of beads 214 may be sourced from a suspension of beads. For example, the channel segment 201 may be connected to a reservoir comprising an aqueous suspension of beads 214. The channel segment 202 may transport the aqueous fluid 212 that includes a plurality of biological particles 216 along the channel segment 202 into junction 210. The plurality of biological particles 216 may be sourced from a suspension of biological particles. For example, the channel segment 202 may be connected to a reservoir comprising an aqueous suspension of biological particles 216.

In some instances, the aqueous fluid 212 in either the first channel segment 201 or the second channel segment 202, or in both segments, can include one or more reagents, as further described below. A second fluid 218 that is immiscible with the aqueous fluid 212 (e.g., oil) can be delivered to the junction 210 from each of channel segments 204 and 206. Upon meeting of the aqueous fluid 212 from each of channel segments 201 and 202 and the second fluid 218 from each of channel segments 204 and 206 at the channel junction 210, the aqueous fluid 212 can be partitioned as discrete droplets 220 in the second fluid 218 and flow away from the junction 210 along channel segment 208. The channel segment 208 may deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 208, where they may be harvested.

[00259] As an alternative, the channel segments 201 and 202 may meet at another junction upstream of the junction 210. At such junction, beads and biological particles may form a mixture that is directed along another channel to the junction 210 to yield droplets 220. The mixture may provide the beads and biological particles in an alternating fashion, such that, for example, a droplet comprises a single bead and a single biological particle.

[00260] Beads, biological particles and droplets may flow along channels at substantially regular flow profiles (e.g., at regular flow rates). Such regular flow profiles may permit a droplet to include a single bead and a single biological particle. Such regular flow profiles may permit the droplets to have an occupancy (e.g., droplets having beads and biological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flow profiles and devices that may be used to provide such regular flow profiles are provided in, for example, U S. Patent Publication No. 2015/0292988, which is entirely incorporated herein by reference. [00261] The second fluid 218 can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 220.

[00262] A discrete droplet that is generated may include an individual biological particle 216. A discrete droplet that is generated may include a barcode or other reagent carrying bead 214. A discrete droplet generated may include both an individual biological particle and a barcode carrying bead, such as droplets 220. In some instances, a discrete droplet may include more than one individual biological particle or no biological particle. In some instances, a discrete droplet may include more than one bead or no bead. A discrete droplet may be unoccupied (e g., no beads, no biological particles).

[00263] In another aspect, in addition to or as an alternative to droplet-based partitioning, biological particles (e.g., cells) may be comprised within (e g., encapsulated within) a particulate material to form a “cell bead”.

[00264] A cell bead can contain a biological particle (e.g., a cell) or macromolecular constituents (e.g., RNA, DNA, proteins, etc.) of a biological particle. A cell bead may include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads. Systems and methods disclosed herein can be applicable to both cell beads (and/or droplets or other partitions) containing biological particles and cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles. Cell beads may be or include a cell, cell derivative, cellular material and/or material derived from the cell in, within, or encased in a matrix, such as a polymeric matrix. In some cases, a cell bead may comprise a live cell. In some instances, the live cell may be capable of being cultured when enclosed in a gel or polymer matrix, or of being cultured when comprising a gel or polymer matrix. In some instances, the polymer or gel may be diffusively permeable to certain components and diffusively impermeable to other components (e.g., macromolecular constituents). [00265] Cell beads can provide certain potential advantages of being more storable and more portable than droplet-based partitioned biological particles. Furthermore, in some cases, it may be desirable to allow biological particles to incubate for a select period of time before analysis, such as in order to characterize changes in such biological particles over time, either in the presence or absence of different stimuli (or reagents).

[00266] Suitable polymers or gels may include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG- acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin. The polymer or gel may comprise any other polymer or gel.

[00267] Encapsulation of biological particles may be performed by a variety of processes. Such processes may combine an aqueous fluid containing the biological particles with a polymeric precursor material that may be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor. The conditions sufficient to polymerize or gel the precursors may comprise any conditions sufficient to polymerize or gel the precursors. Such stimuli can include, for example, thermal stimuli (e.g., either heating or cooling), photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g., through crosslinking, polymerization initiation of the precursor (e.g., through added initiators)), electromagnetic radiation, mechanical stimuli, or any combination thereof.

[00268] In some cases, air knife droplet or aerosol generators may be used to dispense droplets of precursor fluids into gelling solutions in order to form cell beads that include individual biological particles or small groups of biological particles. Likewise, membrane-based encapsulation systems may be used to generate cell beads comprising encapsulated biological particles as described herein. Microfluidic systems of the present disclosure, such as that shown in FIG. 1, may be readily used in encapsulating biological particles (e.g., cells) as described herein. Exemplary methods for encapsulating biological particles (e.g., cells) are also further described in U.S. Patent Application Pub. No. US 2015/0376609 and PCT/US2018/016019, which are hereby incorporated by reference in their entirety. In particular, and with reference to FIG. 1, the aqueous fluid 112 comprising (i) the biological particles 114 and (ii) the polymer precursor material (not shown) is flowed into channel junction 110, where it is partitioned into droplets 118, 120 through the flow of non-aqueous fluid 116. In the case of encapsulation methods, non-aqueous fluid 116 may also include an initiator (not shown) to cause polymerization and/or crosslinking of the polymer precursor to form the bead that includes the entrained biological particles. Examples of polymer precursor/initiator pairs include those described in U.S. Patent Application Publication No. 2014/0378345, which is entirely incorporated herein by reference for all purposes.

[00269] In some cases, encapsulated biological particles can be selectively releasable from the cell bead, such as through passage of time or upon application of a particular stimulus, that degrades the bead sufficiently to allow the biological particles (e.g., cell), or its other contents to be released from the bead, such as into a partition (e.g., droplet). Exemplary stimuli suitable for degradation of the bead are described in U.S. Patent Application Publication No. 2014/0378345, which is entirely incorporated herein by reference for all purposes.

[00270] The polymer or gel may be diffusively permeable to chemical or biochemical reagents. The polymer or gel may be diffusively impermeable to macromolecular constituents of the biological particle. In this manner, the polymer or gel may act to allow the biological particle to be subjected to chemical or biochemical operations while spatially confining the macromolecular constituents to a region of the droplet defined by the polymer or gel.

[00271] The polymer or gel may be functionalized to bind to targeted analytes, such as nucleic acids, proteins, carbohydrates, lipids or other analytes. The polymer or gel may be polymerized or gelled via a passive mechanism. The polymer or gel may be stable in alkaline conditions or at elevated temperature. The polymer or gel may have mechanical properties similar to the mechanical properties of the bead. For instance, the polymer or gel may be of a similar size to the bead. The polymer or gel may have a mechanical strength (e.g. tensile strength) similar to that of the bead. The polymer or gel may be of a lower density than an oil. The polymer or gel may be of a density that is roughly similar to that of a buffer. The polymer or gel may have a tunable pore size. The pore size may be chosen to, for instance, retain denatured nucleic acids. The pore size may be chosen to maintain diffusive permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors. The polymer or gel may be biocompatible. The polymer or gel may maintain or enhance cell viability. The polymer or gel may be biochemically compatible. The polymer or gel may be polymerized and/or depolymerized thermally, chemically, enzymatically, and/or optically.

[00272] The encapsulation of biological particles may constitute the partitioning of the biological particles into which other reagents are co-partitioned. Alternatively or in addition, encapsulated biological particles may be readily deposited into other partitions (e.g., droplets) as described above. [00273] Nucleic acid barcode molecules may be delivered to a partition (e.g., a droplet or well) via a solid support or carrier (e.g., a bead). In some cases, nucleic acid barcode molecules are initially associated with the solid support and then released from the solid support upon application of a stimulus, which allows the nucleic acid barcode molecules to dissociate or to be released from the solid support. In specific examples, nucleic acid barcode molecules are initially associated with the solid support (e.g., bead) and then released from the solid support upon application of a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, and/or a photo stimulus.

[00274] The solid support may be a bead. A solid support, e.g., a bead, may be porous, non- porous, hollow, solid, semi-solid, and/or a combination thereof. Beads may be solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some instances, a solid support, e.g., a bead, may be at least partially dissolvable, disruptable, and/or degradable. In some cases, a solid support, e.g., a bead, may not be degradable. In some cases, the solid support, e.g., a bead, may be a gel bead. A gel bead may be a hydrogel bead. A gel bead may be formed from molecular precursors, such as a polymeric or monomeric species. A semi-solid support, e.g., a bead, may be a liposomal bead. Solid supports, e.g., beads, may comprise metals including iron oxide, gold, and silver. In some cases, the solid support, e.g., the bead, may be a silica bead. In some cases, the solid support, e.g., a bead, can be rigid. In other cases, the solid support, e.g., a bead, may be flexible and/or compressible.

[00275] A partition may comprise one or more unique identifiers, such as barcodes. Barcodes may be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned biological particle. For example, barcodes may be injected into droplets or deposited in microwells previous to, subsequent to, or concurrently with droplet generation or providing of reagents in the microwells, respectively. The delivery of the barcodes to a particular partition allows for the later attribution of the characteristics of the individual biological particle to the particular partition. Barcodes may be delivered, for example on a nucleic acid molecule (e.g., via a nucleic acid barcode molecule), to a partition via any suitable mechanism. Nucleic acid barcode molecules can be delivered to a partition via a bead. Beads are described in further detail below. [00276] In some cases, nucleic acid barcode molecules can be initially associated with the bead and then released from the bead. Release of the nucleic acid barcode molecules can be passive (e.g., by diffusion out of the bead). In addition or alternatively, release from the bead can be upon application of a stimulus which allows the nucleic acid barcode molecules to dissociate or to be released from the bead. Such stimulus may disrupt the bead, an interaction that couples the nucleic acid barcode molecules to or within the bead, or both. Such stimulus can include, for example, a thermal stimulus, photo-stimulus, chemical stimulus (e.g., change in pH or use of a reducing agent(s)), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof.

[00277] Methods and systems for partitioning barcode carrying beads into droplets are provided herein, and in in US. Patent Publication Nos. 2019/0367997 and 2019/0064173, and International Application No. PCT/US20/17785, each of which is herein entirely incorporated by reference for all purposes.

[00278] A bead may be porous, non-porous, solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some instances, a bead may be dissolvable, disruptable, and/or degradable. Degradable beads, as well as methods for degrading beads, are described in PCT/US2014/044398, which is hereby incorporated by reference in its entirety. In some cases, any combination of stimuli, e.g., stimuli described in PCT7US2014/044398 and US Patent Application Pub. No. 2015/0376609, hereby incorporated by reference in its entirety, may trigger degradation of a bead. For example, a change in pH may enable a chemical agent (e.g., DTT) to become an effective reducing agent. [00279] In some cases, a bead may not be degradable. In some cases, the bead may be a gel bead. A gel bead may be a hydrogel bead. A gel bead may be formed from molecular precursors, such as a polymeric or monomeric species. A semi-solid bead may be a liposomal bead. Solid beads may comprise metals including iron oxide, gold, and silver. In some cases, the bead may be a silica bead. In some cases, the bead can be rigid. In other cases, the bead may be flexible and/or compressible. [00280] A bead may be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.

[00281] Beads may be of uniform size or heterogeneous size. Beads may be of uniform size or heterogeneous size. [00282] Beads may also be formed from materials other than polymers, including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and others. In some cases, the bead may comprise covalent or ionic bonds between polymeric precursors (e.g., monomers, oligomers, linear polymers), nucleic acid barcode molecules (e.g., oligonucleotides), primers, and other entities. In some cases, the covalent bonds can be carbon-carbon bonds, thioether bonds, or carbon-heteroatom bonds.

[00283] In some cases, a plurality of nucleic acid barcode molecules may be attached to a bead. The nucleic acid barcode molecules may be attached directly or indirectly to the bead. In some cases, the nucleic acid barcode molecules may be covalently linked to the bead. In some cases, the nucleic acid barcode molecules are covalently linked to the bead via a linker. In some cases, the linker is a degradable linker. In some cases, the linker comprises a labile bond configured to release said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules. In some cases, the labile bond comprises a disulfide linkage. In some cases, a bead may comprise an acrydite moiety, which in certain aspects may be used to attach one or more nucleic acid barcode molecules (e.g., barcode sequence, nucleic acid barcode molecule, barcoded oligonucleotide, primer, or other oligonucleotide) to the bead. Acrydite moieties, as well as their uses in attaching nucleic acid molecules to beads, are described in PCT/US2014/044398, which is hereby incorporated by reference in its entirety.

