Accurate molecular barcoding

Abstract

Compositions and methods are provided for accurate barcoding of nucleic acids. The compositions and methods include a plurality of unique oligonucleotide species comprising unique molecular barcodes. The molecular barcodes have a relatively low G (guanine) content, e.g., each barcode contains less than 50% guanine, or the entire set of barcodes contains, on average, less than 12.5% ​​guanine. The set of barcodes is intended to exhibit reduced bias in amplification and analysis.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 330,500, filed May 2, 2016, which is hereby incorporated by reference in its entirety.

[0002] Array list reference This application has been filed together with a Sequence Listing in electronic format, which is provided in a file named SEQUENCEBDCRI019WO.TXT, created and last modified on April 18, 2017 with a size of 32,588 bytes. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] FIELD OF THE DISCLOSURE Embodiments herein relate generally to compositions and methods for precise barcoding of molecules (eg, nucleic acid molecules). Summary of the Invention [Means for solving the problem]

[0004] Some embodiments include providing a composition comprising at least 1000 unique oligonucleotide species. Each unique oligonucleotide species may comprise a barcode region comprising a molecular barcode comprising at least 7 nucleotides, and the unique oligonucleotide species comprise distinct nucleic acid sequences in the barcode region, and at least one of (a) the composition consists essentially of the unique oligonucleotide species, each molecular barcode having a G content of less than 50%, or (b) the molecular barcodes of all the unique oligonucleotide species in the composition collectively have a G content of 12.5% ​​or less. In some embodiments, the barcode region further comprises a sample barcode comprising at least 3 nucleotides. In some embodiments, each unique oligonucleotide species further comprises a uniform region 3' to the barcode region. In some embodiments, the uniform region further comprises a target specific region 3' to the barcode region, the target specific region comprising at least 10 nucleotides complementary to the target nucleic acid. In some embodiments, the composition consists essentially of the unique oligonucleotide species, each molecular barcode having a G content of less than 50%. In some embodiments, the molecular barcodes of all the unique oligonucleotide species in the composition collectively have a G content of 12.5% ​​or less. In some embodiments, the unique oligonucleotide species are arranged in at least two spatially separated pools, each pool comprising at least 100 unique oligonucleotides of the unique oligonucleotide species, the unique oligonucleotides of the same pool comprising the same sample barcode sequence, and the different unique oligonucleotides of the same pool comprising different molecular barcode sequences. In some embodiments, the sample barcodes of each unique oligonucleotide species have a G content of 50% or less. In some embodiments, the barcode region of each unique oligonucleotide species has a G content of 50% or less. In some embodiments, the molecular barcodes of the unique oligonucleotide species have a G content of less than 12.5% ​​overall. In some embodiments, the barcode region of the unique oligonucleotide species has a G content of 12.5% ​​or less overall.In some embodiments, at least 95% of the unique oligonucleotide species have no Gs in the molecular barcode adjacent to other Gs. In some embodiments, the composition consists essentially of unique oligonucleotide species having no Gs in the molecular barcode adjacent to other Gs. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise a sequence of at least 6 alternating Hs and Ns, with the proviso that each "H" is one of A, C, or T, and each "N" is one of A, G, C, or T. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise the sequence HNHNHNHN, with the proviso that each "H" is one of A, C, or T, and each "N" is one of A, G, C, or T. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN, with the proviso that each "H" is one of A, C, or T, and each "N" is one of A, G, C, or T. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HHHHHHHH, where each "H" is A, C, or T. In some embodiments, each unique oligonucleotide species comprises a spacer 3' to the barcode region and 5' to the target specific region, said spacer comprising the sequence HNHNHNHN, where each "H" is A, C, or T, and where each "N" is A, G, C, or T. In some embodiments, each unique oligonucleotide species comprises a spacer 3' to the barcode region and 5' to the target specific region, said spacer comprising the sequence HHHHHHH, where each "H" is A, C, or T. In some embodiments, the target specific region comprises an oligo dT sequence. In some embodiments, each unique oligonucleotide species has a molecular barcode 3' to the sample barcode. In some embodiments, each unique oligonucleotide species has a sample barcode 3' to the molecular barcode.In some embodiments, each oligonucleotide species has a length of at least 24 nucleotides. In some embodiments, each oligonucleotide species has a length of 24-140 nucleotides. In some embodiments, the composition comprises at least 6,500 unique oligonucleotide species. In some embodiments, the composition comprises at least 65,000 unique oligonucleotide species. In some embodiments, the composition comprises at least 2 oligonucleotides of the same unique oligonucleotide species. In some embodiments, no two oligonucleotides of the composition are the same unique oligonucleotide species. In some embodiments, the composition comprises at least 48 pools. In some embodiments, the unique oligonucleotide species of each pool are immobilized to the substrate such that the sample barcodes, but not the molecular barcodes, of the oligonucleotide species immobilized to each substrate are identical. In some embodiments, the substrate comprises discrete regions of the surface such that the surface may comprise two or more substrates. In some embodiments, the substrate comprises beads. In some embodiments, each of the unique oligonucleotide species further comprises an adapter configured to immobilize the unique oligonucleotide to the substrate, the barcode region being 3' to the adapter. In some embodiments, the uniform region comprises a target specific region comprising a sequence flanking an immune cell receptor variable region coding sequence or an immunoglobulin variable region coding sequence. In some embodiments, the immune cell receptor variable region coding sequence is a T cell receptor variable region coding sequence, a B cell receptor variable region coding sequence, or a combination thereof. In some embodiments, a kit is provided that includes a composition comprising at least 1000 unique oligonucleotide species. The kit may further include a primer configured to hybridize to the opposite side of the variable region from the side on which the target specific region is located and to hybridize to the complement of the strand to which the target specific region hybridizes, such that the primer is configured to amplify the variable region together with the target specific region. In some embodiments, the primer and the target specific region are configured to amplify at least 1 kb of nucleic acid comprising the variable region.In some embodiments, the primers of the kit are part of a composition that includes the unique oligonucleotide species. In some embodiments, the primers of the kit are part of another composition that is separate from the composition that includes the unique oligonucleotide species.

[0005] Some embodiments include providing a method for specifically barcoding multiple nucleic acids from two or more samples. Each sample may include a nucleic acid. The method may include contacting each sample with a pool comprising at least 100 unique oligonucleotide species under conditions in which each sample is contacted with a spatial separation from the other samples. Each unique oligonucleotide species may include a barcode region comprising a molecular barcode comprising at least 7 nucleotides. The unique polynucleotide species of each pool may include the same sample barcode and may include different molecular barcodes. The method may apply at least: (a) the unique oligonucleotide species contacted with the sample consist essentially of unique oligonucleotide species, each molecular barcode having a G content of less than 50%, or (b) the molecular barcodes of all the unique oligonucleotide species collectively have a G content of 12.5% ​​or less. The method may include hybridizing a target-specific region of at least some of the oligonucleotides of the unique oligonucleotide species to at least some of the nucleic acids of the sample. The method may include extending the hybridized oligonucleotides to generate strands comprising oligonucleotides of the unique oligonucleotide species and sequences complementary to the target. However, for each sample, the strands comprise the same sample barcode and different molecular barcodes, and for different samples, the molecular barcodes are different. In some embodiments, the barcode region further comprises a sample barcode comprising at least 3 nucleotides. In some embodiments, each unique oligonucleotide species further comprises a uniform region 3' to the barcode region. In some embodiments, the uniform region further comprises a target specific region 3' to the barcode region, the target specific region comprising at least 10 nucleotides complementary to the target nucleic acid. In some embodiments, the unique oligonucleotide species contacted with the sample consists essentially of unique oligonucleotide species, each molecular barcode having a G content of less than 50%. In some embodiments, the molecular barcodes of all unique oligonucleotide species collectively have a G content of 12.5% ​​or less.In some embodiments, the method further comprises ascertaining the nucleic acid sequence of the strand comprising the oligonucleotide of the unique oligonucleotide species and the sequence complementary to the target. In some embodiments, at least 100 unique oligonucleotide species of each pool are immobilized to a substrate such that the unique oligonucleotide species immobilized to a given substrate comprise the same sample barcode and such that the different unique oligonucleotide species immobilized to the substrate comprise different molecular barcodes. In some embodiments, each sample barcode has a G content of 50% or less. In some embodiments, the molecular barcodes of the unique oligonucleotide species have an overall G content of less than 12.5%. In some embodiments, the barcode region of the unique oligonucleotide species has an overall G content of 12.5% ​​or less. In some embodiments, at least 95% of the unique oligonucleotide species have no Gs of the molecular barcode adjacent to other Gs. In some embodiments, each pool consists essentially of unique oligonucleotide species where no Gs of the molecular barcode adjacent to other Gs. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise a sequence of alternating H and N, a total of at least 6 occurrences. with each "H" being A, C, or T, and each "N" being A, G, C, or T. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise the sequence HNHNHNHN, with each "H" being A, C, or T, and each "N" being A, G, C, or T. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN, with each "H" being A, C, or T, and each "N" being A, G, C, or T. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HHHHHHHH, with each "H" being A, C, or T.In some embodiments, each unique oligonucleotide comprises a spacer 3' to the barcode region and 5' to the target specific region, said spacer comprising the sequence HNHNHNHN, wherein each "H" is A, C, or T, and each "N" is A, G, C, or T. In some embodiments, each unique oligonucleotide comprises a spacer 3' to the barcode region and 5' to the target specific region, said spacer comprising the sequence HHHHHHH, wherein each "H" is A, C, or T. In some embodiments, at least one pool comprises at least two oligonucleotides of the same unique oligonucleotide species. In some embodiments, a pool does not comprise two oligonucleotides of the same unique oligonucleotide species. In some embodiments, the target specific region comprises an oligo dT sequence. In some embodiments, each unique oligonucleotide species has a molecular barcode 3' to the sample barcode. In some embodiments, each unique oligonucleotide species has a sample barcode 3' to the molecular barcode. In some embodiments, each unique oligonucleotide species has a length of at least 24 nucleotides. In some embodiments, each unique oligonucleotide species has a length of 24-140 nucleotides. In some embodiments, each pool comprises at least 6,500 unique oligonucleotide species. In some embodiments, each pool comprises at least 65,000 unique oligonucleotide species. In some embodiments, at least 48 unique samples are contacted with each unique pool. In some embodiments, at least 99% of the samples are each no more than one cell. In some embodiments, the unique oligonucleotide species of each pool are immobilized to a substrate such that the sample barcodes, but not the molecular barcodes of the unique oligonucleotide species immobilized to each of the plurality of substrates, are identical. In some embodiments, the substrate comprises spatially separated surface regions such that substrates of different pools comprise different spatially separated surface regions. In some embodiments, the substrate comprises beads.In some embodiments, each of the unique oligonucleotide species further comprises an adaptor configured to immobilize the unique oligonucleotide to the substrate, and the barcode region is 3' to the adaptor. In some embodiments, the uniform region comprises a target specific region comprising a sequence flanking a sequence encoding a variable region of an immune cell receptor or an immunoglobulin. In some embodiments, the variable region is of a T cell receptor or a B cell receptor, or a combination thereof. In some embodiments, the method further comprises contacting an extension comprising an oligonucleotide of the unique oligonucleotide species and a sequence complementary to the target with a primer configured to hybridize to the opposite side of the variable region from the side on which the target specific region is located and to hybridize to the complement of the strand to which the target specific region hybridizes. Thus, the method may comprise amplifying a sequence encoding a variable region of a T cell receptor, a B cell receptor, or an immunoglobulin. In some embodiments, the method amplifies at least 1 kb of a sequence comprising a variable region coding sequence.

[0006] Some embodiments include providing a method of making a composition comprising unique oligonucleotides. The method may include providing a plurality of different sample barcodes, each comprising at least 3 nucleotides. The method may include providing a plurality of different molecular barcodes, each comprising at least 7 nucleotides. The method may include synthesizing a plurality of unique oligonucleotide species, wherein each unique oligonucleotide species comprises a barcode region comprising a sample barcode and a molecular barcode, and wherein at least one of (a) the plurality of unique oligonucleotide species consists essentially of unique oligonucleotide species, each molecular barcode having a G content of less than 50%, or (b) the molecular barcodes of all of the plurality of unique oligonucleotide species collectively have a G content of 12.5% ​​or less. The method may include disposing the unique oligonucleotide species in spatially separated pools, wherein each pool comprises a plurality of unique oligonucleotide species, wherein each unique oligonucleotide species of the same pool comprises the same sample barcode sequence and wherein different unique oligonucleotide species of the same pool comprise different molecular barcode sequences, and wherein each pool comprises at least 1000 unique oligonucleotide species. In some embodiments, each unique oligonucleotide species further comprises a uniform region 3' to the barcode region. In some embodiments, the uniform region further comprises a target specific region 3' to the barcode region, the target specific region comprising at least 10 nucleotides complementary to the target nucleic acid. In some embodiments, the plurality of unique oligonucleotide species consists essentially of unique oligonucleotide species, each molecular barcode having a G content of less than 50%. In some embodiments, the molecular barcodes of all of the plurality of unique oligonucleotide species collectively have a G content of 12.5% ​​or less. In some embodiments, each molecular barcode has a G content of 50% or less. In some embodiments, each sample barcode has a G content of 50% or less. In some embodiments, the sample barcodes of the unique oligonucleotide species collectively have a G content of 12.5% ​​or less.In some embodiments, the molecular barcodes of the unique oligonucleotide species have an overall G content of less than 12.5%. In some embodiments, the barcode region of the unique oligonucleotide species has an overall G content of 12.5% ​​or less. In some embodiments, at least 95% of the unique oligonucleotide species have no Gs in the molecular barcode adjacent to other Gs. In some embodiments, the plurality of unique oligonucleotide species consists essentially of unique oligonucleotide species having no Gs in the molecular barcode adjacent to other Gs. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise a sequence of at least 6 alternating Hs and Ns, with the proviso that each "H" is any one of A, C, or T, and each "N" is any one of A, G, C, or T. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotides comprise the sequence HNHNHNHN, with the proviso that each "H" is any one of A, C, or T, and each "N" is any one of A, G, C, or T. In some embodiments, each molecular barcode of a unique oligonucleotide comprises the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T. In some embodiments, each molecular barcode of a unique oligonucleotide comprises the sequence HHHHHHHH, where each "H" is A, C, or T. In some embodiments, each unique oligonucleotide species comprises a spacer 3' to the barcode region and 5' to the target specific region, said spacer comprising the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T. In some embodiments, each unique oligonucleotide species comprises a spacer 3' to the barcode region and 5' to the target specific region, said spacer comprising the sequence HHHHHHHH, where each "H" is A, C, or T. In some embodiments, the target specific region comprises an oligo dT sequence.In some embodiments, each unique oligonucleotide species has a molecular barcode 3' to the sample barcode. In some embodiments, each unique oligonucleotide species has a sample barcode 3' to the molecular barcode. In some embodiments, each unique oligonucleotide species has a length of at least 24 nucleotides. In some embodiments, each unique oligonucleotide species has a length of 24-140 nucleotides. In some embodiments, each pool comprises at least 6,500 unique oligonucleotide species. In some embodiments, each pool comprises at least 65,000 unique oligonucleotide species. In some embodiments, at least 48 pools are created. In some embodiments, the method further comprises immobilizing the unique oligonucleotide species of each pool to a substrate such that the sample barcodes but not the molecular barcodes of the oligonucleotide species immobilized to each of the plurality of substrates are identical. In some embodiments, the substrate comprises discrete surface regions. In some embodiments, the substrate comprises beads. In some embodiments, the unique oligonucleotide species are disposed in spatially separated pools simultaneously with said synthesis. In some embodiments, the unique oligonucleotide species are disposed in spatially separated pools subsequent to said synthesis. In some embodiments, the uniform region comprises a target specific region comprising sequences flanking an immune cell receptor or immunoglobulin variable region coding sequence. In some embodiments, the immune cell receptor variable region coding sequence is a T cell receptor variable region coding sequence, a B cell receptor variable region coding sequence, or a combination thereof. In some embodiments, the kit further comprises a primer configured to hybridize to the side of the variable region coding sequence opposite the side on which the target specific region is located and to hybridize to the complement of the strand to which the target specific region hybridizes, thus amplifying the variable region coding sequence along with the target specific region.

[0007] Some embodiments include an oligonucleotide comprising a barcode region 3' to an adapter region. The barcode region can comprise a molecular barcode comprising at least 7 nucleotides, and the molecular barcode has a G content of 50% or less. In some embodiments, the oligonucleotide further comprises a sample barcode comprising at least 3 nucleotides. In some embodiments, the oligonucleotide further comprises a uniform region 3' to the barcode region. In some embodiments, the uniform region comprises a target specific region comprising at least 10 nucleotides complementary to a target nucleic acid. In some embodiments, the oligonucleotide further comprises an adapter region 5' to the barcode region. In some embodiments, the uniform region comprises a target specific region comprising a sequence flanking an immune cell receptor or immunoglobulin variable region coding sequence. In some embodiments, the immune cell receptor variable region coding sequence is a T cell receptor variable region coding sequence, a B cell receptor variable region coding sequence, or a combination thereof. In some embodiments, the kit further comprises a primer configured to hybridize to the side of the variable region coding sequence opposite to the target specific region and to the complement of the strand to which the target specific region hybridizes, thus amplifying the variable region coding sequence along with the target specific region. [Brief description of the drawings]

[0008] [Figure 1A-C] Figure 1A is a schematic diagram illustrating an oligonucleotide species that includes a barcode region 5' to a uniform region according to some embodiments of the present specification. Figure 1B is a schematic diagram illustrating an oligonucleotide species that includes a barcode region 5' to an oligo dT sequence according to some embodiments of the present specification. Figure 1C is a schematic diagram illustrating an oligonucleotide species that includes a barcode region 5' to a gene specific region according to some embodiments of the present specification.

[0009] [Figure 2A-B]2A is a schematic diagram illustrating a barcode region including a molecular barcode 5' to a sample barcode, which may constitute a barcode region of a unique oligonucleotide species according to some embodiments herein. FIG. 2B is a schematic diagram illustrating a barcode region including a sample barcode 5' to a molecular barcode, which may constitute a barcode region of a unique oligonucleotide species according to some embodiments herein.

[0010] [Figure 3A-D] 3A-3H are diagrams illustrating various configurations of oligonucleotide species according to some embodiments herein. FIG. 3A illustrates a baseline oligonucleotide. FIG. 3B illustrates an oligonucleotide having a base spacer on the 3' side of the barcode region. FIG. 3C illustrates an oligonucleotide having a 5-base spacer on the 3' side of the barcode region. FIG. 3D illustrates an oligonucleotide in which the positions of the molecular barcode (represented by "NNNNNNNN") and sample barcode in the barcode region are swapped compared to FIG. 3A so that the molecular barcode is on the 5' side of the sample barcode. FIG. 3E illustrates an oligonucleotide in which the positions of the molecular barcode (represented by "NNNNNNNN") and sample barcode in the barcode region are swapped compared to FIG. 3A so that the molecular barcode is on the 5' side of the sample barcode and includes a 3-base spacer. FIG. 3F illustrates an oligonucleotide in which the positions of the molecular barcode (represented by "NNNNNNNN") and sample barcode in the barcode region are swapped so that the molecular barcode is on the 5' side of the sample barcode and includes a 5-base spacer. Figure 3G illustrates an oligonucleotide whose molecular barcode comprises the sequence HHHHHHHH, where each H is A, C, or T, and where each H can be the same or different from any other H. Figure 3H illustrates an oligonucleotide whose molecular barcode comprises the sequence HNHNHNHN, where each H is A, C, or T, and where each H can be the same or different from any other H.

[0011] [Figure 3E-H] Same as above.

[0012] [Figure 4A] FIG. 4A illustrates nucleotide usage in a sampling of conventional unique oligonucleotide species.

[0013] [Figure 4B] FIG. 4B illustrates nucleotide usage in a sampling of conventional unique oligonucleotide species.

[0014] [Figure 4C] FIG. 4C illustrates nucleotide usage in a sampling of conventional unique oligonucleotide species.

[0015] [Figure 5A] Figure 5A illustrates the nucleic acid sequences of oligonucleotide species constructed according to some embodiments herein. The SEQ ID NOs assigned in the right-most columns of Figures 5A-5D refer to polynucleotide sequences including "anchor", "1st bc", "2nd bc", "spacer" (if any), and "5→3' recognition sequence" as indicated. Each "anchor" in Figures 5A-5D has the polynucleotide sequence of SEQ ID NO:97.

[0016] [Figure 5B] FIG. 5B illustrates the nucleic acid sequences of oligonucleotide species constructed according to some embodiments herein.

[0017] [Figure 5C] FIG. 5C illustrates the nucleic acid sequences of oligonucleotide species constructed according to some embodiments herein.

[0018] [Figure 5D]FIG. 5D illustrates the nucleic acid sequences of oligonucleotide species constructed according to some embodiments herein.

[0019] [Figure 6] FIG. 6 is a graph of error correction by individual in an analysis using unique oligonucleotide species according to some embodiments herein.

[0020] [Figure 7] FIG. 7 is a graph of error correction by individual and filtered MI crossover in an analysis using unique oligonucleotide species according to some embodiments herein.

[0021] [Figure 8] FIG. 8 is a schematic illustrating, without being limited by any theory, amplification errors that may result from oligonucleotides with high G content. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] According to some embodiments herein, methods and compositions are described for accurate barcoding and analysis of nucleic acids. In some embodiments, individual nucleic acids of a sample can be associated with a unique barcode (e.g., a "molecular barcode") so that the individual nucleic acids of the sample can be quantified by amplification and sequence analysis. Without being limited by any theory, it is contemplated that biases favoring or unfavorable to the presentation, amplification, or nature of certain types of barcode sequences can interfere with the quantification and analysis of individual nucleic acids of a sample (a possible source of bias in some amplification events is illustrated diagrammatically in FIG. 8). Some embodiments herein describe the composition and characteristics of unique oligonucleotide species that include barcodes. However, the unique oligonucleotide species are configured to minimize barcode-related biases and provide accurate analysis of nucleic acids. Without being limited by any theory, it is contemplated that a barcode region that includes features such as a guanosine (G) content of less than 50% and / or the absence of two consecutive "G"s in the barcode region can minimize biases that may otherwise confound the quantification and / or analysis of nucleic acids of a sample. Optionally, individual nucleic acid molecules of a given sample can also be associated with a "sample barcode" so that barcode-associated nucleic acids can be later pooled for efficient batch analysis of nucleic acids from two or more samples, e.g., by next-generation sequencing.

[0023] nucleic acid

[0024] Various nucleic acids are described in accordance with some embodiments herein. For example, the oligonucleotide species, the sample, and / or the target may comprise a nucleic acid.

[0025] As used herein, "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. A nucleic acid may comprise nucleotides. A nucleic acid may be exogenous or endogenous to a cell. A nucleic acid may be present in a cell-free environment. A nucleic acid may comprise, consist of, or consist essentially of a gene or a fragment thereof. A nucleic acid may comprise, consist of, or consist essentially of DNA. A nucleic acid may comprise, consist of, or consist essentially of RNA. A nucleic acid may include one or more analogs (e.g., modified backbones, sugars, or nucleobases). Some examples of analogs include, but are not limited to, 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" may be used interchangeably.

[0026] As used herein, "upstream" (and variations of this root word) refers to a relative 5' position on a nucleic acid (e.g., 5' relative to a reference position). As used herein, "downstream" (and variations of this root word) refers to a relative 3' position on a nucleic acid (e.g., 3' relative to a reference position). For example, as shown in FIG. 2A, a "sample barcode" is 3' to a "molecular barcode" and is understood to be downstream of the molecular "barcode". For example, as shown in FIG. 2B, a "sample barcode" is 5' to a "molecular barcode" and is understood to be upstream of the molecular "barcode".

[0027] Nucleic acids may include one or more modifications (e.g., base modifications, backbone modifications) to provide nucleic acids with new or improved characteristics (e.g., improved stability). Nucleic acids may include nucleic acid affinity tags. Nucleosides include, consist of, or consist essentially of base-sugar combinations. The base portion of a nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides include, consist of, or consist essentially of nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. In nucleosides that include a pentofuranosyl sugar, the phosphate group can be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate group can covalently link adjacent nucleosides to each other to form a linear polymeric compound. The respective ends of this linear polymeric compound can then be further linked to form a circular compound. However, linear compounds are generally preferred. Additionally, linear compounds may have internal nucleotide base complementarity and therefore fold to produce fully or partially double-stranded compounds. Within nucleic acids, the phosphate groups can usually be referred to as forming the internucleoside backbone of the nucleic acid. The linkages or backbone of the nucleic acid can be 3'→5' phosphodiester linkages.

[0028] Nucleic acids may contain modified backbones and / or modified internucleoside linkages. Modified backbones may include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones that contain a phosphorus atom may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylenephosphonates and 5'-alkylenephosphonates, chiral phosphonates, phosphinates, phosphoramidates such as 3'-aminophosphoramidates and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates that typically have 3'-5' linkages, 2'-5' linkage analogs, and those that have reverse polarity where one or more internucleotide linkages are 3'→3', 5'→5', or 2'→2' linkages.

[0029] Nucleic acids may contain polynucleotide backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These may include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0030] Nucleic acids may include nucleic acid mimetics. The term "mimetic" includes, for example, polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, where the replacement of only the furanose ring can be referred to as being a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety can be retained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar backbone of a polynucleotide can be replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotides can be retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. The backbone in a PNA compound can include two or more linked aminoethylglycine units, giving the PNA an amide-containing backbone. The heterocyclic base moiety can be directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone.

[0031] The nucleic acid may include a morpholino backbone structure. For example, the nucleic acid may include a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, the phosphodiester linkage may be replaced by a phosphorodiamidate or other non-phosphodiester internucleoside linkage.

[0032] Nucleic acids can include linked morpholino units (i.e., morpholino nucleic acids) having heterocyclic bases attached to the morpholino ring. Linking groups can link morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds can have less undesirable interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds within the morpholino class can be linked using different linking groups. A further class of polynucleotide mimetics can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose rings normally present in nucleic acid molecules can be replaced with cyclohexenyl rings. CeNA DMT-protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to the native complexes. Further modifications can include locked nucleic acids (LNAs) in which a 2'-hydroxyl group is attached to the 4' carbon atom of the sugar ring to form a 2'-C,4'-C-oxymethylene bond to form a bicyclic sugar moiety. The linkage comprises, consists of, or consists essentially of a methylene (-CH2-), group (where n is 1 or 2) bridging the 2' oxygen atom and the 4' carbon atom. LNAs and LNA analogs can exhibit very high duplex thermal stability with complementary nucleic acids (Tm=+3 to +10°C), stability against 3'-exonuclease degradation, and good solubility.

[0033] Nucleic acids may also, in some embodiments, include modifications or substitutions of nucleobases (often simply referred to as "bases"). As used herein, "unmodified" or "natural" nucleobases may include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, These may include cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deaadenine. Modified nucleobases may include tricyclic pyrimidines, e.g., G-clamps such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (Hpyrido(3',':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0034] As used herein, the term "sample" refers to a composition that includes a target. Samples suitable for analysis by the methods, devices, and systems of the present disclosure include, but are not limited to, cells, single cells, tissues, organs, or organisms. In some embodiments, the sample includes a raw or unprocessed sample, such as a whole cell, a whole cell population, or a whole tissue. In some embodiments, the sample includes an isolated cell or cell extract or a nucleic acid-containing fraction thereof, such as an isolated nucleic acid, or a composition that includes enriched or isolated nucleic acid. In some embodiments, the sample includes a fixed tissue, cell, or a nucleic acid-containing fraction thereof. In some embodiments, the sample includes a frozen tissue, cell, or a nucleic acid-containing fraction thereof. In some embodiments, the sample includes a solution that includes nucleic acid. In some embodiments, the sample includes a solution that includes nucleic acid. In some embodiments, the sample includes a solid form of nucleic acid, such as lyophilized nucleic acid.