[00284] For example, precursors (e.g., monomers, cross-linkers) that are polymerized to form a bead may comprise acrydite moieties, such that when a bead is generated, the bead also comprises acrydite moieties. The acrydite moieties can be attached to a nucleic acid molecule, e.g., a nucleic acid barcode molecule described herein.

[00285] In some cases, precursors comprising a functional group that is reactive or capable of being activated such that it becomes reactive can be polymerized with other precursors to generate gel beads comprising the activated or activatable functional group. The functional group may then be used to attach additional species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads. Exemplary precursors comprising functional groups are described in PCT/US2014/044398, which is hereby incorporated by reference in its entirety.

[00286] Species may be encapsulated in beads during bead generation (e.g., during polymerization of precursors). Such species may or may not participate in polymerization. See, e.g., PCT/US2014/044398, which is hereby incorporated by reference in its entirety. Such species may include, for example, nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleic acid amplification reaction (e.g., primers, polymerases, dNTPs, co-factors (e.g., ionic co-factors), buffers) including those described herein, reagents for enzymatic reactions (e.g., enzymes, co-factors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion, and/or reagents for template preparation (e.g., tagmentation) for one or more sequencing platforms (e.g., Nextera® for Illumina®). Such species may include one or more enzymes described herein, including without limitation, polymerase, reverse transcriptase, restriction enzymes (e.g., endonuclease), transposase, ligase, proteinase K, DNAse, etc. Such species may include one or more reagents described elsewhere herein (e.g., lysis agents, inhibitors, inactivating agents, chelating agents, stimulus).

[00287] Other non- limiting examples of labile bonds that may be coupled to a precursor or bead are described in PCT/US2014/044398, which is hereby incorporated by reference in its entirety. A nucleic acid barcode molecule may contain one or more barcode sequences. A plurality of nucleic acid barcode molecules may be coupled to a bead. The one or more barcode sequences may include sequences that are the same for all nucleic acid molecules coupled to a given bead and/or sequences that are different across all nucleic acid molecules coupled to the given bead. The nucleic acid molecule may be incorporated into the bead.

[00288] Nucleic acid barcode molecules can comprise one or more functional sequences for coupling to an analyte or analyte tag such as a reporter oligonucleotide. Such functional sequences can include, e.g., a template switch oligonucleotide (TSO) sequence, a primer sequence (e.g., a poly T sequence, or a nucleic acid primer sequence complementary to a target nucleic acid sequence and/or for amplifying a target nucleic acid sequence, a random primer, and a primer sequence for messenger RNA).

[00289] In some cases, the nucleic acid barcode molecule can further comprise a unique molecular identifier (UMI). In some cases, the nucleic acid barcode molecule can comprise one or more functional sequences, for example, for attachment to a sequencing flow cell, such as, for example, a P5 sequence (or a portion thereof) for Illumina® sequencing. In some cases, the nucleic acid barcode molecule or derivative thereof (e.g., oligonucleotide or polynucleotide generated from the nucleic acid molecule) can comprise another functional sequence, such as, for example, a P7 sequence (or a portion thereof) for attachment to a sequencing flow cell for Illumina sequencing. In some cases, the nucleic acid molecule can comprise an R1 primer sequence for Illumina sequencing. In some cases, the nucleic acid molecule can comprise an R2 primer sequence for Illumina sequencing. In some cases, a functional sequence can comprise a partial sequence, such as a partial barcode sequence, partial anchoring sequence, partial sequencing primer sequence (e.g., partial R1 sequence, partial R2 sequence, etc.), a partial sequence configured to attach to the flow cell of a sequencer (e.g., partial P5 sequence, partial P7 sequence, etc.), or a partial sequence of any other type of sequence described elsewhere herein. A partial sequence may contain a contiguous or continuous portion or segment, but not all, of a full sequence, for example. In some cases, a downstream procedure may extend the partial sequence, or derivative thereof, to achieve a full sequence of the partial sequence, or derivative thereof.

[00290] Examples of such nucleic acid molecules (e.g., oligonucleotides, polynucleotides, etc.) and uses thereof, as may be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Patent Pub. Nos. 2014/0378345 and 2015/0376609, each of which is entirely incorporated herein by reference.

[00291] FIG. 8 illustrates an example of a barcode carrying bead. A nucleic acid molecule 802, e.g., a nucleic acid barcode molecule such as an oligonucleotide, can be coupled to a bead 804 by a releasable linkage 806, such as, for example, a disulfide linker. The same bead 804 may be coupled (e.g., via releasable linkage) to one or more other nucleic acid molecules 818, 820. The nucleic acid molecule 802 may be or comprise a barcode. As noted elsewhere herein, the structure of the barcode may comprise a number of sequence elements. The nucleic acid molecule 802 may comprise a functional sequence 808 that may be used in subsequent processing. For example, the functional sequence 808 may include one or more of a sequencer specific flow cell attachment sequence (e.g., a P5 sequence for Illumina® sequencing systems) and a sequencing primer sequence (e.g., a R1 primer for Illumina® sequencing systems). The nucleic acid molecule 802 may comprise a barcode sequence 810 for use in barcoding the sample (e.g., DNA, RNA, protein, etc.). In some cases, the barcode sequence 810 can be bead-specific such that the barcode sequence 810 is common to all nucleic acid molecules (e.g., including nucleic acid molecule 802) coupled to the same bead 804. Alternatively or in addition, the barcode sequence 810 can be partition-specific such that the barcode sequence 810 is common to all nucleic acid molecules coupled to one or more beads that are partitioned into the same partition. The nucleic acid molecule 802 may comprise a specific priming sequence 812, such as an mRNA specific priming sequence (e.g., poly-T sequence), a targeted priming sequence, and/or a random priming sequence. The nucleic acid molecule 802 may comprise an anchoring sequence 814 to ensure that the specific priming sequence 812 hybridizes at the sequence end (e.g., of the mRNA). For example, the anchoring sequence 814 can include a random short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longer sequence, which can ensure that a poly-T segment is more likely to hybridize at the sequence end of the poly-A tail of the mRNA.

[00292] The nucleic acid molecule 802 may comprise a unique molecular identifying sequence 816 (e.g., unique molecular identifier (UMI)). In some cases, the unique molecular identifying sequence 816 may comprise from about 5 to about 8 nucleotides. Alternatively, the unique molecular identifying sequence 816 may compress less than about 5 or more than about 8 nucleotides. The unique molecular identifying sequence 816 may be a unique sequence that varies across individual nucleic acid molecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g., bead 804). In some cases, the unique molecular identifying sequence 816 may be a random sequence (e.g., such as a random N-mer sequence). For example, the UMI may provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA. As will be appreciated, although FIG. 8 shows three nucleic acid molecules 802, 818, 820 coupled to the surface of the bead 804, an individual bead may be coupled to any number of individual nucleic acid molecules, for example, from one to tens to hundreds of thousands or even millions of individual nucleic acid molecules. The respective barcodes for the individual nucleic acid molecules can comprise both common sequence segments or relatively common sequence segments (e.g., 808, 810, 812, etc.) and variable or unique sequence segments (e.g., 816) between different individual nucleic acid molecules coupled to the same bead.

[00293] In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can be co-partitioned along with a barcode bearing bead 804. The barcoded nucleic acid molecules 802, 818, 820 can be released from the bead 804 in the partition. By way of example, in the context of analyzing sample RNA, the poly-T segment (e.g., 812) of one of the released nucleic acid molecules (e.g., 802) can hybridize to the poly-A tail of a mRNA molecule. Reverse transcription may result in a cDNA transcript of the mRNA, but which transcript includes each of the sequence segments 808, 810, 816 of the nucleic acid molecule 802. Because the nucleic acid molecule 802 comprises an anchoring sequence 814, it will more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA. Within any given partition, all of the cDNA transcripts of the individual mRNA molecules may include a common barcode sequence segment 810. However, the transcripts made from the different mRNA molecules within a given partition may vary at the unique molecular identifying sequence 812 segment (e.g., UMI segment). Beneficially, even following any subsequent amplification of the contents of a given partition, the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the biological particle (e.g., cell). As noted above, the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-T primer sequence is described, other targeted or random priming sequences may also be used in priming the reverse transcription reaction. Likewise, although described as releasing the barcoded oligonucleotides into the partition, in some cases, the nucleic acid molecules bound to the bead (e.g., gel bead) may be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents.

[00294] FIG.25 illustrates another example of a barcode carrying bead. Referring to FIG. 25, a nucleic acid barcode molecule 405, such as an oligonucleotide, can be coupled to a bead 404 by a releasable linkage 406, such as, for example, a disulfide linker. The nucleic acid barcode molecule 405 may comprise a first capture sequence 460. The same bead 404 may be coupled (e.g., via releasable linkage) to one or more other nucleic acid molecules 403, 407 comprising other capture sequences. The nucleic acid barcode molecule 405 may be or comprise a barcode. As noted elsewhere herein, the structure of the barcode may comprise a number of sequence elements, such as a functional sequence 408 (e.g., flow cell attachment sequence, sequencing primer sequence, etc.), a barcode sequence 410 (e.g., bead-specific sequence common to bead, partition-specific sequence common to partition, etc.), and a unique molecular identifier 412 (e.g., unique sequence within different molecules attached to the bead), or partial sequences thereof. The capture sequence 460 may be configured to attach to a corresponding capture sequence 465. In some instances, the corresponding capture sequence 465 may be coupled to another molecule that may be an analyte or an intermediary carrier. For example, as illustrated in FIG. 25, the corresponding capture sequence 465 is coupled to a guide RNA molecule 462 comprising a target sequence 464, wherein the target sequence 464 is configured to attach to the analyte. Another oligonucleotide molecule 407 attached to the bead 404 comprises a second capture sequence 480 which is configured to attach to a second corresponding capture sequence 485. As illustrated in FIG. 25, the second corresponding capture sequence 485 is coupled to an antibody 482. In some cases, the antibody 482 may have binding specificity to an analyte (e.g., surface protein). Alternatively, the antibody 482 may not have binding specificity. Another oligonucleotide molecule 403 attached to the bead 404 comprises a third capture sequence 470 which is configured to attach to a third corresponding capture sequence 475. As illustrated in FIG. 25, the third corresponding capture sequence 475 is coupled to a molecule 472. The molecule 472 may or may not be configured to target an analyte. The other oligonucleotide molecules 403, 407 may comprise the other sequences (e.g., functional sequence, barcode sequence, UMI, etc.) described with respect to oligonucleotide molecule 405. While a single oligonucleotide molecule comprising each capture sequence is illustrated in FIG. 25, it will be appreciated that, for each capture sequence, the bead may comprise a set of one or more oligonucleotide molecules each comprising the capture sequence. For example, the bead may comprise any number of sets of one or more different capture sequences. Alternatively or in addition, the bead 404 may comprise other capture sequences. Alternatively or in addition, the bead 404 may comprise fewer types of capture sequences (e.g., two capture sequences). Alternatively or in addition, the bead 404 may comprise oligonucleotide molecule(s) comprising a priming sequence, such as a specific priming sequence such as an mRNA specific priming sequence (e.g., poly-T sequence), a targeted priming sequence, and/or a random priming sequence, for example, to facilitate an assay for gene expression.. In such cases, further processing may be performed, in the partitions or outside the partitions (e.g., in bulk). For instance, the RNA molecules on the beads may be subjected to reverse transcription or other nucleic acid processing, additional adapter sequences may be added to the barcoded nucleic acid molecules, or other nucleic acid reactions (e.g., amplification, nucleic acid extension) may be performed. The beads or products thereof (e.g., barcoded nucleic acid molecules) may be collected from the partitions, and/or pooled together and subsequently subjected to clean up and further characterization (e.g., sequencing).

[00295] The operations described herein may be performed at any useful or convenient step. For instance, the beads comprising nucleic acid barcode molecules may be introduced into a partition (e.g., well or droplet) prior to, during, or following introduction of a sample into the partition. The nucleic acid molecules of a sample may be subjected to barcoding, which may occur on the bead (in cases where the nucleic acid molecules remain coupled to the bead) or following release of the nucleic acid barcode molecules into the partition. In cases where analytes from the sample are captured by the nucleic acid barcode molecules in a partition (e.g., by hybridization), captured analytes from various partitions may be collected, pooled, and subjected to further processing (e.g., reverse transcription, adapter attachment, amplification, clean up, sequencing). For example, in cases wherein the nucleic acid molecules from the sample remain attached to the bead, the beads from various partitions may be collected, pooled, and subjected to further processing (e.g., reverse transcription, adapter attachment, amplification, clean up, sequencing). In other instances, one or more of the processing methods, e.g., reverse transcription, may occur in the partition. For example, conditions sufficient for barcoding, adapter attachment, reverse transcription, or other nucleic acid processing operations may be provided in the partition and performed prior to clean up and sequencing.

[00296] In some instances, a bead may comprise a capture sequence or binding sequence configured to bind to a corresponding capture sequence or binding sequence. In some instances, a bead may comprise a plurality of different capture sequences or binding sequences configured to bind to different respective corresponding capture sequences or binding sequences. For example, a bead may comprise a first subset of one or more capture sequences each configured to bind to a first corresponding capture sequence, a second subset of one or more capture sequences each configured to bind to a second corresponding capture sequence, a third subset of one or more capture sequences each configured to bind to a third corresponding capture sequence, and etc. A bead may comprise any number of different capture sequences. In some instances, a bead may comprise at least 2, 3, 4,

5, 6, 7, 8, 9, 10 or more different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences, respectively. Alternatively or in addition, a bead may comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, or 2 different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences. In some instances, the different capture sequences or binding sequences may be configured to facilitate analysis of a same type of analyte. In some instances, the different capture sequences or binding sequences may be configured to facilitate analysis of different types of analytes (with the same bead). The capture sequence may be designed to attach to a corresponding capture sequence. Beneficially, such corresponding capture sequence may be introduced to, or otherwise induced in, an biological particle (e.g., cell, cell bead, etc.) for performing different assays in various formats (e.g., barcoded antibodies comprising the corresponding capture sequence, barcoded MHC dextramers comprising the corresponding capture sequence, barcoded guide RNA molecules comprising the corresponding capture sequence, etc.), such that the corresponding capture sequence may later interact with the capture sequence associated with the bead. In some instances, a capture sequence coupled to a bead (or other support) may be configured to attach to a linker molecule, such as a splint molecule, wherein the linker molecule is configured to couple the bead (or other support) to other molecules through the linker molecule, such as to one or more analytes or one or more other linker molecules.

[00297] In some cases, a species (e.g., oligonucleotide molecules comprising barcodes) that are attached to a solid support (e.g., a bead) may comprise a U-excising element that allows the species to release from the bead. In some cases, the U-excising element may comprise a single-stranded DNA (ssDNA) sequence that contains at least one uracil. The species may be attached to a solid support via the ssDNA sequence containing the at least one uracil. The species may be released by a combination of uracil-DNA glycosylase (e.g., to remove the uracil) and an endonuclease (e.g., to induce an ssDNA break). If the endonuclease generates a 5’ phosphate group from the cleavage, then additional enzyme treatment may be included in downstream processing to eliminate the phosphate group, e.g., prior to ligation of additional sequencing handle elements, e.g., Illumina full P5 sequence, partial P5 sequence, full R1 sequence, and/or partial R1 sequence.

[00298] The barcodes that are releasable as described herein may sometimes be referred to as being activatable, in that they are available for reaction once released. Thus, for example, an activatable barcode may be activated by releasing the barcode from a bead (or other suitable type of partition described herein). Other activatable configurations are also envisioned in the context of the described methods and systems.

[00299] The nucleic acid barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecules (e.g., oligonucleotides). The nucleic acid barcode sequences can include from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the length of a barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides may be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they may be separated into two or more separate subsequences that are separated by 1 or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length. In some cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[00300] The co-partitioned nucleic acid molecules can also comprise other functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles. These sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying nucleic acids (e.g., mRNA, the genomic DNA) from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences. Other mechanisms of co-partitioning oligonucleotides may also be employed, including, e.g., coalescence of two or more droplets, where one droplet contains oligonucleotides, or microdispensing of oligonucleotides (e.g., attached to a bead) into partitions, e.g., droplets within microfluidic systems.

[00301] In an example, beads are provided that each include large numbers of the above described nucleic acid barcode molecules releasably attached to the beads, where all of the nucleic acid barcode molecules attached to a particular bead will include a common nucleic acid barcode sequence, but where a large number of diverse barcode sequences are represented across the population of beads used. In some embodiments, hydrogel beads, e.g., comprising polyacrylamide polymer matrices, are used as a solid support and delivery vehicle for the nucleic acid barcode molecules into the partitions, as they are capable of carrying large numbers of nucleic acid barcode molecules, and may be configured to release those nucleic acid molecules upon exposure to a particular stimulus, as described elsewhere herein. In some cases, the population of beads provides a diverse barcode sequence library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences, or more. In some cases, the population of beads provides a diverse barcode sequence library that includes about 1,000 to about 10,000 different barcode sequences, about 5,000 to about 50,000 different barcode sequences, about 10,000 to about 100,000 different barcode sequences, about 50,000 to about 1,000,000 different barcode sequences, or about 100,000 to about 10,000,000 different barcode sequences.

[00302] Additionally, each bead can be provided with large numbers of nucleic acid (e.g., oligonucleotide) molecules attached. In particular, the number of molecules of nucleic acid molecules including the barcode sequence on an individual bead can be at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules, or more. In some embodiments, the number of nucleic acid molecules including the barcode sequence on an individual bead is between about 1,000 to about 10,000 nucleic acid molecules, about 5,000 to about 50,000 nucleic acid molecules, about 10,000 to about 100,000 nucleic acid molecules, about 50,000 to about 1,000,000 nucleic acid molecules, about 100,000 to about 10,000,000 nucleic acid molecules, about 1,000,000 to about 1 billion nucleic acid molecules. [00303] Nucleic acid molecules of a given bead can include identical (or common) barcode sequences, different barcode sequences, or a combination of both. Nucleic acid molecules of a given bead can include multiple sets of nucleic acid molecules. Nucleic acid molecules of a given set can include identical barcode sequences. The identical barcode sequences can be different from barcode sequences of nucleic acid molecules of another set. In some embodiments, such different barcode sequences can be associated with a given bead. [00304] Moreover, when the population of beads is partitioned, the resulting population of partitions can also include a diverse barcode library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. Additionally, each partition of the population can include at least about 1,000 nucleic acid barcode molecules, at least about 5,000 nucleic acid barcode molecules, at least about 10,000 nucleic acid barcode molecules, at least about 50,000 nucleic acid barcode molecules, at least about 100,000 nucleic acid barcode molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid barcode molecules, at least about 5,000,000 nucleic acid barcode molecules, at least about 10,000,000 nucleic acid barcode molecules, at least about 50,000,000 nucleic acid barcode molecules, at least about 100,000,000 nucleic acid barcode molecules, at least about 250,000,000 nucleic acid barcode molecules and in some cases at least about 1 billion nucleic acid barcode molecules.

[00305] In some cases, the resulting population of partitions provides a diverse barcode sequence library that includes about 1,000 to about 10,000 different barcode sequences, about 5,000 to about 50,000 different barcode sequences, about 10,000 to about 100,000 different barcode sequences, about 50,000 to about 1,000,000 different barcode sequences, or about 100,000 to about 10,000,000 different barcode sequences. Additionally, each partition of the population can include between about 1,000 to about 10,000 nucleic acid barcode molecules, about 5,000 to about 50,000 nucleic acid barcode molecules, about 10,000 to about 100,000 nucleic acid barcode molecules, about 50,000 to about 1,000,000 nucleic acid barcode molecules, about 100,000 to about 10,000,000 nucleic acid barcode molecules, about 1,000,000 to about 1 billion nucleic acid barcode molecules. [00306] In some cases, it may be desirable to incorporate multiple different barcodes within a given partition, either attached to a single or multiple beads within the partition. For example, in some cases, a mixed, but known set of barcode sequences may provide greater assurance of identification in the subsequent processing, e.g., by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition. [00307] The nucleic acid molecules (e.g., oligonucleotides) may be releasable from the beads upon the application of a particular stimulus to the beads. In some cases, the stimulus may be a photo-stimulus, e.g., through cleavage of a photo-labile linkage that releases the nucleic acid molecules. In other cases, a thermal stimulus may be used, where elevation of the temperature of the beads environment will result in cleavage of a linkage or other release of the nucleic acid molecules from the beads. In still other cases, a chemical stimulus can be used that cleaves a linkage of the nucleic acid molecules to the beads, or otherwise results in release of the nucleic acid molecules from the beads. In one case, such compositions include the polyacrylamide matrices described above for encapsulation of biological particles, and may be degraded for release of the attached nucleic acid molecules through exposure to a reducing agent, such as DTT.

[00308] In accordance with certain aspects, biological particles may be partitioned along with lysis reagents in order to release the contents of the biological particles within the partition. In such cases, the lysis agents can be contacted with the biological particle suspension concurrently with, or immediately prior to, the introduction of the biological particles into the partitioning junction/droplet generation zone (e.g., junction 210), such as through an additional channel or channels upstream of the channel junction. In accordance with other aspects, additionally or alternatively, biological particles may be partitioned along with other reagents, as will be described further below.

[00309] Examples of lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc. (St Louis, MO), as well as other commercially available lysis enzymes. Other lysis agents may additionally or alternatively be copartitioned with the biological particles to cause the release of the biological particle’s contents into the partitions. For example, in some cases, surfactant-based lysis solutions may be used to lyse cells, although these may be less desirable for emulsion based systems where the surfactants can interfere with stable emulsions. In some cases, lysis solutions may include non-ionic surfactants such as, for example, TritonX-100 and Tween 20. In some cases, lysis solutions may include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanical cellular disruption may also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of biological particles that may be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a given size, following cellular disruption.

[00310] Alternatively or in addition to the lysis agents co-partitioned with the biological particles described above, other reagents can also be co-partitioned with the biological particles, including, for example, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids. In addition, in the case of encapsulated biological particles, the biological particles may be exposed to an appropriate stimulus to release the biological particles or their contents from a co-partitioned microcapsule. For example, in some cases, a chemical stimulus may be co-partitioned along with an encapsulated biological particle to allow for the degradation of the microcapsule and release of the cell or its contents into the larger partition. In some cases, this stimulus may be the same as the stimulus described elsewhere herein for release of nucleic acid molecules (e.g., oligonucleotides) from their respective microcapsule (e.g., bead). In alternative aspects, this may be a different and non-overlapping stimulus, in order to allow an encapsulated biological particle to be released into a partition at a different time from the release of nucleic acid molecules into the same partition.

[00311] Additional reagents may also be co-partitioned with the biological particles, such as endonucleases to fragment a biological particle’s DNA, DNA polymerase enzymes and dNTPs used to amplify the biological particle’s nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments. Other enzymes may be co-partitioned, including without limitation, polymerase, transposase, ligase, proteinase K, DNAse, etc. Additional reagents may also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching.

[00312] In some cases, template switching can be used to increase the length of a cDNA. In some cases, template switching can be used to append a predefined nucleic acid sequence to the cDNA. In an example of template switching, cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA in a template independent manner. Switch oligos can include sequences complementary to the additional nucleotides, e.g., polyG. The additional nucleotides (e.g., polyC) on the cDNA can hybridize to the additional nucleotides (e.g., polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Template switching is further described in PCT/US2017/068320, which is hereby incorporated by reference in its entirety. Template switching oligonucleotides may comprise a hybridization region and a template region. Template switching oligonucleotides are further described in PCT/US2017/068320, which is hereby incorporated by reference in its entirety.

[00313] Once the contents of the cells are released into their respective partitions, the macromolecular components (e.g., macromolecular constituents of biological particles, such as RNA, DNA, or proteins) contained therein may be further processed within the partitions. In accordance with the methods and systems described herein, the macromolecular component contents of individual biological particles can be provided with unique identifiers such that, upon characterization of those macromolecular components they may be attributed as having been derived from the same biological particle or particles. The ability to attribute characteristics to individual biological particles or groups of biological particles is provided by the assignment of unique identifiers specifically to an individual biological particle or groups of biological particles. Unique identifiers, e.g., in the form of nucleic acid barcodes can be assigned or associated with individual biological particles or populations of biological particles, in order to tag or label the biological particle’s macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological particle’s components and characteristics to an individual biological particle or group of biological particles. [00314] In some aspects, this is performed by co-partitioning the individual biological particle or groups of biological particles with the unique identifiers, such as described above (with reference to FIGS. 1-7). In some aspects, the unique identifiers are provided in the form of nucleic acid molecules (e.g., oligonucleotides) that comprise nucleic acid barcode sequences that may be attached to or otherwise associated with the nucleic acid contents of individual biological particle, or to other components of the biological particle, and particularly to fragments of those nucleic acids. The nucleic acid molecules are partitioned such that as between nucleic acid molecules in a given partition, the nucleic acid barcode sequences contained therein are the same, but as between different partitions, the nucleic acid molecule can, and do have differing barcode sequences, or at least represent a large number of different barcode sequences across all of the partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, although in some cases, two or more different barcode sequences may be present.