[0035] Unique Oligonucleotide Species As used in the compositions, methods, and oligonucleotides of some embodiments herein, a "unique oligonucleotide species" refers to an oligonucleotide (e.g., DNA, RNA) having a sequence that differs from another unique oligonucleotide species by at least one base. The unique oligonucleotide species of the compositions of some embodiments herein may share certain structural features or morphologies, but may have different nucleic acid sequences from each other. The unique oligonucleotide species may be single-stranded or double-stranded. The composition may include more than one unique oligonucleotide species, for example, 100, 1000, 6500, or 65,000 different unique oligonucleotide species. Optionally, a composition that includes a unique oligonucleotide species may also include more than one oligonucleotide of the same unique oligonucleotide species. As an example, a composition may include two unique oligonucleotide species: ACTT-X and TCTT-X, where "X" is the same sequence in both unique oligonucleotide species. A composition may include two copies of an oligonucleotide with the sequence ACTT-X and one copy of an oligonucleotide with the sequence TCTT-X.

[0036] The oligonucleotide species of the compositions, methods, and oligonucleotides of some embodiments herein may include a barcode region and a uniform region as described herein. The barcode region may vary between unique oligonucleotide species to provide diversity in the unique oligonucleotide species population, while the uniform region remains the same. The barcode region may include a molecular index as described herein. The molecular index may be configured to minimize bias, for example, by minimizing the G content so that the unique oligonucleotide species in the unique oligonucleotide species population do not have a molecular index with a G content of more than 50% and / or the sequence "GG" does not appear in the molecular index (e.g., there are no two consecutive Gs). Optionally, the barcode region includes a sample index. The sample index may be configured such that the sample indexes of the unique oligonucleotides of a given pool may be the same, but the molecular indexes may be different. Thus, when multiple samples are analyzed, the sample index may represent which sample each oligonucleotide corresponds to. Thus, after the unique oligonucleotide species bind to the target, the unique oligonucleotide species may be pooled and the sequences may be analyzed. In some embodiments, the sample index is 5' to the molecular index. In some embodiments, the molecular index is 5' to the sample index. Optionally, the unique oligonucleotide species comprises an adaptor. The adaptor can be positioned 5' to the barcode region. In some embodiments, the adaptor is configured to immobilize the unique oligonucleotide species to a substrate.

[0037] Without being limited by any theory, it is contemplated that the unique oligonucleotide species configured according to some embodiments herein may provide accurate analysis and reduced, minimal, or no bias sequencing results, for example, by minimizing the G content of the molecular barcode or barcode region and / or by minimizing the G content near the uniform region. By way of example, reduced bias may be observed by reduced noise and / or increased sensitivity in detecting the number of individual molecules of the target nucleic acid in a sample (see, for example, Figures 6-7). In some embodiments, reduced bias may be observed as reduced noise (see Figure 6), for example, as a smaller standard error in the number of target nucleic acid molecules detected in a sample. In some embodiments, reduced bias may be observed as increased sensitivity (see Figure 7), for example, as detection of a greater number of individual molecules of the target nucleic acid in a sample (e.g., missing fewer target nucleic acid molecules). Thus, in some embodiments, the compositions and methods provide quantification of target nucleic acid molecules in a sample with low noise, e.g., with a relative standard error of less than 30%, e.g., less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less than .01%, inclusive of the ranges between any two of the recited values. In some embodiments, the compositions and methods provide quantification of target nucleic acid molecules in a sample with high sensitivity, e.g., with a sensitivity (measured as a percentage of the actual number of individual target nucleic acids in the sample that are detected), e.g., with a sensitivity of at least 70%, 75%, 80%, 85%, 90%, 85%, 96%, 97%, 98%, 99%, 99.9%, inclusive of the ranges between any two of the recited values. In some embodiments, a method or composition described herein reduces bias by increasing sensitivity, decreasing the relative standard error, or increasing sensitivity and decreasing standard error compared to a composition or method comprising a unique oligonucleotide species consistent with FIG. 3A.

[0038] FIG. 3A illustrates a conventional oligonucleotide design. Note that the molecular index sequence NNNNNNNN (where each N is A, G, C, or T, and any two of N may be the same or different from each other) may contain two or more consecutive Gs and / or have a G content of more than 50%. For example, a subset of a population of unique oligonucleotide species consistent with FIG. 3A may be G-rich. Without being limited by any theory, it is contemplated that at least some unique oligonucleotide species in a population based on the configuration of FIG. 3A may be biased in preference. Another example of a "baseline" molecular index that may be subject to bias is the sequence "BBBBBBV" (where B is C, G, or T, and V is A, C, or G). Example configurations of unique oligonucleotide species according to some embodiments herein are illustrated in FIG. 3B-3H. As shown in Figures 3B-C, it is possible to reduce the G-richness along the region by adding either TTT or TTTTT after the molecular index according to some embodiments herein (the length of the non-G spacer can be variable and can include any one or more non-G nucleotides). In Figure 3D, the sample index and molecular positions are swapped (so that the molecular index is 5' to the sample index) so that the potentially highly G-rich MI in at least a subset of the unique oligonucleotide species is not adjacent to the potentially G-rich gene-specific target region. In Figures 3E-F, it is possible to further minimize the potential G-rich region by adding either TTT or TTTTT to the configuration illustrated in Figure 3D. Without being limited by any theory, it is noted that sample barcodes can often have a low G content (e.g., to be "non-G rich"). It is further noted that the length of the non-G spacer can be variable and can be any non-G nucleotide).In the configurations of Figures 3G-H, the molecular barcode includes "HHHHHHHH" or "HNHNHNHN" (wherein H is A, C, or T, and any two Hs may be the same or different from each other, and any two Ns may be the same or different from each other).

[0039] In some embodiments, each of the plurality of unique oligonucleotide species (e.g., each of the unique oligonucleotide species) in the compositions or methods is at least 24 nucleotides in length, e.g., at least 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 280, 290, 305, 310, 320, 335, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 550, 550, 550, 550, 550, 55 120, 125, 130, 135 or 140 nucleotides in length, e.g., 24-140, 24-135, 24-130, 24-125, 24-120, 24-115, 24-110, 24-105, 24-100, 24-95, 24-90, 24-85, 24-80, 24-75, 24-70, 24-65, 24-60, 24-55, 24-50, 24-45, 24-40, 25-140, 25-135, 25-130, 25-125, 25-120, 25-115, 25-110, 25-105, 25-100, 25-95, 25-90, 25-85, 25~80, 25~75, 25~70, 25~65, 25~60, 25~55, 25~50, 25~45, 25~40, 27~140, 27~135, 27~130, 27~125, 27~120, 27~115, 27~110, 27~105, 27~100, 27~95, 27 ~90, 27~85, 27~80, 27~75, 27~70, 27~65, 27~60, 27~55, 27~50, 27~45, 27~40, 30~140, 30~135, 30~130, 30~125, 30~120, 30~115, 30~110, 30~105, 30~10 0, 30~95, 30~90, 30~85, 30~80, 30~75, 30~70, 30~65, 30~60, 30~55, 30~50, 30~45, 30~40, 35~140, 35~135, 35~130, 35~125, 35~120, 35~115, 35~110, 35 ~105, 35~100, 35~95, 35~90, 35~85, 35~80, 35~75, 35~70, 35~65, 35~60, 35~55, 35~50, 35~45, 35~40, 40~140, 40~135, 40~130, 40~125, 40~120, 40~115,Optionally, the different unique oligonucleotide species in the composition or method have lengths different from each other. Optionally, all of the unique oligonucleotide species in the composition or method have lengths identical to each other. Optionally, some unique oligonucleotide species in the composition or method are the same length as each other, while some unique oligonucleotide species are different lengths from each other.

[0040] In some embodiments, the uniform region comprises, consists of, or consists essentially of a 5' to 3' amplification sequence for a target nucleic acid or class of target nucleic acids (this amplification sequence may also be referred to as a "target specific" region). For example, if the target nucleic acid comprises an mRNA, the uniform region may comprise oligo dT. For example, if the target nucleic acid comprises a variable region of a T cell receptor, the uniform region may comprise sequences flanking the variable region of the T cell receptor mRNA. In some embodiments, the uniform region comprises at least 10 nucleotides complementary to the target nucleic acid, e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides complementary to the target, including the ranges between any two of the listed values, e.g., 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 11-30, 11-29, 11-28, 11-2 and 7, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21, 11-20, 12-30, 12-29, 12-28, 12-27, 12-26, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, or 20-21 nucleotides. In some embodiments, the uniform region comprises a target specific region, e.g., an antibody, that hybridizes to a sequence flanking a sequence encoding the variable region of an immune cell receptor, e.g., a T cell receptor, a B cell receptor, or an immunoglobulin variable region. Note that because B cell receptors comprise membrane-bound immunoglobulins, target specific regions specific for immunoglobulin variable region coding sequences are typically suitable for amplifying B cell receptors as well as secreted immunoglobulins (e.g., antibodies).It is noted that both options clarify that the amplification of membrane-bound immunoglobulins (B cell receptors) as well as secreted immunoglobulins (antibodies) is contemplated herein. It will be understood that, as used herein, when a primer or uniform region includes a target specific region that includes a "flanking sequence" (and variations of this root word, e.g., "flanking sequence") of an immune cell receptor and / or an immunoglobulin variable region, the target specific region is understood to include at least one of: (i) a sequence that hybridizes downstream (3') of the variable region coding sequence and specifically hybridizes to a strand of the coding sequence, and thus configured to generate a strand that includes the reverse complement of the variable region coding sequence when extended in the 5'→3' direction, or (ii) a sequence that hybridizes upstream (5') of the variable region coding sequence and specifically hybridizes to a strand that is complementary to the strand of the coding sequence, and thus configured to generate a strand that includes the variable region coding sequence when extended in the 5'→3' direction. Thus, the flanking sequence can be configured to amplify the variable region coding sequence with a suitable priming partner (e.g., the flanking sequence on the other side of the variable region). It will be understood that the flanking sequence does not necessarily have to end or start exactly where the coding sequence begins or ends. Thus, there may be an intervening sequence between the hybridization site of the flanking sequence and the variable region coding sequence itself. Although the flanking sequence generally hybridizes to a sequence outside the variable region coding sequence to amplify a wide range of possible variable region coding sequences, it will be understood that in some embodiments, e.g., when a subset of possible variable regions is of interest, the variable region "flanking sequence" further comprises some sequence of the variable region itself. However, "flanking sequence" as used herein does not require a single sequence flanking both sides of the variable region.More precisely, when flanking sequences are described in the context of the compositions, methods, and oligonucleotides of several embodiments herein, it will be understood that 5' and 3' sequences comprising primer pairs suitable for amplifying variable region-encoding sequences are expressly contemplated.

[0041] In some embodiments, the unique oligonucleotide species comprises a uniform region that includes a barcode region as described herein and a target specific region that includes a sequence flanking an immune cell receptor and / or immunoglobulin variable region coding sequence. A second oligonucleotide primer can also be provided on the other side of the variable region coding sequence to amplify the variable region sequence along with the target specific region of the uniform region. In some embodiments, the uniform region comprises a target specific region located 3' to the barcode region that includes a sequence flanking an immunoglobulin variable region (and thus flanking a B cell receptor variable region and also a corresponding antibody variable region), for example, a sequence flanking a variable region of an immunoglobulin heavy chain locus, a sequence flanking a variable region of an immunoglobulin (light chain) kappa locus, or a sequence flanking a variable region of an immunoglobulin (light chain) lambda locus. In some embodiments, the uniform region comprises a target specific region positioned 3' to the barcode region that includes sequences flanking at least one of the variable region of the T cell receptor alpha chain, the variable region of the T cell receptor beta chain, the variable region of the T cell receptor gamma chain, or the T cell receptor delta chain.

[0042] In some embodiments, the kit comprises a composition comprising a unique oligonucleotide species as described herein, each of which comprises a uniform sequence flanking a variable region coding sequence of an immune cell receptor or an immunoglobulin as described herein. In some embodiments, the kit further comprises an oligonucleotide primer configured to hybridize to the opposite strand on the other side of the variable region coding sequence compared to the uniform region, thus amplifying the variable region sequence along with the target specific region of the uniform region. In some embodiments, the amplified sequence is at least 1 kb, e.g., comprises at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb of variable coding sequence, including the ranges between any two of the recited values.

[0043] It is noted that some immune cell receptor or immunoglobulin variable region encoding nucleic acids may be more than 1 kb in length. For example, a T cell receptor variable region sequence may include a CDR3 coding sequence that ends more than 1 kb away from where the CDR1 coding sequence begins. Without being limited by theory, it is noted that some conventional and next-generation sequencing methods, such as synthetic sequencing, are typically limited to short reads that are much shorter than 1 kb. Thus, it is contemplated that the methods and compositions and kits according to some embodiments herein may be useful for barcoding and analyzing immune cell receptor and / or immunoglobulin variable region encoding nucleic acids that would otherwise be incompatible with single-read sequencing of less than 1 kb. Thus, in some embodiments, the unique oligonucleotide species comprises sequences flanking immune cell receptor or immunoglobulin variable region coding sequences and is configured to amplify at least 1 kb of sequence including the variable region coding sequence, e.g., at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb of sequence, including the ranges between any two of the recited values.