[00315] The nucleic acid barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecules (e.g., oligonucleotides). The nucleic acid barcode sequences can include from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the length of a barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides may be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they may be separated into two or more separate subsequences that are separated by 1 or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length. In some cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[00316] The co-partitioned nucleic acid molecules can also comprise other functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles. These sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences. Other mechanisms of co partitioning oligonucleotides may also be employed, including, e.g., coalescence of two or more droplets, where one droplet contains oligonucleotides, or microdispensing of oligonucleotides into partitions, e.g., droplets within microfluidic systems.

[00317] In some aspects, provided are systems and methods for controlled partitioning. Droplet size may be controlled by adjusting certain geometric features in channel architecture (e.g., microfluidics channel architecture). For example, an expansion angle, width, and/or length of a channel may be adjusted to control droplet size.

[00318] FIG. 4 shows an example of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets. A channel structure 400 can include a channel segment 402 communicating at a channel junction 406 (or intersection) with a reservoir 404. The reservoir 404 can be a chamber. Any reference to “reservoir,” as used herein, can also refer to a “chamber.”

In operation, an aqueous fluid 408 that includes suspended beads 412 may be transported along the channel segment 402 into the junction 406 to meet a second fluid 410 that is immiscible with the aqueous fluid 408 in the reservoir 404 to create droplets 416, 418 of the aqueous fluid 408 flowing into the reservoir 404. At the junction 406 where the aqueous fluid 408 and the second fluid 410 meet, droplets can form based on factors such as the hydrodynamic forces at the junction 406, flow rates of the two fluids 408, 410, fluid properties, and certain geometric parameters (e g., w, ho, a, etc.) of the channel structure 400. A plurality of droplets can be collected in the reservoir 404 by continuously injecting the aqueous fluid 408 from the channel segment 402 through the junction 406

[00319] In some instances, the aqueous fluid 408 can have a substantially uniform concentration or frequency of beads 412. The beads 412 can be introduced into the channel segment 402 from a separate channel (not shown in FIG. 4). The frequency of beads 412 in the channel segment 402 may be controlled by controlling the frequency in which the beads 412 are introduced into the channel segment 402 and/or the relative flow rates of the fluids in the channel segment 402 and the separate channel. In some instances, the beads can be introduced into the channel segment 402 from a plurality of different channels, and the frequency controlled accordingly.

[00320] In some instances, the aqueous fluid 408 in the channel segment 402 can comprise biological particles (e.g., described with reference to FIGS. 1 and 2). In some instances, the aqueous fluid 408 can have a substantially uniform concentration or frequency of biological particles. As with the beads, the biological particles can be introduced into the channel segment 402 from a separate channel. The frequency or concentration of the biological particles in the aqueous fluid 408 in the channel segment 402 may be controlled by controlling the frequency in which the biological particles are introduced into the channel segment 402 and/or the relative flow rates of the fluids in the channel segment 402 and the separate channel. In some instances, the biological particles can be introduced into the channel segment 402 from a plurality of different channels, and the frequency controlled accordingly. In some instances, a first separate channel can introduce beads and a second separate channel can introduce biological particles into the channel segment 402. The first separate channel introducing the beads may be upstream or downstream of the second separate channel introducing the biological particles.

[00321] The second fluid 410 can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets.

[00322] In some instances, the second fluid 410 may not be subjected to and/or directed to any flow in or out of the reservoir 404. For example, the second fluid 410 may be substantially stationary in the reservoir 404. In some instances, the second fluid 410 may be subjected to flow within the reservoir 404, but not in or out of the reservoir 404, such as via application of pressure to the reservoir 404 and/or as affected by the incoming flow of the aqueous fluid 408 at the junction 406. Alternatively, the second fluid 410 may be subjected and/or directed to flow in or out of the reservoir 404. For example, the reservoir 404 can be a channel directing the second fluid 410 from upstream to downstream, transporting the generated droplets.

[00323] Systems and methods for controlled partitioning are described further in PCT/US2018/047551, which is hereby incorporated by reference in its entirety.

[00324] In some embodiments, following the generation of barcoded nucleic acid molecules according to methods disclosed herein, subsequent operations that can be performed can include generation of amplification products, purification ( e.g ., via solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations may occur in bulk (e.g, outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations

[00325] A variety of applications require the evaluation of the presence and quantification of different biological particle or organism types within a population of biological particles, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g., in tracing contamination or the like. [00326] In the methods and systems described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more cell features may be used to characterize cells and/or cell features. In some instances, cell features include cell surface features. Cell surface features may include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features may include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof.

[00327] A labelling agent may include, but is not limited to, a protein (e.g., an antigen), a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi- specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The labelling agents can include (e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g., a first cell surface feature) may have coupled thereto a first reporter oligonucleotide, while a labelling agent that is specific to a different cell feature (e.g., a second cell surface feature) may have a different reporter oligonucleotide coupled thereto. For a description of exemplary labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub. 20190367969, which are each incorporated by reference herein in their entirety.

[00328] In a particular example, a library of potential cell feature labelling agents may be provided associated with nucleic acid reporter molecules, e.g., where a different reporter oligonucleotide sequence is associated with each labelling agent capable of binding to a specific cell feature. In some aspects, different members of the library may be characterized by the presence of a different oligonucleotide sequence label, e.g., an antibody capable of binding to a first type of protein may have associated with it a first known reporter oligonucleotide sequence, while an antibody capable of binding to a second protein (i.e., different than the first protein) may have a different known reporter oligonucleotide sequence associated with it. Prior to partitioning, the cells may be incubated with the library of labelling agents, that may represent labelling agents to a broad panel of different cell features, e.g., receptors (e.g., BCRs, TCRs), proteins, etc., and which include their associated reporter oligonucleotides. Unbound labelling agents may be washed from the cells, and the cells may then be co-partitioned (e.g., into droplets or wells) along with partition-specific barcode oligonucleotides (e.g., attached to a bead, such as a gel bead) as described elsewhere herein. As a result, the partitions may include the cell or cells, as well as the bound labelling agents and their known, associated reporter oligonucleotides.

[00329] In other instances, e.g., to facilitate sample multiplexing, a labelling agent that is specific to a particular cell feature may have a first plurality of the labelling agent (e.g., an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labelling agent coupled to a second reporter oligonucleotide. In this way, different samples or groups can be independently processed and subsequently combined together for pooled analysis (e.g., partition- based barcoding as described elsewhere herein). See, e.g., U.S. Pat. Pub. 20190323088, which is hereby incorporated by reference its entirety.

[00330] In some aspects, these reporter oligonucleotides may comprise nucleic acid barcode sequences that permit identification of the labelling agent which the reporter oligonucleotide is coupled to. The selection of oligonucleotides as the reporter may provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g., using sequencing or array technologies.

[00331] Attachment (coupling) of the reporter oligonucleotides to the labelling agents may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides may be covalently attached to a portion of a labelling agent (such a protein, e.g., an antibody or antibody fragment) using chemical conjugation techniques (e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, which is entirely incorporated herein by reference for all purposes. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552, which is entirely incorporated herein by reference for all purposes. Furthermore, click reaction chemistry such as a Methyltetrazine-PEG5-NHS Ester reaction, a TCO-PEG4-NHS Ester reaction, or the like, may be used to couple reporter oligonucleotides to labelling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art may be used to couple reporter oligonucleotides to labelling agents as appropriate. In another example, a labelling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide comprising a barcode sequence that identifies the label agent. For instance, the labelling agent may be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that comprises a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labelling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the labelling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide may be attached to the labeling agent through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein. In some instances, the reporter oligonucleotides described herein may include one or more functional sequences that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, a sequencing primer or primer biding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[00332] In some cases, the labelling agent can comprise a reporter oligonucleotide and a label. A label can be fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide). In some cases, a label is conjugated to a first oligonucleotide that is complementary (e.g., hybridizes) to a sequence of the reporter oligonucleotide.

[00333] FIG. 11 describes exemplary labelling agents (1110, 1120, 1130) comprising reporter oligonucleotides (1140) attached thereto. Labelling agent 1110 (e.g., any of the labelling agents described herein) is attached (either directly, e.g., covalently attached, or indirectly) to reporter oligonucleotide 1140. Reporter oligonucleotide 1140 may comprise barcode sequence 1142 that identifies labelling agent 1110. Reporter oligonucleotide 1140 may also comprise one or more functional sequences that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, or a sequencing primer or primer biding sequence (such as an Rl, R2, or partial R1 or R2 sequence). [00334] Referring to FIG. 11, in some instances, reporter oligonucleotide 1140 conjugated to a labelling agent (e.g., 1110, 1120, 1130) comprises a primer sequence 1141, a barcode sequence that identifies the labelling agent (e.g., 1110, 1120, 1130), and functional sequence 1143. Functional sequence 1143 may be configured to hybridize to a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule 1190 (not shown), such as those described elsewhere herein. In some instances, nucleic acid barcode molecule 1190 is attached to a support (e.g., a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1190 may be attached to the support via a releasable linkage (e.g., comprising a labile bond), such as those described elsewhere herein. In some instances, reporter oligonucleotide 1140 comprises one or more additional functional sequences, such as those described above.

[00335] In some instances, the labelling agent 1110 is a protein or polypeptide (e.g., an antigen or prospective antigen) comprising reporter oligonucleotide 1140. Reporter oligonucleotide 1140 comprises barcode sequence 1142 that identifies polypeptide 1110 and can be used to infer the presence of, e.g., a binding partner of polypeptide 1110 (i.e., a molecule or compound to which the polypeptide binds). In some instances, the labelling agent 1110 is a lipophilic moiety (e.g., cholesterol) comprising reporter oligonucleotide 1140, where the lipophilic moiety is selected such that labelling agent 1110 integrates into a membrane of a cell or nucleus. Reporter oligonucleotide 1140 comprises barcode sequence 1142 that identifies lipophilic moiety 1110 which in some instances is used to tag cells (e.g., groups of cells, cell samples, etc.) for multiplex analyses as described elsewhere herein. In some instances, the labelling agent is an antibody 1120 (or an epitope binding fragment thereof) comprising reporter oligonucleotide 1140. Reporter oligonucleotide 1140 comprises barcode sequence 1142 that identifies antibody 1120 and can be used to infer the presence of, e.g., a target of antibody 1120 (i.e., a molecule or compound to which antibody 1120 binds). In other embodiments, labelling agent 1130 comprises an MHC molecule 1131 comprising peptide 1132 and reporter oligonucleotide 1140 that identifies peptide 1132. In some instances, the MHC molecule is coupled to a support 1133. In some instances, support 1133 is streptavidin (e.g., MHC molecule 1131 may comprise biotin). In other embodiments, support 1133 is a polysaccharide, such as dextran. In some instances, reporter oligonucleotide 1140 may be directly or indirectly coupled to MHC labelling agent 1130 in any suitable manner, such as to MCH molecule 1131, support 1133, or peptide 1132. In some embodiments, labelling agent 1130 comprises a plurality of MHC molecules, i.e., is an MHC multimer, which may be coupled to a support (e.g., 1133). There are many possible configurations of Class I and/or Class II MHC multimers that can be utilized with the compositions, methods, and systems disclosed herein, e.g., MHC tetramers, MHC pentamers (MHC assembled via a coiled-coil domain, e.g., Pro5® MHC Class I Pentamers, (Prolmmune, Ltd.), MHC octamers,

MHC dodecamers, MHC decorated dextran molecules (e.g., MHC Dextramer® (Immudex)), etc.

For a description of exemplary labelling agents, including antibody and MHC -based labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429, U.S. 10,954,562, U.S. Pat. Pub. 20190367969 and U.S. patent application serial number 63/135,514 filed January 8, 2021, which are each incorporated by reference herein in their entirety.

[00336] In some instances, analysis of one or more analytes (e.g., using the labelling agents described herein) comprises a workflow as generally depicted in FIG. 12A. For example, in some embodiments, cells are contacted with one or more reporter oligonucleotide 1220 conjugated labelling agents 1210 (e.g., polypeptide (e.g., antigen), antibody, or pMHC molecule or complex) and optionally further processed prior to barcoding. Optional processing steps may include one or more washing and/or cell sorting steps. In some instances, a cell bound to labelling agent 1210 (e.g., polypeptide, antibody, or pMHC molecule or complex) conjugated to oligonucleotide 1220 and support 1230 (e.g., a bead, such as a gel bead) comprising nucleic acid barcode molecule 1290 are partitioned into a partition amongst a plurality of partitions (e.g., a droplet of a droplet emulsion or a well of a micro/nanowell array). In some instances, the partition comprises at most a single cell bound to labelling agent 1210. In some embodiments, nucleic acid barcode molecule 1290 is attached to support 1230 via a releasable linkage 1240 (e.g., comprising a labile bond) as described elsewhere herein.

[00337] With continued reference to FIG. 12A, in some instances, reporter oligonucleotide 1220 conjugated to labelling agent 1210 (e.g., polypeptide, an antibody, pMHC molecule such as an MHC multimer, etc.) comprises a first adapter sequence 1211 (e.g., a primer sequence), a barcode sequence 1212 that identifies the labelling agent 1210 (e.g., the polypeptide, antibody, or peptide of a pMHC molecule or complex), and an adapter sequence 1213. Adapter sequence 1213 may be configured to hybridize to a complementary sequence, such as a complementary sequence 1223 present on a nucleic acid barcode molecule 1290, such as those described elsewhere herein. In some instances, nucleic acid barcode molecule 1290 is attached to a support 1230 (e.g., ahead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1290 may be attached to support 1230 via a releasable linkage 1240 (e.g., comprising a labile bond), such as those described elsewhere herein. In some instances, oligonucleotide 1220 comprises one or more additional functional sequences, such as those described above.

[00338] In some instances, analysis of multiple analytes (e.g., RNA and one or more analytes using labelling agents described herein) comprises a workflow as generally depicted in FIGS. 12A- C. Cells are contacted with labeling agents and processed as generally described above and depicted in FIG. 12A. For example, sequence 1213 may then be hybridized to complementary sequence 1223 to generate (e.g., via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and reporter barcode sequence 1212 (or a reverse complement thereof). Referring to FIGS. 12B-C, in some instances, nucleic acid molecules derived from a cell (such as RNA molecules) can be similarly processed to append the cell (e.g., partition-specific) barcode sequence 1222 to these molecules or derivatives thereof (e.g., cDNA molecules). For example, referring to FIG. 12B, in some embodiments, primer 1250 comprises a sequence complementary to a sequence of RNA molecule 1260 (such as an RNA encoding for a BCR sequence) from a cell. In some instances, primer 1250 comprises one or more adapter sequences 1251 that are not complementary to RNA molecule 1260. In some instances, primer 1250 comprises a poly-T sequence. In some instances, primer 1250 comprises a sequence complementary to a target sequence in an RNA molecule. In some instances, primer 1250 comprises a sequence complementary to a region of an immune molecule, such as the constant region of a TCR or BCR sequence. Primer 1250 is hybridized to RNA molecule 1260 and cDNA molecule 1270 is generated in a reverse transcription reaction. In some instances, the reverse transcriptase enzyme is selected such that several non-templated bases 1280 (e.g., a poly-C sequence) are appended to the cDNA. Nucleic acid barcode molecule 1290 comprises a sequence 1224 complementary to the non-templated bases, and the reverse transcriptase performs a template switching reaction onto nucleic acid barcode molecule 1290 to generate a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and a sequence of cDNA 1270 (or a portion thereof). In another example, referring to FIG. 12C, in some embodiments, nucleic acid barcode molecule 1290 comprises sequence 1223 complementary to a sequence of RNA molecule 1260 from a cell. In some instances, sequence 1223 comprises a sequence specific for an RNA molecule. In some instances, sequence 1223 comprises a poly-T sequence. In some instances, sequence 1223 comprises a sequence specific for an RNA molecule. In some instances, sequence 1223 comprises a sequence complementary to a region of an immune molecule, such as the constant region of a TCR or BCR sequence. Sequence 1223 is hybridized to RNA molecule 1260 and a cDNA molecule 1270 is generated in a reverse transcription reaction generating a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and a sequence of cDNA 1270 (or a portion thereof). Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g., to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g., U.S. Pat. Pub. 20180105808, which is hereby incorporated by reference in its entirety. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform.

[00339] In some embodiments, analysis of multiple analytes (e.g., RNA and one or more analytes using labelling agents described herein) comprises a workflow as generally depicted in FIGS. 13A- C. For example, in some embodiments, cells are contacted with one or more reporter oligonucleotide 1220 conjugated labelling agents 1210 (e.g., polypeptide, antibody, or pMHC molecule or complex) and optionally further processed prior to barcoding. Optional processing steps may include one or more washing and/or cell sorting steps. In some instances, a cell bound to labelling agent 1210 (e.g., polypeptide (e.g., antigen), antibody, or pMHC molecule or complex) conjugated to oligonucleotide 1220 and support 1330 (e.g., a bead, such as a gel bead) comprising nucleic acid barcode molecules 1310 and 1320 comprising common barcode sequence 1314 are partitioned into a partition amongst a plurality of partitions (e.g., a droplet of a droplet emulsion or a well of a micro/nanowell array). In some instances, the partition comprises at most a single cell bound to labelling agent 1210. In some embodiments, nucleic acid barcode molecules 1310 and 1320 are attached to support 1230 via a releasable linkage 1340 (e.g., comprising a labile bond) as described elsewhere herein. Nucleic acid barcode molecule 1310 may comprise adapter sequence 1311, barcode sequence 1312 and adapter sequence 1313. Nucleic acid barcode molecule 1320 may comprise adapter sequence 1321, barcode sequence 1312, and adapter sequence 1323, wherein adapter sequence 1323 comprises a different sequence than adapter sequence 1313. In some instances, adapter 1311 and adapter 1321 comprise the same sequence. In some instances, adapter 1311 and adapter 1321 comprise different sequences. Although support 1330 is shown comprising nucleic acid barcode molecules 1310 and 1320, any suitable number of barcode molecules comprising common barcode sequence 1312are contemplated herein. For example, in some embodiments, support 1330 further comprises nucleic acid barcode molecule 1350. Nucleic acid barcode molecule 1350 may comprise adapter sequence 1351, barcode sequence 1312 and adapter sequence 1353, wherein adapter sequence 1353 comprises a different sequence than adapter sequence 1313 and 1323. In some instances, nucleic acid barcode molecules (e.g., 1310, 1320,

1550) comprise one or more additional functional sequences, such as a UMI or other sequences described herein.

[00340] Subsequent to partitioning, referring to FIG. 13B, in some embodiments, sequence 1213 is hybridized to complementary sequence 1313 of nucleic acid barcode molecule 1310 to generate (e.g., via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1312 (or a reverse complement thereof) and reporter barcode sequence 1212 (or a reverse complement thereof). Nucleic acid molecules derived from a cell (such as RNA molecules) can be similarly processed to append the cell (e.g., partition-specific) barcode sequence 1312 to these molecules or derivatives thereof (e.g., cDNA molecules). For example, referring to FIG. 13C, in some embodiments, nucleic acid barcode molecule 1320 comprises sequence 1323 complementary to a sequence of RNA molecule 1260 from a cell. In some instances, sequence 1323 comprises a poly-T sequence. In other instances, sequence 1323 comprises a sequence complementary to a target sequence in an RNA molecule. In some instances, sequence 1323 comprises a sequence complementary to a region of an immune molecule, such as the constant region of a TCR or BCR sequence. Sequence 1323 is hybridized to RNA molecule 1260 and a barcoded cDNA molecule is generated in a reverse transcription reaction comprising cell (e.g., partition specific) barcode sequence 1323 (or a reverse complement thereof) and a cDNA sequence corresponding to mRNA 1260 (or a portion thereof). Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g., to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g., U.S. Pat. Pub. 20180105808, which is hereby incorporated by reference in its entirety. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform. Nucleic acid sequences of interest can be identified from the sequence data. Such nucleic acid sequences of interest can be enriched from the barcoded nucleic acid molecules or derivatives generated therefrom, according to methods disclosed herein.

[00341] As described herein, one or more processes may be performed in a partition, which may be a well. The well may be a well of a plurality of wells of a substrate, such as a microwell of a microwell array or plate, or the well may be a microwell or microchamber of a device (e.g., microfluidic device) comprising a substrate. The well may be a well of a well array or plate, or the well may be a well or chamber of a device (e.g., fluidic device). In some embodiments, a well of a fluidic device is fluidically connected to another well of the fluidic device. Accordingly, the wells or microwells may assume an “open” configuration, in which the wells or microwells are exposed to the environment (e.g., contain an open surface) and are accessible on one planar face of the substrate, or the wells or microwells may assume a “closed” or “sealed” configuration, in which the microwells are not accessible on a planar face of the substrate. In some instances, the wells or microwells may be configured to toggle between “open” and “closed” configurations. For instance, an “open” microwell or set of microwells may be “closed” or “sealed” using a membrane (e.g., semi- permeable membrane), an oil (e.g., fluorinated oil to cover an aqueous solution), or a lid, as described elsewhere herein.

[00342] The well may have a volume of less than 1 milliliter (mL). For instance, the well may be configured to hold a volume of at most 1000 microliters (pL), at most 100 pL, at most 10 pL, at most 1 pL, at most 100 nanoliters (nL), at most 10 nL, at most 1 nL, at most 100 picoliters (pL), at most 10 (pL), or less. The well may be configured to hold a volume of about 1000 pL, about 100 pL, about 10 pL, about 1 pL, about 100 nL, about 10 nL, about 1 nL, about 100 pL, about 10 pL, etc.

The well may be configured to hold a volume of at least 10 pL, at least 100 pL, at least 1 nL, at least 10 nL, at least 100 nL, at least 1 pL, at least 10 pL, at least 100 pL, at least 1000 pL, or more. The well may be configured to hold a volume in a range of volumes listed herein, for example, from about 5 nL to about 20 nL, from about 1 nL to about 100 nL, from about 500 pL to about 100 pL, etc. The well may be of a plurality of wells that have varying volumes and may be configured to hold a volume appropriate to accommodate any of the partition volumes described herein.

[00343] A well may comprise any of the reagents described herein, or combinations thereof.

These reagents may include, for example, barcode molecules, enzymes, adapters, and combinations thereof. The reagents may be physically separated from a sample (e.g., a cell, cell bead, or cellular components, e.g., proteins, nucleic acid molecules, etc.) that is placed in the well. This physical separation may be accomplished by containing the reagents within, or coupling to, a bead that is placed within a well. The physical separation may also be accomplished by dispensing the reagents in the well and overlaying the reagents with a layer that is, for example, dissolvable, meltable, or permeable prior to introducing the polynucleotide sample into the well. This layer may be, for example, an oil, wax, membrane (e.g., semi-permeable membrane), or the like. The well may be sealed at any point, for example, after addition of the bead, after addition of the reagents, or after addition of either of these components. The sealing of the well may be useful for a variety of purposes, including preventing escape of beads or loaded reagents from the well, permitting select delivery of certain reagents (e.g., via the use of a semi-permeable membrane), for storage of the well prior to or following further processing, etc.

[00344] Once sealed, the well may be subjected to conditions for further processing of a cell (or cells) in the well. For instance, reagents in the well may allow further processing of the cell, e.g., cell lysis, as further described herein. Alternatively, the well (or wells such as those of a well-based array) comprising the cell (or cells) may be subjected to freeze-thaw cycling to process the cell (or cells), e.g., cell lysis. The well containing the cell may be subjected to freezing temperatures (e.g., 0°C, below 0°C, -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, - 60°C, -65°C, -70°C, -80°C, or -85°C). Freezing may be performed in a suitable manner, e.g., sub- zero freezer or a dry ice/ethanol bath. Following an initial freezing, the well (or wells) comprising the cell (or cells) may be subjected to freeze-thaw cycles to lyse the cell (or cells). In one embodiment, the initially frozen well (or wells) are thawed to a temperature above freezing (e.g.,

4°C or above, 8°C or above, 12°C or above, 16°C or above, 20°C or above, room temperature, or 25°C or above). In another embodiment, the freezing is performed for less than 10 minutes (e.g., 5 minutes or 7 minutes) followed by thawing at room temperature for less than 10 minutes (e.g., 5 minutes or 7 minutes). This freeze-thaw cycle may be repeated a number of times, e.g., 2, 3, 4 or more times, to obtain lysis of the cell (or cells) in the well (or wells). In one embodiment, the freezing, thawing and/or freeze/thaw cycling is performed in the absence of a lysis buffer.

Additional disclosure related to freeze-thaw cycling is provided in WO2019165181A1, which is incorporated herein by reference in its entirety.

[00345] A well may comprise free reagents and/or reagents encapsulated in, or otherwise coupled to or associated with, beads, or droplets.