[0044] Barcode AreaAccording to some embodiments of the compositions, methods, and oligonucleotides herein, the barcode region comprises a nucleic acid sequence useful for identifying a nucleic acid, for example, an amplicon or reverse transcript derived from a target nucleic acid from a sample or a single target nucleic acid of a sample. For example, two mRNA transcripts from a sample are reverse transcribed and barcoded such that the nucleic acid corresponding to the first mRNA comprises a first barcode and the nucleic acid corresponding to the second mRNA comprises a second barcode. Upon sequencing (or other analysis), information about individual mRNAs in a sample, for example, copy number, can be ascertained even after amplification. However, when a large population of mRNAs is stochastically labeled to more favorably present some barcodes (e.g., due to stability, amplification efficiency, etc.), bias can be introduced and the ability to quantify the nucleic acid of the sample can be distorted. Thus, according to some embodiments herein, each unique oligonucleotide species in a population can comprise a unique barcode region. The greater the diversity of barcodes, the greater the diversity of unique oligonucleotide species, and the greater the likelihood that a particular barcode sequence is associated with only one target nucleic acid of a sample. The barcode region may be located, for example, 5' to the uniform region of the oligonucleotide species. In some embodiments, the barcode region comprises a molecular barcode as described herein. Optionally, the barcode region comprises a molecular barcode and a sample barcode as described herein. Optionally, the barcode region comprises a molecular barcode 5' to the sample barcode. In some embodiments, a method or composition comprising a unique oligonucleotide species comprising a molecular barcode as described herein reduces bias by increasing sensitivity, by decreasing relative standard error, or by increasing sensitivity and decreasing standard error. As used herein, "molecular barcode" can also be referred to as "molecular barcode", "molecular index (MI)", or unique molecular identifier (UMI).As used herein, a "sample barcode" can also be referred to as a "sample index" (SI).

[0045] The barcode region may include a molecular barcode. The molecular barcode may include a unique sequence, so that when multiple sample nucleic acids (which may be identical and / or different from each other) are associated one-to-one with the molecular barcode, different sample nucleic acids can be distinguished from each other by the molecular barcode. Thus, even if a sample contains two nucleic acids with the same sequence, each of these two nucleic acids can be labeled with a different molecular barcode, so that the nucleic acid in the population can be quantified even after amplification. The molecular barcode may include at least 5 nucleotides, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 9 , 44, 45, 46, 47, 48, 49, or 50 nucleotides, e.g., 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6- 13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8 The nucleic acid sequence of the molecular barcode may comprise a nucleic acid sequence of 10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides. In some embodiments, the nucleic acid sequence of the molecular barcode comprises a unique sequence, e.g., such that each unique oligonucleotide species in the composition comprises a different molecular barcode.In some embodiments, two or more unique oligonucleotide species may contain the same molecular barcode, but still be different from each other. For example, if a unique oligonucleotide species contains a sample barcode, each unique oligonucleotide species with a particular sample barcode may contain a different molecular barcode. In some embodiments, a composition comprising a unique oligonucleotide species comprises a molecular barcode diversity of at least 1000 different molecular barcodes, thus at least 1000 unique oligonucleotide species. In some embodiments, a composition comprising a unique oligonucleotide species comprises a molecular barcode diversity of at least 6,500 different molecular barcodes, thus at least 6,500 unique oligonucleotide species. In some embodiments, a composition comprising a unique oligonucleotide species comprises a molecular barcode diversity of at least 65,000 different molecular barcodes, thus at least 65,000 unique oligonucleotide species.

[0046] Without being limited by any theory, molecular barcodes with low G content (e.g., 50% G or less, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G) are contemplated to minimize bias (e.g., minimize potential bias in preferentially amplifying barcodes with higher G content) when using compositions or pools of unique oligonucleotide species to barcode a population of nucleic acids. It is noted that conventional barcoding methods typically exhibit a bias in favor of higher G content. For example, Figures 4A, 4B, and 4C illustrate sample nucleotide usage of conventional molecular barcodes of compositions comprising multiple unique molecular barcodes for ES32, TRAC (Figure 4A), ES32 TRBC (Figure 4B), and ES32 oligo dT (Figure 4C). That is, if anything, conventional molecular barcodes and barcode regions designed without considering certain guidelines provided herein may contain a higher G content than would be expected by random chance. In some embodiments, methods or compositions comprising unique oligonucleotide species comprising molecular barcodes as described herein reduce bias by increasing sensitivity, by decreasing relative standard error, or by increasing sensitivity and decreasing standard error.

[0047] In some embodiments, all of the compositions or molecular barcodes of the compositions used in the methods described herein that contain unique oligonucleotide species are, overall, 50% G or less, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, including the ranges between any two of the recited values. , less than 5%G, or less than 2.5%G, or 0%G, e.g., 2.5-50%G, 2.5-45%G, 2.5-40%G, 2.5-35%G, 2.5-30%G, 2.5-25%G, 2.5-20%G, 2.5-15%G, 2.5-10%G, 2.5-7.5%G, 2.5-5%G, 5-50%G, 5-45%G, 5-40%G, 5-35%G, 5-30%G, 5-25%G, 5-20%G, 5-15% G, 5~10%G, 5~7.5%G, 7.5~50%G, 7.5~45%G, 7.5~40%G, 7.5~35%G, 7.5~30%G, 7.5~25%G, 7.5~20%G, 7.5~15%G, 7.5 ~10%G, 10~50%G, 10~45%G, 10~40%G, 10~35%G, 10~30%G, 10~25%G, 10~20%G, 10~15%G, 10~12.5%G, 12.5~50%G, 12 With a G content of .5-45%G, 12.5-40%G, 12.5-35%G, 12.5-30%G, 12.5-25%G, 12.5-20%G, 12.5-15%G, 15-50%G, 15-45%G, 15-40%G, 15-35%G, 15-30%G, 15-25%G, 15-20%G, 20-50%G, 20-45%G, 20-40%G, 20-35%G, 20-30%G, or 20-25%G. "All of the molecular barcodes of a composition of unique oligonucleotide species have, in the aggregate, a G content of..." means that when the total G content across all of the molecular barcodes in the entire composition is calculated (e.g., a population of at least 10, 50, 100, 200, 500, 1000, 2000, 5000, 6500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, or 65,000 unique oligonucleotide species), this total G content across the barcodes can be less than the recited value or within the recited range.Although it may still be possible for individual unique oligonucleotide species to have molecular barcodes with a G content above the indicated value or outside the indicated range, the total nucleotide content of the unique oligonucleotide species of the composition may be less than the indicated value or within the indicated range. In some embodiments, all of the molecular barcodes in a composition comprising at least 1000 unique oligonucleotide species have a G content of less than or equal to 50% G overall, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, inclusive of ranges between any two of the recited values. In some embodiments, all of the molecular barcodes in a composition comprising at least 6500 unique oligonucleotide species have a G content overall of 50% G or less, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, inclusive of ranges between any two of the recited values. In some embodiments, all of the molecular barcodes in a composition comprising at least 65,000 unique oligonucleotide species have an overall G content of 50% G or less, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, inclusive of ranges between any two of the recited values. In some embodiments, all of the barcode regions of the compositions described herein have an overall G content of less than 50% as described herein.

[0048] In some embodiments, in a composition comprising unique oligonucleotide species (or such a composition used in a method), the composition has no more than 50% G, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5%, inclusive of any range between any two of the recited values. Less than G or 0%G, e.g. 2.5-50%G, 2.5-45%G, 2.5-40%G, 2.5-35%G, 2.5-30%G, 2.5-25%G, 2.5-20%G, 2.5-15%G, 2.5-10%G, 2.5-7.5%G, 2.5-5%G, 5-50%G, 5-45%G, 5-40%G, 5-35%G, 5-30%G, 5-25%G, 5-20%G, 5-15%G, 5-10%G, 5-7.5%G, 7.5-5%G, 0%G, 7.5~45%G, 7.5~40%G, 7.5~35%G, 7.5~30%G, 7.5~25%G, 7.5~20%G, 7.5~15%G, 7.5~10%G, 10~50%G, 10~45%G, 10~ 40%G, 10~35%G, 10~30%G, 10~25%G, 10~20%G, 10~15%G, 10~12.5%G, 12.5~50%G, 12.5~45%G, 12.5~40%G, 12.5~35%G, 1 A composition may consist of or consist essentially of unique oligonucleotide species with a molecular barcode G content of 2.5-30% G, 12.5-25% G, 12.5-20% G, 12.5-15% G, 15-50% G, 15-45% G, 15-40% G, 15-35% G, 15-30% G, 15-25% G, 15-20% G, 20-50% G, 20-45% G, 20-40% G, 20-35% G, 20-30% G, or 20-25% G. By "a composition consisting of or consisting essentially of unique oligonucleotide species each having a molecular barcode G content less than..." it is meant that each or essentially each of the unique oligonucleotide species in the composition, population, or pool has a molecular barcode G content less than the indicated value or outside the indicated range.That is, in a composition, population, or pool "consisting essentially of" unique oligonucleotide species, each having a designated G content, an analytically insignificant portion of the unique oligonucleotides in the composition may have molecular barcodes with a G content above the designated value or outside the recited range. For example, an analytically insignificant portion of the unique oligonucleotides may be, or may not exceed, or be less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% of the unique oligonucleotides in the composition. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 1000 unique oligonucleotides comprise molecular barcodes with a G content greater than 50%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 50%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 6500 unique oligonucleotides comprise molecular barcodes that have a G content greater than 50%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 50%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 65,000 unique oligonucleotides comprise molecular barcodes that have a G content greater than 50%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 50%.In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 1000 unique oligonucleotides comprises a molecular barcode with a G content of greater than 25%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content of greater than 25%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition of at least 6500 unique oligonucleotides comprises a molecular barcode with a G content of greater than 25%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 25%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition, population, or pool of at least 65,000 unique oligonucleotides comprise molecular barcodes that have a G content greater than 25%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 25%. Optionally, none of the molecular barcodes of the unique oligonucleotide species of the composition as a whole have a G content greater than 50% G. For example, all of the molecular barcodes of the unique oligonucleotide species have a G content of less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, inclusive of ranges between any two of the listed values.

[0049] In some embodiments, a composition described herein (or such composition used in a method) consists of or consists essentially of unique oligonucleotide species each comprising less than 50% barcode region G content as described herein. In some embodiments, in compositions comprising unique oligonucleotide species, the composition comprises no more than 50% G, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, inclusive of ranges between any two of the recited values. , 2.5~50%G, 2.5~45%G, 2.5~40%G, 2.5~35%G, 2.5~30%G, 2.5~25%G, 2.5~20%G, 2.5~15%G, 2.5~10%G, 2.5~7.5%G, 2 .5~5%G, 5~50%G, 5~45%G, 5~40%G, 5~35%G, 5~30%G, 5~25%G, 5~20%G, 5~15%G, 5~10%G, 5~7.5%G, 7.5~50%G, 7.5~45% G, 7.5~40%G, 7.5~35%G, 7.5~30%G, 7.5~25%G, 7.5~20%G, 7.5~15%G, 7.5~10%G, 10~50%G, 10~45%G, 10~40%G, 10~3 5%G, 10~30%G, 10~25%G, 10~20%G, 10~15%G, 10~12.5%G, 12.5~50%G, 12.5~45%G, 12.5~40%G, 12.5~35%G, 12.5~30 %G, 12.5-25%G, 12.5-20%G, 12.5-15%G, 15-50%G, 15-45%G, 15-40%G, 15-35%G, 15-30%G, 15-25%G, 15-20%G, 20-50%G, 20-45%G, 20-40%G, 20-35%G, 20-30%G, or 20-25%G."A composition consisting of or consisting essentially of unique oligonucleotide species each having a barcode region G content less than ..." means that each or essentially each of the unique oligonucleotide species in the composition, population, or pool has a molecular barcode G content less than the indicated value or outside the indicated range. That is, in a composition, population, or pool that "consists essentially of" unique oligonucleotide species each having an indicated G content, an analytically insignificant portion of the unique oligonucleotides in the composition may have a barcode region with a G content greater than the indicated value or outside the recited range. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 1000 unique oligonucleotides comprise a barcode region with a G content greater than 50%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 50%, including the ranges between any two of the recited values. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 6500 unique oligonucleotides comprise a barcode region having a G content greater than 50%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 50%, including the ranges between any two of the recited values. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 65,000 unique oligonucleotides comprise a barcode region having a G content greater than 50%.For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 50%, including the range between any two of the recited values. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 1000 unique oligonucleotides comprise a barcode region having a G content greater than 25%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 25%, including the ranges between any two of the recited values. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 6500 unique oligonucleotides comprise a barcode region having a G content greater than 25%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 25%, including the ranges between any two of the recited values. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 65,000 unique oligonucleotides comprise a barcode region having a G content greater than 25%. For example, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than 0.0001% of the unique oligonucleotide species have a G content greater than 25%, including the range between any two of the recited values. Optionally, none of the barcode regions of the unique oligonucleotide species of the composition as a whole has a G content greater than 50% G.For example, all barcode regions of unique oligonucleotide species have a G content of less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, including ranges between any two of the listed values.