[00346] The wells may be provided as a part of a kit. For example, a kit may comprise instructions for use, a microwell array or device, and reagents (e.g., beads). The kit may comprise any useful reagents for performing the processes described herein, e.g., nucleic acid reactions, barcoding of nucleic acid molecules, sample processing (e.g., for cell lysis, fixation, and/or permeabilization).

[00347] As described elsewhere herein, the nucleic acid barcode molecules and other reagents may be contained within a bead, or droplet. These beads, or droplets may be loaded into a partition (e.g., a microwell) before, after, or concurrently with the loading of a cell, such that each cell is contacted with a different bead, or droplet. This technique may be used to attach a unique nucleic acid barcode molecule to nucleic acid molecules obtained from each cell. Alternatively or in addition to, the sample nucleic acid molecules may be attached to a support. For instance, the partition (e.g., microwell) may comprise a bead which has coupled thereto a plurality of nucleic acid barcode molecules. The sample nucleic acid molecules, or derivatives thereof, may couple or attach to the nucleic acid barcode molecules on the support. The resulting barcoded nucleic acid molecules may then be removed from the partition, and in some instances, pooled and sequenced. In such cases, the nucleic acid barcode sequences may be used to trace the origin of the sample nucleic acid molecule. For example, polynucleotides with identical barcodes may be determined to originate from the same cell or partition, while polynucleotides with different barcodes may be determined to originate from different cells or partitions.

[00348] The samples or reagents may be loaded in the wells or microwells using a variety of approaches. The samples (e.g., a cell, cell bead, or cellular component) or reagents (as described herein) may be loaded into the well or microwell using an external force, e.g., gravitational force, electrical force, magnetic force, or using mechanisms to drive the sample or reagents into the well, e.g., via pressure-driven flow, centrifugation, optoelectronics, acoustic loading, electrokinetic pumping, vacuum, capillary flow, etc. In certain cases, a fluid handling system may be used to load the samples or reagents into the well. The loading of the samples or reagents may follow a Poissonian distribution or a non-Poissonian distribution, e.g., super Poisson or sub-Poisson. The geometry, spacing between wells, density, and size of the microwells may be modified to accommodate a useful sample or reagent distribution; for instance, the size and spacing of the microwells may be adjusted such that the sample or reagents may be distributed in a super- Poissonian fashion.

[00349] In some instances, the wells can comprise nucleic acid barcode molecules attached thereto. The nucleic acid barcode molecules may be attached to a surface of the well (e.g., a wall of the well). The nucleic acid barcode molecules may be attached to a droplet or bead that has been partitioned into the well. The nucleic acid barcode molecule (e.g., a partition barcode sequence) of one well may differ from the nucleic acid barcode molecule of another well, which can permit identification of the contents contained with a single partition or well. In some cases, the nucleic acid barcode molecule can comprise a spatial barcode sequence that can identify a spatial coordinate of a well, such as within the well array or well plate. In some cases, the nucleic acid barcode molecule can comprise a unique molecular identifier for individual molecule identification. In some instances, the nucleic acid barcode molecules may be configured to attach to or capture a nucleic acid molecule within a sample or cell distributed in the well. For example, the nucleic acid barcode molecules may comprise a capture sequence that may be used to capture or hybridize to a nucleic acid molecule (e.g., RNA, DNA) within the sample. In some instances, the nucleic acid barcode molecules may be releasable from the microwell. In some instances, the nucleic acid barcode molecules may be releasable from the bead or droplet. For instance, the nucleic acid barcode molecules may comprise a chemical cross-linker which may be cleaved upon application of a stimulus (e.g., photo-, magnetic, chemical, biological, stimulus). The nucleic acid barcode molecules, which may be hybridized or configured to hybridize to a sample nucleic acid molecule, may be collected and pooled for further processing, which can include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In some instances nucleic acid barcode molecules attached to a bead or droplet in a well may be hybridized to sample nucleic acid molecules, and the bead with the sample nucleic acid molecules hybridized thereto may be collected and pooled for further processing, which can include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In such cases, the unique partition barcode sequences may be used to identify the cell or partition from which a nucleic acid molecule originated.

[00350] Characterization of samples within a well may be performed. Such characterization can include, in non-limiting examples, imaging of the sample (e.g., cell, cell bead, or cellular components) or derivatives thereof. Characterization techniques such as microscopy or imaging may be useful in measuring sample profiles in fixed spatial locations. For instance, when cells are partitioned, optionally with beads, imaging of each microwell and the contents contained therein may provide useful information on cell doublet formation (e.g., frequency, spatial locations, etc.), cell-bead pair efficiency, cell viability, cell size, cell morphology, expression level of a biomarker (e.g., a surface marker, a fluorescently labeled molecule therein, etc.), cell or bead loading rate, number of cell-bead pairs, etc. In some instances, imaging may be used to characterize live cells in the wells, including, but not limited to: dynamic live-cell tracking, cell-cell interactions (when two or more cells are co-partitioned), cell proliferation, etc. Alternatively or in addition to, imaging may be used to characterize a quantity of amplification products in the well.

[00351] In operation, a well may be loaded with a sample and reagents, simultaneously or sequentially. When cells or cell beads are loaded, the well may be subjected to washing, e.g., to remove excess cells from the well, microwell array, or plate. Similarly, washing may be performed to remove excess beads or other reagents from the well, microwell array, or plate. In the instances where live cells are used, the cells may be lysed in the individual partitions to release the intracellular components or cellular analytes. Alternatively, the cells may be fixed or permeabilized in the individual partitions. The intracellular components or cellular analytes may couple to a support, e.g., on a surface of the microwell, on a solid support (e.g., bead), or they may be collected for further downstream processing. For instance, after cell lysis, the intracellular components or cellular analytes may be transferred to individual droplets or other partitions for barcoding. Alternatively, or in addition to, the intracellular components or cellular analytes (e.g., nucleic acid molecules) may couple to a bead comprising a nucleic acid barcode molecule; subsequently, the bead may be collected and further processed, e.g., subjected to nucleic acid reaction such as reverse transcription, amplification, or extension, and the nucleic acid molecules thereon may be further characterized, e.g., via sequencing. Alternatively, or in addition to, the intracellular components or cellular analytes may be barcoded in the well (e.g., using a bead comprising nucleic acid barcode molecules that are releasable or on a surface of the microwell comprising nucleic acid barcode molecules). The barcoded nucleic acid molecules or analytes may be further processed in the well, or the barcoded nucleic acid molecules or analytes may be collected from the individual partitions and subjected to further processing outside the partition. Further processing can include nucleic acid processing (e.g., performing an amplification, extension) or characterization (e.g., fluorescence monitoring of amplified molecules, sequencing). At any convenient or useful step, the well (or microwell array or plate) may be sealed (e.g., using an oil, membrane, wax, etc.), which enables storage of the assay or selective introduction of additional reagents.

Examples

Example 1: Amplification of a target cDNA of the cDNA library

[00352] A cDNA library member of a cDNA library (e.g., a whole transcriptome barcoded gene expression library) can be identified as comprising a nucleic acid sequence of interest. For example, a cDNA library can be identified as corresponding to an antibody that has a desired activity, or can specifically bind or neutralize an antigen (e.g., using the labelling agents described elsewhere herein). In particular embodiments, the cDNA library member is identified as comprising the nucleic acid sequence with high confidence if the library member comprises a UMI that has been validated according to a method disclosed herein (e.g., a method disclosed in Example 6).

[00353] cDNA can be enriched using a PCR protocol. For example, a cDNA library can be incubated with a primer pair, a polymerase, nucleotides, and buffer. A primer pair can comprise a first primer having a sequence at least partially complementary to the barcode and/or UMI of a member of the cDNA library, and a second primer at least partially complementary to a sequence that is complementary to a sequence downstream of the barcode and/or UMI (e.g., in the constant region). cDNA can be subjected to thermocycling for between 15 cycles and 40 cycles, until the cDNA is enriched.

[00354] Example 2: Further enrichment of a target cDNA

[00355] A cDNA can be further enriched from the library, for example to increase the abundance of sequences of interest as compared to other cDNA molecules in the library and/or enriched product of Example 1.

[00356] Further enrichment can be performed using a second PCR reaction. For example, the enriched cDNA from Example 1 can be incubated with a primer pair, a polymerase, nucleotides, and buffer. A primer pair can comprise a first primer at least partially complementary to a portion of the V(D)J region of one of the cDNA library, and a second primer at least partially complementary to a sequence that is complementary to a sequence downstream of the portion of the V(D)J sequence (e.g., in the constant region). cDNA can be subjected to thermocycling for between 15 cycles and 40 cycles, until the cDNA is enriched.

Example 3: Nested PCR

[00357] A double (e.g., nested) PCR strategy can be employed for the enrichment of a nucleic acid sequence of interest. An example of a nested PCR scheme is illustrated in FIG. 10. In this example, a cDNA molecule of interest is illustrated which comprises a first read sequence, a barcode sequence (identified as a “lOx barcode sequence”), which may be a partition-specific barcode), a unique molecular identifier sequence (UMI), a template switch oligonucleotide (TSO), a V sequence, a D sequence, a J sequence, a constant (C) sequence, and a second read sequence. Primers can be designed to enrich the sequence for an antibody (i.e., the V, D, J, and C sequences) using a double PCR strategy. Such a double PCR strategy can employ a first enrichment step and a second enrichment step. The outer F (forward) and outer R (reverse) primers can be primers employed for a first PCR enrichment step to enrich one of a plurality of cDNA molecules comprising barcodes. The outer F primer can comprise a sequence complementary to an identification sequence described herein (e.g., an identification sequence comprising the barcode sequence or portion thereof and/or the UMI sequence or portion thereof), and the outer R primer can comprise a sequence complementary to the complement of the second read sequence. The inner F (forward) and inner R (reverse) primers can be employed for a second enrichment step to further enrich the product of the first enrichment step. The inner F primer can be complementary to the V sequence, and optionally part of the TSO sequence. The inner R primer can be complementary to the C sequence and the J sequence. In some cases, the inner F and inner R primers can include non-binding handles that can allow cloning into a vector or enable pairing of sequences, for example using overlap extension. [00358] An example of primer design scheme for the first enrichment step and second enrichment step is provided in FIG. 15. Here, the primers shown for a first enrichment step can be used for a first PCR reaction, and primers shown for a second enrichment step can be used for a second PCR reaction.

Example 4: Producing a clonable sequence.

[00359] A sequence of a nucleic acid sequence of interest can be extracted to yield a clonable sequence. For example, primers can be designed to yield a clonable sequence (e.g., a sequence coding for an amino acid fragment) from enriched cDNA (e.g., the enriched library from Example 3). An example of primer design that can yield a clonable sequence is provided in FIG. 16. This can be accomplished, for example, by utilizing a forward primer that is V gene specific (e.g., specific to a V sequence) and a reverse primer specific to a constant sequence. The resultant nucleic acid molecule, shown in the bottom panel, can be cloned into a vector for expression or analysis. The expression vector may be configured to comprise a constant region sequence (or a portion thereof) such that, when cloned into the expression vector, the enriched V(D)J molecules (such as a paired light and heavy antibody chain) can be expressed as a fully functional immune molecule (e.g., comprising a full, intact constant region).

Example 5: Cloning an enriched nucleic acid sequence.

[00360] B cells (e.g., single B cells) can be captured, (e.g., partitioned with a barcoded bead), for example using techniques provided herein. The interior of cells can be accessed, for example by lysing or permeablizing the cells, and RNA of the cells can be reverse transcribed to generate barcoded cDNA from the RNA sequences. See, e.g., FIG. 12B or FIG. 13C and accompanying text. This can be performed, for example, by 2 rounds of targeted amplification; the first or second amplifications or the full-length unfragmented cDNA can be used in the following step(s). In some implementations, the partition can comprise a cell barcode and TSO sequence. In some implementations, the partition can comprise a cell barcode and a UMI sequence. In some implementations, a partition can comprise a cell barcode, UMI sequence as provided herein, and a TSO sequence as provided herein. [00361] The resulting nucleic acid sequence (e.g., full length or a fragment thereof) can be sequenced. Sequencing can yield one or more paired heavy and light chain sequences (e.g., heavy and light chain sequence pairs) associated with a specific cell barcode. Some of the input cDNA subject to targeted amplification can be saved for later use (e.g., for capture of a specific input cDNA or other use).

[00362] One or more probes can be designed to target one or more V(D)J junction regions, which can comprise highly unique nucleotide sequences 60-150 base pairs in length. See, e.g., FIG. 19. Similarly, one or more probes can be designed to target the corresponding cell barcodes, or cell barcode and a chosen UMI sequence. In some example embodiments, the one or more probes may be designed to target cell barcodes having valid unique molecular identifiers as determined in the manner described herein. These probes can be captured for example using a streptavidin/biotinylation approach, where the probes can be are annealed to the cDNA, and fragments not annealing to the probes can be washed away. In some embodiments, other suitable capture techniques can also be employed. In some cases, probes can be fluorescent, which can enable droplet sorting. In some such cases, the addition of probe reagents and annealing to existing nucleic acid(s) in the droplet can enable selection of droplet(s) of interest for further amplification or cloning. In some cases, a hydrogel can be selectively formed in a droplet containing a probe of interest. Such a hydrogen can be used as part of an enrichment step. Probes can be used to target specific V genes or J genes in addition to or without junction-specific probes.

[00363] Using the retained DNA of interest (containing sequences which the probes successfully annealed to), specific heavy and light chains can be amplified, for example by one or more rounds of PCR or linear amplification. Amplification can comprise targeting with forward primers the against one or more of the cell barcode, UMI, 5' UTR, and leader sequence; one or more of the cell barcode, UMI, 5' UTR, the cell barcode, UMI, the cell barcode, and the 5' UTR, or a region of the V gene (such as the framework region) and with reverse primers the constant region of the targeted antibodies, or a combination thereof. A primer can comprise overlap extension linkers to physically connect the targeted heavy and light chains, or to introduce restriction or Gibson assembly sites for optimized cloning. In some example embodiments, the primers may be designed to target the cell barcodes associated with unique molecular identifiers that have been validated in accordance with the methods disclosed herein. [00364] In some cases, for example if the number of antibodies being targeted is large, a set of unique overlap extension or linker molecules can be designed in a plate-based reaction. Such overlap extension or linker molecules can be used to introduce clone-specific molecular tags.

Example 6: UMI validation.

[00365] In some example embodiments, multiple read sequences may cover a nucleic acid sequence of interest associated with a specific cell barcode (e.g., a V(D)J sequence). A V(D)J sequencing library was generated using the lOx 5Ύ2 Single Cell Immune Profiling kit per manufacturer’s instructions and sequenced at high depth. A contig sequence corresponding to a V(D)J sequence of an antibody heavy chain associated with a cell barcode was determined using the Enel one software tool. Unique molecular identifiers associated with at least a portion of the contig sequence and the corresponding cell barcode sequence were validated, for example, by the validation engine 1502, in accordance with various methods disclosed herein. For example, referring to FIG. 14C, one or more read sequences were aligned to a contig sequence corresponding to a V(D)J sequence of an antibody associated with a cell barcode. The unique molecular identifier (UMI) associated with these read sequences was validated if the read sequences identified the nucleic acid base occupying each position in the V(D)J sequence of the antibody heavy chain with sufficient confidence. For instance, when one or more read sequences provided a same base call for a position within the V(D)J sequence, that position was validated when the base call matched the type of nucleic acid base occupying the position in the corresponding contig sequence and the total quality score associated with the base calls exceeds a threshold value. Alternatively, when the read sequences provided different base calls for the same position within the V(D)J sequence, the position was not validated if the base calls having the highest total quality score did not match the type of nucleic acid base occupying the position in the contig sequence and the highest total quality score exceeds the second highest total quality score by a threshold value.

[00366] FIG. 14G depicts examples of results of unique molecular identifier (UMI) validation, in accordance with some example embodiments. In the case of the unique molecular identifiers associated with a V(D)J sequence of the antibody associated with the cell barcode, the validation engine 1502 was able to validate nine unique molecular identifiers but five unique molecular identifiers failed to validate. The five invalid unique molecular identifiers are shown in FIG. 14G. According to FIG. 14G, manual analysis of the sequence reads associated with the five invalid unique molecular identifiers revealed several discrepancies between the UMI-associated sequences and the contig sequence. These discrepancies include truncations, base substitutions, deletions, and other errors). Thus, as shown in FIG. 14G, the validation engine 1502 implementing the methods for unique molecular identifier (UMI) validation provided herein successfully filtered out those unique molecular identifiers associated with damaged library members.

Example 7: Specific enrichment of BCR sequences from a pooled cDNA library.

Sample selection. A library of BCR enriched product from cells selected by PE+/APC+ gate was used as template in the nested PCR reactions. Two negative controls were included to verify product specificity: BCR enriched product from antigen negative cells from the same donor as the sample used to generate the target clones and BCR enriched product from purified B cells from a different donor.

Clone selection for amplification:

[00367] Nested PCR reactions were performed on the BCR enriched product (and negative controls) to enrich for sequences of four antigen-specific clonotypes, e.g., antibody sequences, in the library. The antigen-specific clonotypes were selected as belonging to one of four categories: (1) expanded clonotype with multiple unique subclones (Clone A) (2); expanded clonotype with a single unique subclone (Clone B); (3) single cell clonotype with many valid UMIs (Clone C); and (4)

Single cell clonotypes with few valid UMIs (Clone D). UMIs were validated according to the methods described herein, e.g., in Example 6.

Primer design:

[00368] Commercially available software (Geneious Prime, primer3) was used to generate the primer sequences to be free of typical sequence weakness (such as hairpin Tm, self dimer Tm, and pair dimer Tm). Primers for the nested PCR reaction were designed to target: (1) in an outer reaction, the cell barcode and UMI (forward primer) and isotype and J region (reverse primer); and (2) in an inner reaction, the leader peptide or FWR1 (forward primer) and CDR3 /junction (potentially extending into the J region, if necessary; reverse primer). Primer pairs were selected based on compatibility of the inner and outer pairs.

[00369] Default settings for Geneious Prime 2021.1.1 using primer3 Tm settings were as described in Santa Lucia et al. 1998 and salt correction settings as described in Owczarzy et al. 2004 were used. Monovalent, divalent, oligo, and dNTP concentrations were set to 50 mM, 1.5 mM, 50 nM, and 0.6 mM respectively. The minimum size allowed for each primer was 18 nucleotides, with a maximum of 27, and an optimal length of 20 nucleotides. The minimum, maximum, and optimum Tms for each primer were set to 57, 60, and 63°C. The allowed GC% content minimum, maximum, and optimum were set to 20, 80, and 50%. The maximum permitted dimer Tm was 47°C. The maximum permitted Tm difference was 100°C.

Amplification reactions:

[00370] Outer primer sequences targeting Cell Barcode + UMI and framework 4 regions were used for the first round of PCR for each of the 4 clones described above. Each amplification reaction contained lOnM of each primer, luL BCR enriched product, 25uM betaine (to increase polymerase processivity), and 50uL 2X hot start high fidelity PCR master mix in a total volume of lOOuL. These were amplified for 10 cycles total, with annealing temperatures appropriate to the primer pair used (51-54C) and a 1 minute 72C extension. Reactions were cleaned up using 0.6X SPRIselect, and the entire volume was put into the second PCR. This reaction included lOnM of each inner primer (targeting leader and framework 4/constant regions), the amplified product from the first PCR, 25uM betaine, and 50uL 2X hot start high fidelity PCR master mix in a total volume of lOOuL. These were amplified for 10 cycles total, with annealing temperatures appropriate to the primer pair used (54C) and a 1 minute 72C extension. Reactions were cleaned up using 0.6X SPRIselect.

[00371] Nested PCR product was run on BioA and/or Labchip to assess product size and specificity. This process confirmed specific product in only antigen positive B cells for clones B (Fig. 21B), C (Fig. 21C), and D (Fig. 21D). The product for clone C was more varied in size and appeared in negative controls as well, suggesting more non-specific amplification for this clone. Results also showed several products for clone A, which was expected given clonotype A was associated with multiple unique subclones (FIG. 21 A).

[00372] In addition, a single step PCR using the conditions and primers described or the second step of the nested PCR was run and analyzed as before. Significantly more nonspecific product was observed for all clones tested, demonstrating the advantages of the nested approach in improving specificity. See, for example, Fig. 20A and B.

Sequencing: [00373] Finally, full length enriched clone sequences were converted into sequencing libraries using the commercially available Prism library prep kit from IDT and sequenced on a paired end 300bp MiSeq run to assay sequence purity.

[00374] FIG. 22 shows sequencing results of the enrichment products following nested amplification for a nucleic acid sequence of interest from a pooled barcoded cDNA library, e.g., a target nucleic acid sequence encoding a fragment of a BCR produced from Clone A (an expanded clonotype with multiple subclonotypes), when the forward outer primer lacked sufficient specificity. The consensus region from positions 254-284 depict the cell barcode + UMI region (indicated by circling) targeted by the forward outer primer. As shown, the consensus for the cell barcode + UMI had several variant positions, indicating poor forward outer primer specificity for the selected barcode/UMI combination. The results indicate retrieval of off-target sequences, due to off-target binding of cDNA library members having multiple cell barcode/UMI combinations.

[00375] FIG. 23 shows sequencing results of the enrichment products following nested amplification for a nucleic acid sequence of interest from a pooled barcoded cDNA library, e.g., a target nucleic acid sequence encoding a fragment of a BCR produced from Clone C (a single cell clone with many valid UMIs), when the forward outer primer lacked sufficient specificity. As shown (circled in consensus sequence), the cell barcode + UMI region largely lacked consensus, indicating poor forward outer primer specificity for the selected barcode/UMI combination. The consensus for the BCR sequence of interest had two variant positions in the CDR3 region, indicating retrieval of off-target sequences, due to binding of cDNA library members having multiple cell barcode/UMI combinations.

[00376] Similarly, sequencing results of the enrichment products produced from Clone D (a single cell clone with few valid UMIs) following nested amplification from the pooled barcoded cDNA library indicated that the forward outer primer lacked specificity for the cell barcode + UMI combination, and retrieved off-target sequences (data not shown).

[00377] FIG. 24 shows sequencing results of the enrichment products following nested amplification for a nucleic acid sequence of interest from a pooled barcoded cDNA library, e.g., a target nucleic acid sequence encoding a fragment of a BCR produced from Clone B (an expanded clonotype with a single unique subclone), when the forward outer primer bound with sufficient specificity to the cell barcode and UMI. As shown (circled in consensus sequence), the consensus for the cell barcode + UMI region had a single variant position, demonstrating that the forward outer primer bound with sufficient specificity to the cell barcode and UMI. Also as shown, the consensus for the BCR fragment had no variant positions, indicating successful retrieval of the full sequence of interest from the barcoded cDNA library with the nested amplification approach when the forward outer primer bound with sufficient specificity to the cell barcode and UMI.

[00378] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIM WHAT IS CLAIMED IS:

1. A system, comprising: a data processor; and a memory storing instructions, which when executed by the data processor, result in operations comprising: aligning, to a contig sequence, each read sequence of a plurality of read sequences associated with a unique molecular identifier, the aligning being based at least on a subsequence in each read sequence and a matching subsequence in the contig sequence; validating, based at least on the plurality of read sequences aligned to the contig sequence, a first position of a plurality of positions in a nucleic acid sequence of interest in the contig sequence, the first position being validated based on at least one of (i) a first base type occupying the first position in at least one of the plurality of read sequences matching a second base type occupying the first position in the contig sequence and (ii) a first quality score of the first base type exceeding a threshold value; and in response to validating the plurality of positions in the nucleic acid sequence of interest in the contig sequence, validating the unique molecular identifier.

2. A system, comprising: a data processor; and a memory storing instructions, which when executed by the data processor, result in operations comprising: aligning a plurality of read sequences associated with a unique molecular identifier to a contig sequence comprising a nucleic acid sequence of interest, wherein a read sequence of the plurality of read sequences is aligned to the contig sequence by aligning a subsequence of the read sequence to a matching subsequence in the nucleic acid sequence of interest; for a first position in the contig that corresponds to the nucleic acid sequence of interest, determining a quality score for each possible base type at the first position in the contig, based on quality scores of base types identified in positions of the plurality of reads that align to the first position in the contig; validating the first position in the contig based on at least one of (i) a first base type having a highest quality score matching a base type occupying the first position in the contig and (ii) the highest quality score exceeding a second highest quality score associated with a second base type occupying the first position in the contig by a threshold value; and validating the unique molecular identifier in response to validating each position in the contig that corresponds to the nucleic acid sequence of interest.

3. The system of claims 1 or 2, wherein the first base type occupies the first position in a first read sequence and a second read sequence of the plurality of read sequences, and wherein the first quality score comprises a value that is representative of a respective quality scores of the first base type in each of the first read sequence and the second read sequence.