[0050] In some embodiments, the composition (or such composition used in the method) consists of or consists essentially of unique oligonucleotide species each having a molecular barcode that includes a sequence of at least three repeats, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the listed values, of the doublet "HN" (wherein each "H" is either A, C, or T, and "N" is either A, G, C, or T). Examples of multiple repeats of the doublet "HN" include HN, HNHN, HNHNHN, and HNHNHNHN. Note that while the formula "HN" describes a constraint on base content, not all H or all N need to be identical or different. For example, if the molecular barcodes of the unique oligonucleotide species in a composition include HNHNHN, one molecular barcode may include the sequence ACTGCA, while another may include the sequence TAACTA, and another may include the sequence AGACAC. Note that the doublet "HN" of any number of repeats may have a G content of 50% or less. In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise a molecular barcode that includes at least 3 repeats of the doublet "HN", e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, including the ranges between any two of the recited values. In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise a molecular barcode that comprises at least 3 repeats of the doublet "HN", for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the recited values.In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise a molecular barcode that comprises at least 3 repeats of the doublet "HN", e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the enumerated values. In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise a molecular barcode that comprises at least 3 repeats of the doublet "HN", e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the enumerated values. In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise a molecular barcode that comprises at least 3 repeats of the doublet "HN", e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the enumerated values. In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise a molecular barcode that comprises at least 3 repeats of the doublet "HN", e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the enumerated values. In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise a molecular barcode that comprises at least 3 repeats of the doublet "HN", e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the recited values.In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise a molecular barcode that comprises at least 3 repeats of the doublet "HN", e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the enumerated values. In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise a molecular barcode that comprises at least 3 repeats of the doublet "HN", e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, inclusive of the ranges between any two of the enumerated values. In some embodiments, the composition consists of or consists essentially of at least 1000, 6500, or 65,000 unique oligonucleotide species, each having a molecular barcode comprising the sequence HNHNHN. In some embodiments, the composition consists of or consists essentially of at least 1000, 6500, or 65,000 unique oligonucleotide species, each having a molecular barcode comprising the sequence HNHNHNHN. In some embodiments, at least 95%, 99%, or 99.9% of the barcode regions of the compositions described herein comprise at least three repeats of the doublet "HN" as described herein. Without being limited by any theory, it is noted that a relatively large number of available nucleotide sequences of molecular barcodes can be useful in barcoding a population of target nucleic acids from a sample, for example, to increase the diversity of barcodes within a given sequence length along with the likelihood that each target nucleic acid will be uniquely labeled while minimizing the size of the oligonucleotide species. It should be noted that limiting the G content of molecular barcodes and / or barcode regions may limit the diversity of such barcodes and barcode regions as it reduces the number of available nucleotides from which barcodes can be constructed (and the number of available different sequences per length of nucleic acid).Thus, having some Gs in the molecular barcode or barcode region according to various embodiments herein can help to increase diversity, while limiting the G content can help to minimize bias. It is noted that sequences containing repeats of "HN" doublets can be observed to provide low bias, providing a compromise between reducing bias and maintaining a relatively large number of available nucleotide sequences, such that relatively high diversity can be obtained in relatively short sequences with minimal bias (see Example 2 and Figures 6-7). In some embodiments, methods or compositions comprising unique oligonucleotide species comprising molecular barcodes containing repeats of "HN" doublets as described herein reduce bias by increasing sensitivity, by reducing the relative standard error, or by increasing sensitivity and reducing the standard error.

[0051] In some embodiments, a composition (or such composition used in a method) comprises, consists of, or consists essentially of unique oligonucleotide species each comprising a molecular barcode that comprises at least 6 consecutive "H" (wherein each "H" is either A, C, or T), e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the listed values. Note that the formula "H" describes a constraint on base content, but not all H need to be identical or different. For example, if the molecular barcodes of the unique oligonucleotide species in a population each comprised the sequence HHHH, then one of the molecular barcodes of the unique oligonucleotide species could comprise ACTA, while one of the molecular barcodes of the other unique oligonucleotide species could comprise TTAC, and one of the molecular barcodes of the other unique oligonucleotide species could comprise ACAT. In some embodiments, a composition comprises, consists of, or consists essentially of at least 1000 unique oligonucleotide species, at least 95% of which comprise a molecular barcode that includes at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values. In some embodiments, a composition comprises, consists of, or consists essentially of at least 1000 unique oligonucleotide species, at least 99% of which comprise a molecular barcode that includes at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values.In some embodiments, a composition comprises, consists of, or consists essentially of at least 1000 unique oligonucleotide species, at least 99.9% of these unique oligonucleotide species comprise a molecular barcode that includes at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values. In some embodiments, a composition comprises, consists of, or consists essentially of at least 6500 unique oligonucleotide species, at least 95% of these unique oligonucleotide species comprise a molecular barcode that includes at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values. In some embodiments, a composition comprises, consists of, or consists essentially of at least 6500 unique oligonucleotide species, at least 99% of which comprise molecular barcodes that include at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values. In some embodiments, a composition comprises, consists of, or consists essentially of at least 6500 unique oligonucleotide species, at least 99.9% of which comprise molecular barcodes that include at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values.In some embodiments, a composition comprises, consists of, or consists essentially of at least 65,000 unique oligonucleotide species, at least 95% of which comprise molecular barcodes that include at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values. In some embodiments, a composition comprises, consists of, or consists essentially of at least 65,000 unique oligonucleotide species, at least 99% of which comprise molecular barcodes that include at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values. In some embodiments, a composition comprises, consists of, or consists essentially of at least 65,000 unique oligonucleotide species, and at least 99.9% of these unique oligonucleotide species comprise a molecular barcode that comprises at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of any two of the ranges between the enumerated values. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise a sequence of at least 6 alternating H and N occurrences in total, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 alternating H and N occurrences inclusive of any two of the enumerated values. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise the sequence HNHNHNHN. wherein each "H" is any one of A, C, or T, and each "N" is any one of A, G, C, or T. In some embodiments, each molecular barcode of a unique oligonucleotide species comprises the sequence HNHNHNHN.wherein each "H" is A, C, or T, and each "N" is A, G, C, or T. In some embodiments, each molecular barcode of a unique oligonucleotide species comprises the sequence HHHHHHHH, wherein each "H" is A, C, or T.

[0052] In some embodiments, at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or more) of the unique oligonucleotide species of a composition described herein (or such composition used in a method) comprise a barcode region that includes at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values ​​described herein. In some embodiments, at least 99% of the unique oligonucleotide species of a composition described herein comprise a barcode region that includes at least 6 consecutive H, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, inclusive of the ranges between any two of the enumerated values ​​described herein. In some embodiments, at least 99.9% of the unique oligonucleotide species of the compositions described herein comprise a barcode region that includes at least 6 consecutive Hs, for example, at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive Hs, inclusive of the ranges between any two of the recited values ​​described herein.

[0053] In some embodiments, the sample barcodes of each unique oligonucleotide species have a G content of 50% or less, e.g., 50% or less, 40% or less, 25% or less, 20% or less, 12.5% ​​or less, 10% or less, or 5% or less, inclusive of the ranges between any two of the listed values.

[0054] In some embodiments, the barcode region of each unique oligonucleotide species has a G content of 50% or less, e.g., 50% or less, 40% or less, 25% or less, 20% or less, 12.5% ​​or less, 10% or less, or 5% or less, inclusive of ranges between any two of the recited values.

[0055] In some embodiments, the molecular barcodes of the unique oligonucleotide species have an overall G content of less than 12.5%, e.g., less than 12.5%, less than 10%, less than 7.5%, less than 5%, less than 2.5%, or less than 1%, inclusive of the ranges between any two of the recited values.

[0056] In some embodiments, the barcode region of a unique oligonucleotide species has an overall G content of less than 12.5%, e.g., less than 12.5%, less than 10%, less than 7.5%, less than 5%, less than 2.5%, or less than 1%, including ranges between any two of the recited values.

[0057] In some embodiments, at least 95% of the unique oligonucleotide species have no Gs in their molecular barcodes adjacent to other Gs. In some embodiments, at least 99% of the unique oligonucleotide species have no Gs in their molecular barcodes adjacent to other Gs. In some embodiments, all or substantially all of the unique oligonucleotide species have no Gs in their molecular barcodes adjacent to other Gs.

[0058] Each barcode region may optionally include a sample barcode. According to the compositions, methods, and oligonucleotides of some embodiments herein, each unique oligonucleotide species in a pool may include the same sample barcode, but there may be two or more pools each associated with a different sample barcode. Thus, all or essentially all of the unique oligonucleotide species in pool #1 may include sample barcode #1, and all or essentially all of the unique oligonucleotide species in pool #2 may include sample barcode #2. For example, by hybridization and amplification, nucleic acids from a first sample may be associated with unique oligonucleotide species in pool #1, and nucleic acids from a second sample may be associated with unique oligonucleotide species in pool #2. Thus, all or essentially all of the amplified nucleic acids corresponding to the first sample will include sample barcode #1 (but may include different molecular barcodes). And all of the amplified nucleic acids corresponding to the second sample will include sample barcode #2. In some embodiments, there are at least 24, 48, 96, or 192 pools.

[0059] A sample barcode may be at least 3 nucleotides, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides, including the range between any two of the enumerated values, e.g., 3 to 5 nucleotides. 50, 3~45, 3~40, 3~35, 3~30, 3~25, 3~20, 3~15, 3~14, 3~13, 3~12, 3~11, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~5, 3~4, 4~50, 4~45, 4~40, 4~35, 4~30, 4~25, 4~20, 4~15, 4~14, 4~13, 4~12, 4~11, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~50, 5~45, 5~40, 5~35, 5~30, 5~25, 5~20 , 5~15, 5~14, 5~13, 5~12, 5~11, 5~10, 5~9, 5~8, 5~7, 5~6, 6~50, 6~45, 6~40, 6~35, 6~30, 6~25, 6~20, 6~15, 6~14, 6~13, 6~12, 6~11, 6~10, 6~9, 6~8, 6~7, 7~50, 7~45, 7~40, 7~35, 7~30, 7~25, 7~20, 7~15, 7~14, 7~13, 7~12, 7~11, 7~10, 7~9, 7~8, 8~50, 8~4 The nucleic acid sequence of the sample barcode may comprise 0, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides. In some embodiments, the nucleic acid sequence of the sample barcode comprises a unique sequence, e.g., such that each unique oligonucleotide species in the population comprises a different molecular barcode.

[0060] Without being limited by any theory, it is contemplated that a sample barcode with low G content (e.g., less than 50% G, e.g., less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G) can be positioned 3' to a sample barcode with a relatively high G content and 5' to a uniform region (e.g., a target-specific sequence or an oligo dT sequence) to minimize bias by separating the relatively G-rich sample barcode from the uniform region. In some embodiments, the barcode region is 50% G or less, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, e.g., between 2.5 and 50% G, 2.5~45%G, 2.5~40%G, 2.5~35%G, 2.5~30%G, 2.5~25%G, 2.5~20%G, 2.5~15%G, 2.5~10%G, 2.5~7.5%G, 2.5~ 5%G, 5~50%G, 5~45%G, 5~40%G, 5~35%G, 5~30%G, 5~25%G, 5~20%G, 5~15%G, 5~10%G, 5~7.5%G, 7.5~50%G, 7.5 ~45%G, 7.5~40%G, 7.5~35%G, 7.5~30%G, 7.5~25%G, 7.5~20%G, 7.5~15%G, 7.5~10%G, 10~50%G, 10~45%G, 1 0~40%G, 10~35%G, 10~30%G, 10~25%G, 10~20%G, 10~15%G, 10~12.5%G, 12.5~50%G, 12.5~45%G, 12.5~40%G, Include sample barcodes with the following content: 12.5–35% G, 12.5–30% G, 12.5–25% G, 12.5–20% G, 12.5–15% G, 15–50% G, 15–45% G, 15–40% G, 15–35% G, 15–30% G, 15–25% G, 15–20% G, 20–50% G, 20–45% G, 20–40% G, 20–35% G, 20–30% G, or 20–25% G.

[0061] In some embodiments, in a composition comprising a unique oligonucleotide species (or such a composition used in a method), at least 95% of the sample barcodes of the unique oligonucleotides of the composition are less than 50% G, e.g., less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 50 ...40% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 50% G, less than 40% G, less than 40% G Less than 10%G, less than 7.5%G, less than 5%G, or less than 2.5%G, or 0%G, for example, 2.5-50%G, 2.5-45%G, 2.5-40%G, 2.5-35%G, 2.5-30%G, 2.5-25%G, 2.5-20%G, 2.5-15%G, 2.5-10%G, 2.5-7.5%G, 2.5-5%G, 5-50%G, 5-45%G, 5-40%G, 5-35%G, 5-30%G, 5-25%G, 5- 20%G, 5~15%G, 5~10%G, 5~7.5%G, 7.5~50%G, 7.5~45%G, 7.5~40%G, 7.5~35%G, 7.5~30%G, 7.5~25%G, 7.5~20%G, 7.5~ 15%G, 7.5~10%G, 10~50%G, 10~45%G, 10~40%G, 10~35%G, 10~30%G, 10~25%G, 10~20%G, 10~15%G, 10~12.5%G, 12.5~5 With a content of 0%G, 12.5-45%G, 12.5-40%G, 12.5-35%G, 12.5-30%G, 12.5-25%G, 12.5-20%G, 12.5-15%G, 15-50%G, 15-45%G, 15-40%G, 15-35%G, 15-30%G, 15-25%G, 15-20%G, 20-50%G, 20-45%G, 20-40%G, 20-35%G, 20-30%G, or 20-25%G.

[0062] In some embodiments, in a composition comprising a unique oligonucleotide species (or such a composition used in a method), at least 99% of the sample barcodes of the unique oligonucleotides of the composition are 50% G or less, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5%, inclusive of any range between any two of the recited values. %G, less than 10%G, less than 7.5%G, less than 5%G, or less than 2.5%G, or 0%G, e.g., 2.5-50%G, 2.5-45%G, 2.5-40%G, 2.5-35%G, 2.5-30%G, 2.5-25%G, 2.5-20%G, 2.5-15%G, 2.5-10%G, 2.5-7.5%G, 2.5-5%G, 5-50%G, 5-45%G, 5-40%G, 5-35%G, 5-30%G, 5-25%G , 5~20%G, 5~15%G, 5~10%G, 5~7.5%G, 7.5~50%G, 7.5~45%G, 7.5~40%G, 7.5~35%G, 7.5~30%G, 7.5~25%G, 7.5~20%G, 7. 5~15%G, 7.5~10%G, 10~50%G, 10~45%G, 10~40%G, 10~35%G, 10~30%G, 10~25%G, 10~20%G, 10~15%G, 10~12.5%G, 12.5~ have a G content of 50% G, 12.5-45% G, 12.5-40% G, 12.5-35% G, 12.5-30% G, 12.5-25% G, 12.5-20% G, 12.5-15% G, 15-50% G, 15-45% G, 15-40% G, 15-35% G, 15-30% G, 15-25% G, 15-20% G, 20-50% G, 20-45% G, 20-40% G, 20-35% G, 20-30% G, or 20-25% G. That is, within the unique oligonucleotide population, less than 1% of the sample barcodes have a G content greater than 50%. In some embodiments, the unique oligonucleotide species of the composition consist of or consist essentially of unique oligonucleotide species, each having a sample barcode with less than 50% G content, as described herein.