4. The system of claim 3, wherein the value comprises a sum, a mean, a medium, a mode, a maximum, or a minimum.

5. The system of any one of the preceding claims, wherein the first base type occupies the first position in at least a first read sequence of the plurality of read sequences, wherein a third base type occupies the first position in at least a second read sequence of the plurality of read sequences, wherein the first quality score comprises a highest quality score associated with the first position, wherein a second quality score of the third base type comprises a second highest quality score associated with the first position, and wherein the first position is validated further based on the first quality score of the first base type exceeding the second quality score of the third base type by the threshold value.

6. The system of any one of the preceding claims, wherein the first position is invalid based at least on the first position not being covered by any one of the plurality of read sequences.

7. The system of any one of the preceding claims, wherein the first base type and the second base type comprise adenine (A), cytosine (C), guanine (G), or thymine (T).

8. The system of any one of the preceding claims, wherein the first quality score indicates an accuracy and/or a probability of error associated with a base call indicating the first base type.

9. The system of any one of the preceding claims, wherein the first quality score comprises a Phred quality score.

10. The system of any one of the preceding claims, wherein the threshold value comprises 15, 20, 25, or 30.

11. The system of any one of the preceding claims, wherein the threshold value is between 15 and 30.

12. The system of any one of the preceding claims, wherein the operations furthercomprise: validating, based at least on the plurality of read sequences aligned to the contig, a second position of the plurality of positions in the nucleic acid sequence of interest in the contig, the second position being validated based on at least one of (i) a third base type occupying the second position in at least one of the plurality of read sequences matching a fourth base type occupying the second position in the contig and (ii) a second quality score of the third base type exceeding the threshold value.

13. The system of any one of the preceding claims, wherein the subsequence and the matching subsequence each comprise a continuous sequence of bases.

14. The system of any one of the preceding claims, wherein the subsequence and the matching subsequence comprise a longest sequence of matching bases between each read sequence and the contig sequence.

15. The system of any one of the preceding claims, wherein the nucleic acid sequence of interest comprises a variable (V) gene segment sequence and a joining (J) gene segment sequence.

16. The system of claim 15, wherein the first position of the plurality of positions comprising the nucleic acid sequence of interest corresponds to a start of the variable (V) gene segment sequence.

17. The system of any one of claims 14-16, wherein a last position of the plurality of positions comprising the nucleic acid sequence of interest corresponds to an end of the joining (J) gene segment sequence.

18. The system of any one of the preceding claims, wherein the nucleic acid sequence of interest encodes an antigen binding molecule or an antigen binding fragment of the antigen binding molecule.

19. The system of claim 18, wherein the antigen binding molecule or the antigen binding fragment of the antigen binding molecule comprises a T cell receptor (TCR) or a fragment of the T cell receptor.

20. The system of any one of claims 18-19, wherein the antigen binding molecule or the antigen-binding fragment of the antigen binding molecule comprises an antibody or an antigen binding fragment of the antibody.

21. The system of any one of the preceding claims, wherein the validating includes examining one or more positions in the subsequence of each read sequence.

22. The system of claim 21, wherein the validating includes examining one or more additional positions in each read sequence between the subsequence and an indel-free alignment corresponding to a start of each read sequence and/or an end of each read sequence.

23. The system of any one of the preceding claims, wherein the unique molecular identifier comprises one of a plurality of unique molecular identifiers associated with a barcode sequence identifying a cell from which the nucleic acid sequence of interest is derived.

24. The system of claim 23, wherein the contig sequence comprises a consensus sequence in which each position is occupied by a most frequently encountered nucleic acid base at a same position across a plurality of read sequences associated with the barcode.

25. The system of any one of the preceding claims, wherein the unique molecular identifier, or a complement of the unique molecular identifier, is comprised in a complementary deoxyribonucleic acid (cDNA) molecule comprising one or more sequences corresponding to an analyte, and wherein the unique molecular identifier identifies the analyte.

26. The system of claim 25, wherein the complementary deoxyribonucleic acid (cDNA) molecule includes a nucleic acid sequence of a heavy chain and/or a light chain of an antibody expressed by a cell.

27. The system of any one of claims 25-26, wherein the cell is a B cell or a T cell.

28. The system of any one of claims 25-27, wherein the complementary deoxyribonucleic acid (cDNA) molecule includes a template switch oligonucleotide (TSO) sequence, a variable (V) gene segment sequence, a joining (J) gene segment sequence, a diversity (D) sequence, a constant (C) sequence, and a barcode sequence identifying a cell from which the complementary deoxyribonucleic acid (cDNA) molecule is derived.

29. The system of claim 28, wherein the barcode sequence is a partition-specific barcode that is unique to a partition containing a single one of the cell.

30. The system of any one of the preceding claims, wherein the operations further comprise: generating an output corresponding to a result of validating the unique molecular identifier.

31. The system of claim 30, wherein the operations further comprise: generating a user interface displaying, at a client device, at least a portion of the output.

32. The system of any one of claims 30-31, wherein at least a portion of the output is sent, over a wired network and/or a wireless network, to a client device.

33. The system of any one of the preceding claims, wherein the operations further comprise: designing a primer configured to target the validated unique molecular identifier.

34. The system of claim 33, wherein the primer is configured to enrich the nucleic acid sequence of interest associated with the unique molecular identifier.

35. The system of claim 34, wherein the primer enriches the nucleic acid sequence of interest through a complementary base pairing.

36. The system of any one of claims 34-35, wherein the primer is configured to enrich the nucleic acid sequence of interest during a nested polymerase chain reaction (PCR) amplification having a first amplification reaction and a second amplification reaction.

37. The system of claim 36, wherein the first amplification reaction includes using an outer F (forward) primer and an outer R (reverse) primer configured to enrich the nucleic acid sequence of interest associated with the unique molecular identifier and/or a barcode sequence of a cell from which the nucleic acid sequence of interest is derived.

38. The system of claim 37, wherein the outer F primer comprises a sequence complementary to the validated unique molecular identifier and/or the barcode sequence, and wherein the outer R primer comprises a sequence complementary to (i) a complement of one or more of the plurality of read sequences associated with the validated unique molecular identifier, (ii) a portion of the nucleic acid sequence of interest that encodes at least a part of a B cell receptor (BCR) or T cell receptor (TCR) constant sequence, or (iii) a portion of the nucleic acid sequence of interest that encodes a junction (J) region and/or isotype region of the B cell receptor or T cell receptor.

39. The system of any one of claims 36-38, wherein the second amplification reaction includes using an inner F (forward) primer and an inner R (reverse) primer to further enrich a product of the first amplification reaction.

40. The system of claim 39, wherein the inner F (forward) primer is complementary to (i) a variable (V) gene segment sequence of the nucleic acid sequence of interest or (ii) nucleotides of at least a portion of the leader sequence and/or encoding framework region (FWR)l of the BCR or TCR, or fragment thereof, and wherein the inner R (reverse) primer comprises a sequence complementary to (iii) a constant (C) gene segment sequence and a joining (J) gene segment sequence of the nucleic acid sequence of interest or complement thereof, or (iv) at least a portion of the nucleic acid sequence of interest that encodes a complementarity region (CDR)3, a FWR4, a J region, a D region, and/or a V region, or a junction between any one or more thereof, of the BCR or TCR, or fragment thereof (or a complement thereof).

41. The system of claim 40, wherein the inner F (forward) primer is further complementary to at least a portion of a template switch oligonucleotide (TSO) sequence of the nucleic acid sequence of interest.

42. A computer-implemented method comprising: aligning, to a contig sequence, each read sequence of a plurality of read sequences associated with a unique molecular identifier, the aligning being based at least on a subsequence in each read sequence and a matching subsequence in the contig sequence; validating, based at least on the plurality of read sequences aligned to the contig sequence, a first position of a plurality of positions in a nucleic acid sequence of interest in the contig sequence, the first position being validated based on at least one of (i) a first base type occupying the first position in at least one of the plurality of read sequences matching a second base type occupying the first position in the contig sequence and (ii) a first quality score of the first base type exceeding a threshold value; and in response to validating the plurality of positions in the nucleic acid sequence of interest in the contig sequence, validating the unique molecular identifier.

43. A computer-implemented method, comprising: aligning a plurality of read sequences associated with a unique molecular identifier to a contig sequence comprising a nucleic acid sequence of interest, wherein a read sequence of the plurality of read sequences is aligned to the contig sequence by aligning a subsequence of the read sequence to a matching subsequence in the nucleic acid sequence of interest; for a first position in the contig that corresponds to the nucleic acid sequence of interest, determining a quality score for each possible base type at the first position in the contig, based on quality scores of base types identified in positions of the plurality of reads that align to the first position in the contig; validating the first position in the contig based on at least one of (i) a first base type having a highest quality score matching a base type occupying the first position in the contig and (ii) the highest quality score exceeding a second highest quality score associated with a second base type occupying the first position in the contig by a threshold value; and validating the unique molecular identifier in response to validating each position in the contig that corresponds to the nucleic acid sequence of interest.

44. The method of claims 42 or 43, wherein the first base type occupies the first position in a first read sequence and a second read sequence of the plurality of read sequences, and wherein the first quality score comprises a value that is representative of a respective quality scores of the first base type in each of the first read sequence and the second read sequence.

45. The method of claim 44, wherein the value comprises a sum, a mean, a medium, a mode, a maximum, or a minimum.

46. A non-transitory computer readable storing instructions, which when executed by at least one data processor, result in operations comprising: aligning, to a contig sequence, each read sequence of a plurality of read sequences associated with a unique molecular identifier, the aligning being based at least on a subsequence in each read sequence and a matching subsequence in the contig sequence; validating, based at least on the plurality of read sequences aligned to the contig sequence, a first position of a plurality of positions in a nucleic acid sequence of interest in the contig sequence, the first position being validated based on at least one of (i) a first base type occupying the first position in at least one of the plurality of read sequences matching a second base type occupying the first position in the contig sequence and (ii) a first quality score of the first base type exceeding a threshold value; and in response to validating the plurality of positions in the nucleic acid sequence of interest in the contig sequence, validating the unique molecular identifier.

47. A non-transitory computer readable storing instructions, which when executed by at least one data processor, result in operations comprising: aligning a plurality of read sequences associated with a unique molecular identifier to a contig sequence comprising a nucleic acid sequence of interest, wherein a read sequence of the plurality of read sequences is aligned to the contig sequence by aligning a subsequence of the read sequence to a matching subsequence in the nucleic acid sequence of interest; for each position in the contig that corresponds to the nucleic acid sequence of interest, determining a quality score for each possible base type at the position in the contig, based on quality scores of base types identified in positions of the plurality of reads that align to the position in the contig; validating each position in the contig based on at least one of (i) a first base type having a highest quality score matching a base type occupying the position in the contig and (ii) the highest quality score exceeding a second highest quality score associated with a second base type occupying the position in the contig by a threshold value; and validating the unique molecular identifier in response to validating each position in the contig that corresponds to the nucleic acid sequence of interest.

48. A method, comprising: aligning, to a contig sequence, each read sequence of a plurality of read sequences associated with a unique molecular identifier, the aligning being based at least on a subsequence in each read sequence and a matching subsequence in the contig sequence; validating, based at least on the plurality of read sequences aligned to the contig sequence, a first position of a plurality of positions in a nucleic acid sequence of interest in the contig sequence, the first position being validated based on at least one of (i) a first base type occupying the first position in at least one of the plurality of read sequences matching a second base type occupying the first position in the contig sequence and (ii) a first quality score of the first base type exceeding a threshold value; in response to validating the plurality of positions in the nucleic acid sequence of interest in the contig sequence, validating the unique molecular identifier; designing a primer configured to target the validated unique molecular identifier; and using the primer to enrich the nucleic acid sequence of interest associated with the unique molecular identifier.

49. A method, comprising: aligning a plurality of read sequences associated with a unique molecular identifier to a contig sequence comprising a nucleic acid sequence of interest, wherein a read sequence of the plurality of read sequences is aligned to the contig sequence by aligning a subsequence of the read sequence to a matching subsequence in the nucleic acid sequence of interest; for each position in the contig that corresponds to the nucleic acid sequence of interest, determining a quality score for each possible base type at the position in the contig, based on quality scores of base types identified in positions of the plurality of reads that align to the position in the contig; validating each position in the contig based on at least one of (i) a first base type having a highest quality score matching a base type occupying the position in the contig and (ii) the highest quality score exceeding a second highest quality score associated with a second base type occupying the position in the contig by a threshold value; validating the unique molecular identifier in response to validating each position in the contig that corresponds to the nucleic acid sequence of interest; designing a primer configured to target the validated unique molecular identifier; and using the primer to enrich the nucleic acid sequence of interest associated with the unique molecular identifier.