[0063] In some embodiments, the barcode region is positioned 5' to the uniform region without any intervening sequence between the barcode region and the uniform region. In some embodiments, the barcode region is positioned 5' to a spacer that is positioned 5' to the uniform region such that the spacer is between the barcode region and the uniform region. Optionally, the spacer has a low G content (e.g., 50% G or less, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G). A spacer may be at least one nucleotide, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 nucleotides, 1-50, 1-45, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-9, 1-8, 1- 7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-45, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-50, 3-45, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-50, 4-45, 4-35, 4- 30, 4~25, 4~20, 4~15, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~50, 5~45, 5~35, 5~30, 5~25, 5~20, 5~15, 5~10, 5~9, 5~8, 5~7, 5~6, 6~50, 6~45, 6~35, 6~30, 6~25, 6~20, 6~15, 6~10, 6~9, 6~8, 6~7, 7~50, 7~45, 7~35, 7~30, It may have a length of 7 to 25, 7 to 20, 7 to 15, 7 to 10, 7 to 9, 7 to 8, 8 to 50, 8 to 45, 8 to 35, 8 to 30, 8 to 25, 8 to 20, 8 to 15, 8 to 10, 8 to 9, 9 to 50, 9 to 45, 9 to 35, 9 to 30, 9 to 25, 9 to 20, 9 to 15, 9 to 10, 10 to 50, 10 to 45, 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 nucleotides.In some embodiments, the spacer is a spacer that has at least two consecutive non-G nucleotides (represented by "H" (where "H" is A, C, or T)), e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, e.g., 2-20, 2-10, 3-30, 4-40, 5-50, 6-60, 7-80, 8-90, 9-100, 10-110, 11-120, 13-140, 14-150, 15-160, 17-180, 18-200, 19-200, 20-200, 21-220, 22-240, 23-240, 24-260, 25-260, 26-280, 27-290, 28-300, 29-400, 30-400, 31-400, 32-400, 33-400, 34-400, 35-400, 36-400, 37-400, 38-400, 39-500, 41-410, 42-420, 43-430, 44-440, 45-450, 46-460, 47-470, 48-480, 49-500, 50-500, 51-520, 53-540, 54-550, 5, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-15, 6-10, 6-9, 6-8, or 6-7. In some embodiments, the spacer nucleotide is T. For example, the spacer may include a poly-T sequence such as TT, TTT, TTTT, TTTTT, TTTTTT, TTTTTTTT, TTTTTTTTTT. It should be noted that spacers do not necessarily provide diversity, so that in compositions comprising unique oligonucleotide species as described herein, some or all of the unique oligonucleotide species may have the same spacer sequence. Optionally, all of the unique oligonucleotide species of a composition include the same spacer sequence.

[0064] Pools and Pooling Compositions comprising unique oligonucleotide species as described according to the compositions and methods of some embodiments herein can be arranged in spatially separated pools, for example, to allow for the analysis of multiple samples with one sample / pool. In some embodiments, the unique oligonucleotide species are arranged in spatially separated pools, each pool comprising multiple unique oligonucleotides of the unique oligonucleotide species, such that the unique oligonucleotides of the same pool comprise the same sample barcode sequence, and different unique oligonucleotides of the same pool comprise different molecular barcode sequences. As used herein, "spatial separation" (and variations of this root word) means that the target nucleic acid of a sample can hybridize to the unique oligonucleotide species of a pool without substantially cross-reacting with other pools and without the unique oligonucleotide species of the pool substantially hybridizing to the target nucleic acid of other samples. Thus, the sample barcode can identify which pool a given unique oligonucleotide species originated from. Furthermore, by barcoding the target nucleic acid sequence with the unique oligonucleotide species, the sample barcode can identify which pool the barcoded target nucleic acid sequence (or its reverse transcript or amplicon) originated from.

[0065] In some embodiments, the substrate organizes the pools so that they are spatially separated from one another. For example, a multiwell plate can organize the pools so that each pool is in a separate well. For example, each pool can be immobilized on a different bead. Optionally, each well of the multiwell plate can contain a single bead to which a pool of unique oligonucleotide species is immobilized, such that a different pool of unique oligonucleotide species is positioned in each well of the multiwell plate.

[0066] In some embodiments, the unique oligonucleotide species are arranged in at least 2 pools, e.g., at least 2, 3, 4, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 72, 96, 99, 100, 110, 120, 144, 168, or 192 pools, including the ranges between any two of the recited values. Optionally, there may be at least 100 unique oligonucleotides per pool.

[0067] In some embodiments, the unique oligonucleotide species are arranged in at least 2 pools and there are at least 100 unique oligonucleotides / pool, e.g., at least 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 6500, 10,000, or 65,000 unique oligonucleotide species / pool.

[0068] In some embodiments, the unique oligonucleotide species are arranged in at least 24 pools and there are at least 100 unique oligonucleotides / pool, e.g., at least 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 6500, 10,000, or 65,000 unique oligonucleotide species / pool.

[0069] In some embodiments, the unique oligonucleotide species are arranged in at least 48 pools and there are at least 100 unique oligonucleotides / pool, e.g., at least 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 6500, 10,000, or 65,000 unique oligonucleotide species / pool.

[0070] In some embodiments, the unique oligonucleotide species are arranged in at least 96 pools and there are at least 100 unique oligonucleotides / pool, e.g., at least 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 6500, 10,000, or 65,000 unique oligonucleotide species / pool.

[0071] Base material The unique oligonucleotide species according to the compositions and methods of some embodiments herein can be immobilized on a substrate such as a bead, a well of a multi-well plate, an array, etc. For example, all unique oligonucleotide species with the same sample barcode but different molecular barcodes can be immobilized on a substrate such as a single bead or a single well of a multi-well plate. Thus, when a unique oligonucleotide species immobilized on a particular substrate is contacted with a particular sample such as a single cell, the unique oligonucleotide species immobilized on the substrate will be associated with the target nucleic acid of the same sample. If the unique oligonucleotide species immobilized on the same substrate have the same sample barcode while those on other substrates have different sample barcodes, the sample associated with each target nucleic acid can be easily identified (e.g., all unique oligonucleotide species on substrate #1 have sample barcode #1, and all unique oligonucleotide species on substrate #2 have sample barcode #2).

[0072] In some embodiments, such configurations of immobilizing unique oligonucleotide species on a substrate can help improve efficiency and throughput. For example, single cells can be added to wells of a multi-well plate such that no more than one single cell is present in each well. If the unique oligonucleotide species immobilized in a given well (e.g., on a well or on a bead) contains a unique sample barcode, the barcoded target nucleic acid corresponding to a single cell and its reverse transcript and amplicon can be identified and quantified, even if the barcoded reverse transcript or amplicon from multiple samples are pooled.

[0073] Substrates can include any type of solid support. Substrates can refer to a continuous solid or semi-solid surface on which the disclosed method can be performed. Substrates can refer to, for example, arrays, cartridges, chips, devices, and slides. Thus, "solid support" and "substrate" can be used interchangeably.

[0074] The substrate or solid support according to some embodiments herein may include any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar structure composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) to which nucleic acid can be immobilized (e.g., covalently or non-covalently). The substrate or solid support may include discrete particles that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, e.g., cuboid, rectangular, pyramidal, cylindrical, conical, oblate spheroid, disk, etc. A plurality of solid supports spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "beads."

[0075] In some embodiments, multiple substrates can be provided to present a diversity of sample barcodes across the multiple substrates such that unique substrates can include unique sample barcodes.

[0076] Barcoding Methods According to some embodiments herein, a method is described for specifically barcoding nucleic acids from two or more samples. Each sample may comprise a nucleic acid. The method may include contacting each sample with a pool comprising a plurality of unique oligonucleotide species as described herein. Each sample may be contacted with spatial separation from the other samples. The unique polynucleotide species of each pool may comprise the same sample barcode and may comprise different molecular barcodes. The method may include hybridizing target specific regions of at least some of the oligonucleotides of the unique oligonucleotide species to at least some of the nucleic acids of the sample. The method may include extending the hybridized oligonucleotides to generate strands comprising oligonucleotides of the unique oligonucleotide species and sequences complementary to the target regions. Thus, for each sample, the generated strands may comprise the same sample barcode and different molecular barcodes. For different samples, the sample barcodes may be different. In some embodiments, the unique oligonucleotide species contacted with the sample consist of or consist essentially of unique oligonucleotide species, each molecular barcode having less than 50% G content. In some embodiments, the molecular barcodes of all the unique oligonucleotide species collectively have a G content of 12.5% ​​or less. Optionally, each pool comprises at least 100 unique oligonucleotide species, e.g., at least 100, 500, 100, 500, 1000, 2000, 6500, or 65,000 unique oligonucleotide species. Such methods according to some embodiments herein are capable of barcoding nucleic acids of two or more different samples, e.g., single cell samples, to allow analysis and quantification of the nucleic acids of single cells.

[0077] Optionally, each sample is contacted with a single pool of unique oligonucleotide species in a spatially separated manner from the other pools and samples. In some embodiments, the pools are spatially separated by being placed in unique confined spaces, e.g., different wells of a multi-well plate, different test tubes, different microfluidic channels, etc. In some embodiments, the pools are spatially separated by being placed on different surface regions, e.g., reaction dots on an array.

[0078] The oligonucleotides may be contacted with the target nucleic acid of the sample under standard hybridization conditions (e.g., in a standard buffer and at a temperature below the Tm of the portion of the uniform region that is complementary to the target nucleic acid). Optionally, for example, if the target nucleic acid comprises RNA, the target nucleic acid of the sample is reverse transcribed after hybridization to the unique oligonucleotide species to generate DNA (e.g., cDNA). Optionally, the products of the hybridization or reverse transcription reaction are amplified to generate a library of DNA. Amplification may be performed by PCR under standard conditions or by other suitable methods, e.g., isothermal amplification, rolling circle amplification, etc. Optionally, amplification may include amplifying the reverse transcripts or hybridized nucleic acids with a polymerase having 5'→3' activity. Optionally, the amplification products can be further analyzed, for example, by sequencing. Optionally, reverse transcripts or amplicons from different pools can be pooled for sequencing, since different sample barcodes represent corresponding pools (and samples) of each individual barcoded nucleic acid. It should be noted that there may be a bias in favor of G-rich molecular barcodes or unique oligonucleotide species, and that the methods of some embodiments herein may minimize or eliminate such bias. As an example, when quantifying nucleic acids of a sample at the single nucleic acid level, it may be useful to minimize or eliminate such bias that favors the representation of some nucleic acids and may skew the quantification results in favor of certain types of barcodes rather than the representative target nucleic acid of the sample. In some embodiments, the methods comprising the unique oligonucleotide species described herein reduce bias by increasing sensitivity, decreasing the relative standard error, or increasing sensitivity and decreasing the standard error.

[0079] Optionally, the method may further comprise ascertaining the nucleic acid sequence of the strand comprising the oligonucleotide of the unique oligonucleotide species and the sequence complementary to the target. For example, the reverse transcriptase library, amplicon library, and / or cDNA library generated from hybridization and extension of the unique oligonucleotide probes may be sequenced. Any suitable sequencing method known in the art may be used, preferably a high-throughput method. For example, cyclic array sequencing may be utilized using platforms such as Roche454, Illumina Solexa, ABI-SOLiD, ION Torrent, Complete Genomics, Pacific Bioscience, Helicos, Polonator platforms, etc. Sequencing may include MiSeq sequencing. Sequencing may include HiSeq sequencing. If applicable, the unique oligonucleotide species may include an adaptor, e.g., a universal priming site, that mediates high-throughput sequencing. Optionally, barcoded nucleic acids corresponding to two or more different samples (e.g., from two or more different pools) may be pooled or combined for sequencing. Optionally, all of the pools can be combined or pooled for sequencing. Without being limited by any theory, it is noted that sample barcodes can identify corresponding samples (or pools) of combined barcoded nucleic acids, combining or pooling nucleic acids can improve sequencing throughput and / or resource utilization, and accurate barcoding according to some embodiments herein can minimize or eliminate bias even when analyzing large numbers of different nucleic acids.

[0080] In some embodiments, the barcode region of each pool comprises a sample barcode comprising at least 3 nucleotides as described herein, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, including the range between any two of the listed values. Optionally, the sample barcode is positioned 3' to the molecular barcode. Optionally, the molecular barcode is positioned 3' to the sample barcode. Optionally, the unique oligonucleotide species comprises the same sample barcode, which may be a different sample barcode from the unique oligonucleotide species of other pools. Optionally, the sample barcodes have a low G content as described herein, e.g., less than 50% G, e.g., less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G. Optionally, at least 95%, 99%, or 99.9% of the sample barcodes have a low G content as described herein. Optionally, each unique oligonucleotide comprises a spacer 3' to the molecular barcode and 5' to the uniform region as described herein.

[0081] In some embodiments, the unique oligonucleotide species contacted with the sample consists essentially of unique oligonucleotide species, each molecular barcode having less than 50% G content as described herein, e.g., less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G. In some embodiments, the molecular barcodes of at least 95% of the unique oligonucleotide species contacted with the sample comprise molecular barcodes having less than 50% G content as described herein, e.g., less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G content. In some embodiments, at least 99% of the molecular barcodes of the unique oligonucleotide species contacted with the sample comprise molecular barcodes having less than 50% G content as described herein, e.g., less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or a content of 0% G. In some embodiments, all of the molecular barcodes of the unique oligonucleotide species contacted with the sample comprise molecular barcodes having less than 50% G content as described herein, e.g., less than 45% G, less than 40% G, 35% G, 30% G, 25% G, 20% G, 15% G, 12.5% ​​G, 10% G, 7.5% G, 5% G, or 2.5% G, or a content of 0% G.

[0082] In some embodiments, the molecular barcodes of all unique oligonucleotide species have an overall G content of less than 50% G, e.g., less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, inclusive of ranges between any two of the recited values. In some embodiments, the molecular barcodes of all unique oligonucleotide species have an overall G content of 12.5% ​​or less.

[0083] In some embodiments, each unique oligonucleotide species further comprises a uniform region 3' to the barcode region. Optionally, the uniform region comprises at least 10 nucleotides complementary to the target nucleic acid sequence (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides complementary to the target, including the range between any two of the listed values), and such nucleotides are configured for 5'→3' amplification of the target nucleic acid sequence. Optionally, the uniform region comprises an oligo dT sequence.

[0084] In some embodiments, the unique oligonucleotide species of each pool are immobilized to a substrate such that the unique oligonucleotide species immobilized to a given substrate contain the same sample barcode and the different unique oligonucleotide species immobilized to the substrate contain different molecular barcodes. By way of example, the substrate may include wells of a multi-well plate (e.g., 24, 48, and 96 well plate), spots on an array, beads, etc. In some embodiments, at least 100 unique oligonucleotides are immobilized to each substrate. Optionally, all unique oligonucleotide species immobilized to a given substrate (and thus the same pool) contain the same sample barcode. In some embodiments, each sample barcode has a G content of 50% or less, e.g., 50% or less, 40% or less, 25% or less, 20% or less, 12.5% ​​or less, 10% or less, or 5% or less.

[0085] In some embodiments, the molecular barcodes of the unique oligonucleotide species have an overall G content of less than 12.5%, e.g., less than 12.5%, less than 10%, less than 7.5%, less than 5%, less than 2.5%, or less than 1%.

[0086] In some embodiments, the barcode region of a unique oligonucleotide species has an overall G content of 12.5% ​​or less, e.g., 12.5% ​​or less, 10% or less, 7.5% or less, 5% or less, 2.5% or less, or 1% or less.

[0087] In some embodiments, at least 95% of the unique oligonucleotide species do not have any G in their molecular barcode adjacent to another G. For example, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the unique oligonucleotide species do not have any G in their molecular barcode adjacent to another G.

[0088] In some embodiments, each pool consists of or consists essentially of unique oligonucleotide species in which no G in the molecular barcode is adjacent to any other G.

[0089] In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise a total of at least 6 alternating H and N occurrences, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 alternating H and N occurrences, including the ranges between any two of the listed values. By way of example, 6 alternating H and N occurrences may be represented as HNHNHN. Note that each H may be the same or different from any other H, and each N may be the same or different from any other N.

[0090] In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise the sequence HNHNHNHN, where each "H" is any one of A, C, or T, and each "N" is any one of A, G, C, or T. For example, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the molecular barcodes of the unique oligonucleotide species may comprise the sequence HNHNHNHN. Note that each H may be the same or different from any other H, and each N may be the same or different from any other N. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN.

[0091] In some embodiments, at least 99% of the molecular barcodes of a unique oligonucleotide species comprise the sequence HHHHHHHH, where each "H" is one of A, C, or T. For example, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the molecular barcodes of a unique oligonucleotide species may comprise the sequence HHHHHHHH. Note that each H may be the same or different from any other H. In some embodiments, each molecular barcode of a unique oligonucleotide species comprises the sequence HHHHHHHH.

[0092] In some embodiments, each unique oligonucleotide comprises a spacer 3' to the barcode region and 5' to the target specific region. Optionally, the spacer may comprise the sequence HNH, where each "H" is A, C, or T, and each "N" is A, G, C, or T. Optionally, the spacer may comprise the sequence HHH. Optionally, the spacer may comprise the sequence HNHNHNHN. Optionally, the spacer may comprise the sequence HHHHHHHH. Note that each H may be the same or different from any other H, and each N may be the same or different from any other N. In some embodiments, each unique oligonucleotide comprises a spacer 3' to the barcode region and 5' to the target specific region, where the spacer comprises the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T. In some embodiments, the unique oligonucleotide comprises a spacer 3' to the barcode region and 5' to the target specific region, said spacer comprising the sequence HHHHHHHH, where each "H" is one of A, C, or T.

[0093] In some embodiments, at least one pool comprises at least two oligonucleotides of the same unique oligonucleotide species. In some embodiments, a pool does not comprise two oligonucleotides of the same unique oligonucleotide species.

[0094] In some embodiments, the target specific region may comprise a sequence for 5' to 3' amplification of the target nucleic acid. In some embodiments, the target specific region comprises an oligo dT sequence. In some embodiments, the target specific region comprises a sequence flanking a sequence encoding an immune cell receptor or an immunoglobulin variable region.

[0095] In some embodiments, each unique oligonucleotide species has a molecular barcode 3' to the sample barcode. In some embodiments, each unique oligonucleotide species has a sample barcode 3' to the molecular barcode.

[0096] In some embodiments, each unique oligonucleotide species has a length of at least 24 nucleotides, hi some embodiments, each unique oligonucleotide species has a length of between 24 and 140 nucleotides.

[0097] In some embodiments, each pool contains at least 1000 unique oligonucleotide species. In some embodiments, each pool contains at least 6,500 unique oligonucleotide species. In some embodiments, each pool contains at least 65,000 unique oligonucleotide species.

[0098] In some embodiments, at least 48 unique samples are each contacted with a different pool of unique oligonucleotide species. For example, at least 48, 72, 96, 120, 144, 168, or 192 samples may each be contacted with a different pool. For example, each sample may be contacted with a different pool of unique oligonucleotide species in the wells of a multi-well plate.

[0099] In some embodiments, at least 99% of the samples each have less than or equal to 1 cell. For example, at least 99%, 99.5%, 99.9%, or 99.99% of the samples can have less than or equal to 1 cell. By way of example, a solution containing multiple cells can be diluted to an appropriate concentration such that each sample is suitably likely to have less than or equal to 1 cell.

[0100] In some embodiments, the unique oligonucleotide species of each pool are immobilized to a substrate such that the sample barcode, but not the molecular barcode, of the unique oligonucleotide species immobilized to each substrate is the same. Thus, each pool can be identified by a specific sample barcode. Note that different substrates (and thus different pools) can be associated with different sample barcodes. In some embodiments, the substrate comprises spatially separated surface regions such that substrates of different pools comprise different spatially separated surface regions. In some embodiments, the substrate comprises a well of a multi-well plate. In some embodiments, the substrate comprises a bead.

[0101] In some embodiments, the unique oligonucleotide species further comprises an adapter configured to immobilize the unique oligonucleotide to a substrate, and the barcode region is 3' to the adapter. Optionally, each adapter can include a universal priming site, e.g., for use in sequencing.

[0102] Methods for Producing Compositions Comprising Unique Oligonucleotide Species According to some embodiments herein, a method of making a composition comprising unique oligonucleotides is described. The method may include providing a plurality of different sample barcodes as described herein. The method may include providing a plurality of different molecular barcodes as described herein. The method may include synthesizing a plurality of unique oligonucleotide species as described herein, where each unique oligonucleotide species comprises a barcode region comprising a sample barcode and a molecular barcode as described herein. The method may include disposing the plurality of unique oligonucleotide species in spatially separated pools. Each pool may comprise a plurality of unique oligonucleotide species such that unique oligonucleotide species of the same pool comprise the same sample barcode sequence, and different unique oligonucleotide species of the same pool comprise different molecular barcode sequences. Optionally, the unique oligonucleotide species are disposed in the spatially separated pools simultaneously with synthesis. Optionally, the unique oligonucleotide species are disposed in the spatially separated pools after synthesis. Optionally, the composition consists essentially of unique oligonucleotide species, each molecular barcode having less than 50% G content. Optionally, the molecular barcodes of all unique oligonucleotide species collectively have a G content of 12.5% ​​or less. Optionally, the composition consists essentially of unique oligonucleotide species, each molecular barcode having a G-content of less than 50, and the molecular barcodes of all unique oligonucleotide species collectively have a G-content of 12.5% ​​or less. In some embodiments, each spatially separated pool comprises at least 100 unique oligonucleotide species, e.g., at least 100, 200, 300, 400, 500, 1000, 2000, 3000, 5000, 6500, or 65,000 unique oligonucleotide species. In some embodiments, each spatially separated pool comprises at least 1000 unique oligonucleotide species.

[0103] The unique oligonucleotides can be synthesized using any of several suitable methods. In some embodiments, the sample barcode sequence and the molecular barcode sequence are generated in silico, and a unique oligonucleotide species containing both the sample barcode sequence and the molecular barcode sequence is synthesized, for example, using chemical oligonucleotide synthesis. In some embodiments, multiple oligonucleotides containing the same sample barcode sequence are pooled in a spatially separated state and linked, for example, by hybridization and extension or by ligation, to multiple oligonucleotides containing molecular barcodes. Such methods can be performed in multiple spatially separated environments in parallel or sequentially to obtain multiple unique oligonucleotide species.

[0104] Optionally, each unique oligonucleotide species further comprises a uniform region 3' to the barcode region as described herein. Optionally, the uniform region comprises a target specific region. In some embodiments, the target specific region comprises at least 10 nucleotides complementary to the target nucleic acid (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides complementary to the target, including the range between any two of the listed values), and is configured for 5'→3' amplification of the target nucleic acid sequence. Optionally, the uniform region comprises an oligo dT sequence.

[0105] In some embodiments, the plurality of molecular barcodes (and thus the plurality of unique oligonucleotide species) consists essentially of unique oligonucleotide species, each molecular barcode having 50% or less G content as described herein, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G. In some embodiments, the molecular barcodes of at least 95% of the unique oligonucleotide species have a G content of 50% or less as described herein, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or a content of 0% G. In some embodiments, the molecular barcodes of at least 99% of the unique oligonucleotide species have a G content of 50% or less as described herein, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or a content of 0% G. In some embodiments, all of the molecular barcodes of the unique oligonucleotide species have molecular barcodes with 50% or less G content as described herein, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or a content of 0% G. In some embodiments, each molecular barcode of each unique oligonucleotide species has 50% or less G content, e.g., less than 50% G, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or a content of 0% G.

[0106] In some embodiments, the molecular barcodes of all of the unique oligonucleotide species of the plurality of unique oligonucleotide species have a G content of 50% G or less, e.g., less than 50%, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, inclusive of ranges between any two of the listed values. In some embodiments, the molecular barcodes of all of the unique oligonucleotide species of the plurality of unique oligonucleotide species have a G content of 12.5% ​​or less.

[0107] In some embodiments, each sample barcode has a G content of 50% or less as described herein, e.g., less than 50%, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or a content of 0% G.

[0108] In some embodiments, the sample barcodes of all unique oligonucleotide species of the plurality of unique oligonucleotide species have an overall G content of 50% G or less, e.g., less than 50%, less than 45% G, less than 40% G, less than 35% G, less than 30% G, less than 25% G, less than 20% G, less than 15% G, less than 12.5% ​​G, less than 10% G, less than 7.5% G, less than 5% G, or less than 2.5% G, or 0% G, inclusive of ranges between any two of the listed values. In some embodiments, the sample barcodes of all unique oligonucleotide species of the plurality of unique oligonucleotide species have an overall G content of 12.5% ​​or less.

[0109] In some embodiments, at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99%) of the unique oligonucleotide species have no Gs in the molecular barcodes adjacent to other Gs. In some embodiments, the plurality of unique oligonucleotide species consists essentially of unique oligonucleotide species having no Gs in the molecular barcodes adjacent to other Gs.

[0110] In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise the sequence HNHNHNHN, where each "H" is any one of A, C, or T, and each "N" is any one of A, G, C, or T. For example, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the molecular barcodes of the unique oligonucleotide species may comprise the sequence HNHNHNHN. Note that each H may be the same or different from any other H, and each N may be the same or different from any other N. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN.

[0111] In some embodiments, at least 99% of the molecular barcodes of a unique oligonucleotide species comprise a molecular barcode that comprises the sequence HHHHHHHH, where each "H" is one of A, C, or T. For example, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the molecular barcodes of a unique oligonucleotide species may comprise the sequence HHHHHHHH. Note that each H may be the same or different from any other H. In some embodiments, each molecular barcode of a unique oligonucleotide species comprises the sequence HHHHHHHH.

[0112] In some embodiments, each unique oligonucleotide species comprises a spacer 3' to the barcode region and 5' to the target specific region. Optionally, the spacer may comprise the sequence HNH, where each "H" is A, C, or T, and each "N" is A, G, C, or T. Optionally, the spacer may comprise the sequence HHH. Optionally, the spacer may comprise the sequence HNHNHNHN. Optionally, the spacer may comprise the sequence HHHHHHHH. Note that each H may be the same or different from any other H, and each N may be the same or different from any other N. In some embodiments, each unique oligonucleotide species comprises a spacer 3' to the barcode region and 5' to the target specific region, where the spacer comprises the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T. In some embodiments, the unique oligonucleotide comprises a spacer 3' to the barcode region and 5' to the target specific region, wherein the spacer comprises the sequence HHHHHHHH, where each "H" is either A, C, or T.

[0113] In some embodiments, at least one pool comprises at least two oligonucleotides of the same unique oligonucleotide species. In some embodiments, a pool does not comprise two oligonucleotides of the same unique oligonucleotide species.

[0114] In some embodiments, the target specific region may comprise a sequence for 5' to 3' amplification of the target nucleic acid. In some embodiments, the target specific region comprises an oligo dT sequence. In some embodiments, the target specific region comprises a sequence flanking a sequence encoding an immune cell receptor or an immunoglobulin variable region.

[0115] In some embodiments, each unique oligonucleotide species has a molecular barcode 3' to the sample barcode. In some embodiments, each unique oligonucleotide species has a sample barcode 3' to the molecular barcode.

[0116] In some embodiments, each unique oligonucleotide species has a length of at least 24 nucleotides, hi some embodiments, each unique oligonucleotide species has a length of between 24 and 140 nucleotides.

[0117] In some embodiments, each pool contains at least 6,500 unique oligonucleotide species, hi some embodiments, each pool contains at least 65,000 unique oligonucleotide species.

[0118] In some embodiments, the method further comprises immobilizing each of the spatially distinguishable pools of unique oligonucleotide species to a substrate such that the sample barcodes, but not the molecular barcodes, of the unique oligonucleotide species immobilized to each substrate are identical. Thus, each pool is identifiable by a particular sample barcode. Note that different substrates (and thus different pools) can be associated with different sample barcodes. In some embodiments, the substrate comprises discrete surface regions such that substrates of different pools comprise different discrete surface regions. In some embodiments, the substrate comprises a well of a multi-well plate. In some embodiments, the substrate comprises a bead. In some embodiments, the unique oligonucleotide species is immobilized to the substrate by a covalent bond. In some embodiments, the unique oligonucleotide species is immobilized to the substrate by magnetic or electromagnetic forces. In some embodiments, the unique oligonucleotide species is embedded in the substrate for immobilization to the substrate.

[0119] In some embodiments, at least 48 spatially distinguishable pools are generated, for example, at least 48, 72, 96, 120, 144, 168, or 192 spatially distinguishable pools.

[0120] In some embodiments, the unique oligonucleotide species are disposed in spatially separated pools contemporaneously with said synthesis, hi some embodiments, the unique oligonucleotide species are disposed in spatially separated pools subsequent to said synthesis.

[0121] Oligonucleotides In some embodiments, oligonucleotides are described that include a barcode region 3' to the adapter region. The barcode region can include a molecular barcode as described herein, and the molecular barcode has a G content of 50% or less. The molecular barcode can include at least 7 nucleotides.

[0122] In some embodiments, the oligonucleotide further comprises a sample barcode as described herein. The sample barcode may comprise at least 3 nucleotides.

[0123] In some embodiments, the oligonucleotide further comprises a uniform region 3' to the barcode region. In some embodiments, the uniform region comprises a target specific region comprising at least 10 nucleotides complementary to the target nucleic acid as described herein. In some embodiments, the uniform region comprises an oligo dT sequence. In some embodiments, the oligonucleotide further comprises an adapter region 5' to the barcode region. In some embodiments, the oligonucleotide further comprises a spacer 3' to the barcode region and 5' to the uniform region as described herein.

[0124] Alternative Embodiments In some embodiments, a design of accurate and unbiased barcoding of nucleic acid transcripts is provided. The primer design is composed of nucleic acid sequences (DNA, RNA, or LNA) that can target a specific nucleic acid (DNA / RNA) transcript via a gene-specific method or can target a group of many nucleic acid transcripts (e.g., via the polyA tail or other consensus sequence of messenger RNA) (Figure 1A-C). Along with the target sequence, such primers contain two categories of nucleic acid barcodes: 1) molecular barcodes, also called "molecular barcodes", molecular indexes (MI), or unique molecular identifiers (UMI), and 2) sample barcodes, also called sample indexes (SI) (Figure 1A-C). The MI and SI barcodes are used to uniquely label and identify the original target transcript via MI and its sample source (SI) after library preparation for next generation sequencing or other sequence readout and barcode readout methods. In some embodiments, different arrangements of MI and SI within primers are detailed (see, for example, Figures 3A-3H) that ensure unbiased random labeling of target transcripts with barcodes for accurate gene expression profiling (see Fu et al. 2011 PNAS 108:9026-9032, which is hereby incorporated by reference in its entirety for discussion of random labeling).

[0125] In some embodiments, unique primer designs are designed that can reduce "barcode bias," a phenomenon observed in the prior art where barcodes with high G nucleotide content are used more frequently compared to other barcode compositions (Figure 4). In certain cases where conventional primers are used to target and label mRNA transcripts with gene-specific sequences, MIs with high % G content and multiple Gs are preferably observed (Figure 4). Furthermore, without being limited by any theory, within the scope of conventional designs, when SIs that are not associated with the target genes are used, a small amount of PCR error is observed due to cross-reaction of such primers during sample multiplex PCR (Figures 4-5). Such crossover events often occur with primers with high % G content and multiple Gs, likely due to the unique secondary structure of G-rich oligonucleotides called "G-quadruplexes." As such G-rich MI / SI primers can form inter- and intra-molecular complexes with each other, conventional DNA cleanup steps to remove primers (e.g., Ampure XP beads) may not be sufficient. To minimize the occurrence of such events, the present invention provides several designs that reduce the G content of the primers to minimize "barcode bias" and primer crossover (Figures 3A-3H).

[0126] Figures 3A-3H show prior art barcode layout designs (Figure 3A) and novel designs (Figures 3B-H) that minimize bias as described. In Figure 3A, the conventional design allows for either "BBBBBBV" or "NNNNNNNN" (B is C, G, or T, V is A, C, or G, and N is A, T, G, or C) MI compositions. A subset of the conventionally designed MI barcodes can be G-rich. In panels 3B-C, the G-richness is reduced along the region by adding either TTT or TTTTT after the MI (the length of the non-G spacer can be variable and can be any non-G nucleotide). In Figure 3D, the highly G-rich MI is spaced apart to avoid potentially G-rich gene-specific target regions. Compared to Fig. 3A The SI and MI positions are swapped. In Figures 3E-F, the G-rich region is further reduced by adding either TTT or TTTTT to the design of Figure 3D (note that SI is not G-rich and the length of the non-G spacer can be variable and can be any non-G nucleotide). In the designs of Figures 3G-H, MI is designed to not be G-rich with "HHHHHHHH" or "HNHNHNHN" (where H is A, C, or T).

[0127] The G-rich shortcomings of conventional designs are most often observed when gene-specific targeting methods are used. Some embodiments include using new primer designs with gene-specific target panels, such as T cell receptors (TCRs), where primers are used to target TCR-specific sequences. The designs according to some embodiments herein can reduce SI barcode crossovers, thus reducing the "noise" of TCR molecular barcoding assays. EXAMPLES

[0128] Example 1: Accurate analysis of sample target nucleic acid using unique oligonucleotide species 96 formats of unique oligonucleotide species were designed. Their sequences are summarized in Figures 5A-5E (SEQ ID NOs: 1-96). Unique oligonucleotide species formats A01-A12 (SEQ ID NOs: 1-12) served as controls in which the molecular barcode ("2nd bc") was NNNNNNNN. Thus, the molecular barcodes of these control unique oligonucleotide species did not have any constraints or limitations regarding G content. Unique oligonucleotide species formats B01-B12 (SEQ ID NOs: 13-24) contained a spacer of sequence TTT on the 3' side of the molecular barcode ("2nd bc") and on the 5' side of the uniform region ("5'→3' recognition sequence"). Unique oligonucleotide species formats C01-C12 (SEQ ID NOs: 25-36) contained a spacer of sequence TTTTT on the 3' side of the molecular barcode ("2nd bc") and on the 5' side of the uniform region ("5'→3' recognition sequence"). The unique oligonucleotide species formats D01 to D12 (SEQ ID NOs: 37 to 48) contained a molecular barcode ("first bc") on the 5' side of the sample barcode ("second bc"). The unique oligonucleotide species formats E01 to E12 (SEQ ID NOs: 49 to 60) contained a molecular barcode ("first bc") on the 5' side of the sample barcode ("second bc") and a molecular barcode ("first bc") on the 3' side of the barcode region and in the uniform region. 5 The unique oligonucleotide species formats F01 to F12 (SEQ ID NOs: 61 to 72) contained a molecular barcode ("first bc") on the 5' side of the sample barcode ("second bc") and a spacer of sequence TTT on the 3' side of the barcode region and on the 3' side of the uniform region. 5Each of the unique oligonucleotide seed formats G01-G12 (SEQ ID NOs: 73-84) contained a molecular barcode ("second bc") that included the sequence HHHHHHHH (wherein each "H" is A, C, or T, and any two "H" nucleotides may be the same or different). Each of the unique oligonucleotide seed formats H01-H12 (SEQ ID NOs: 85-96) contained a molecular barcode ("second bc") that included the sequence HNHNHNHN (wherein each "H" is A, C, or T, and any two "H" nucleotides may be the same or different, and each "N" may be A, G, C, or T, and any two "N" nucleotides may be the same or different).

[0129] Amplification and analysis of target T cell receptor nucleic acid sequences was performed as follows: Precise™ TCR encoding plates (BD Cellular Research Inc.) were thawed at room temperature and briefly centrifuged to collect 5 μL of reagent in each well. The plates were placed in a 96-well rack and the seal was removed very carefully on ice, avoiding disturbance of the reagents in the wells. Cells were sorted or added directly to the 96-well encoding plate (1 cell / well). The plate was sealed and mixed by briefly vortexing (5-10 s), then briefly centrifuged (approximately 1000 rpm x 10 s). Sorting sample plates can be optionally stored at -80°C until ready to begin the Precise™ protocol. For reverse transcription, the plate was incubated at 65°C for 3 min, cooled to 4°C, and then placed on ice for 5 min. The reaction master mix was as follows:

[0130] [Table 1]

[0131] 5 μL of RT MM was pipetted into each well. Each plate was sealed and the reverse transcriptase program was run as follows: 42° C. for 30 minutes, 80° C. for 5 minutes, 4° C. rest.

[0132] cDNA purification was performed using the following protocol: Centrifuge the plate (1000 rpm x 10 s). Combine all reactions into a single 2 mL tube. Add 8 μL of Precise DBP mix to the pooled cDNA tube. Gently mix to bring the buffer into solution. Add an equivalent volume of AMPure XP beads to the single tube containing the pooled RT reaction products (1x volume of pooled PCR products). Mix the sample and beads by pipetting up and down. Incubate the bead mix for 5 min at room temperature. Open the cap to ensure there is no disturbance of the beads. Place the tube on a magnet and wait for the liquid and beads to separate. The liquid should be completely clear before removal. This takes 5 min. Carefully remove and discard only the liquid while keeping the tube on the magnet. Gently rinse the beads once with 2 mL of 70% EtOH / 30% H2O while keeping the tube on the magnetic stand. Prepare fresh EtOH solution daily to avoid evaporation. Care may be taken to avoid disturbing or resuspending the beads during the rinsing steps. Immediately remove all residual EtOH solution using a pipette. Tap the tube while it is on the magnet to collect as much EtOH as possible at the bottom of the tube. Remove the tube from the magnet to elute the product from the beads. Introduce 68 μL of Elution Buffer into the tube with the beads. Wet the beads with Elution Buffer by tilting the tube. Mix thoroughly and wait 1 minute. Return the tube to the magnet. Wait until the solution is clear, typically <5 minutes. Recover the purified PCR product solution by carefully pipetting the solution off the beads. Introduce the clear liquid product (68 μL) into a new 1.5 mL tube.

[0133] PCR N1 amplification (target amplification) was performed using the following protocol: Prepare PCR mix (200 µL) with the pre-amplification region according to Table 2.

[0134] [Table 2]

[0135] Distribute this 200 μL of reaction mix into 4 PCR tubes. Run 20 cycles of the following PCR conditions: 3 min annealing time at 60C, 1 min extension time at 68C (approximately 2 hours). Combine all PCR products into one sample tube. Add 160 μL of Ampure beads (0.8x), mix thoroughly by pipetting, and incubate for 5 min at room temperature. Place the tube on a magnet and wait for the liquid and beads to separate. The liquid should be completely clear before removing. This takes 5 min. Remove and discard only the liquid. Gently rinse the tube once with 1 mL of 70% EtOH / 30% H2O while still on the magnetic stand. Do not resuspend the beads during the rinsing step. Immediately remove all residual EtOH using a pipette. Tap the tube while still on the magnet to collect as much EtOH as possible at the bottom of the tube. Elute the PCR product in 50 μL of elution buffer.

[0136] PCR N1 amplification (adapter and indexing PCR) was performed using the following protocol: Prepare PCR mix (50 µL) according to Table 3.

[0137] [Table 3]

[0138] Run 25 cycles using the following PCR conditions: annealing time 3 min at 60 C, extension time 1 min at 68 C. Purify twice with 0.8x Ampure and elute in 30 μL elution buffer.

[0139] Figure 6 shows the error correction for the average of the sequencing results (by individual TCRa CDR3). In Figure 6, the Y-axis plots the number of different molecular barcodes detected for the TCRa.

[0140] Results are shown for reactions with 50 pg of target RNA (represented as "50" in FIG. 6, e.g., A50, B50, C50, D50, E50, F50, G50) and negative controls incorporating 0 pg of target RNA (represented as "0" in FIG. 6, e.g., A0, B0, C0, D0, E0, F0, G0). Thus, 0 pg serves as a detector of assay noise leaking from the negative controls and 50 pg samples. In reactions incorporating target RNA, the unique oligonucleotide designs B01-B12 ("B50"), C01-C12 ("C50"), D01-D12 ("D50"), E01-E12 ("E50"), F01-F12 ("F50"), G01-G12 ("G50"), and H01-H12 ("H50") according to some embodiments herein had lower error rates than the conventional format A01-A12 ("A50"). Notably, in this experimental TRAC, format "H" had the highest sensitivity with low noise and behaved better than the original barcoded primer (A).

[0141] Thus, compositions comprising unique oligonucleotide species that include a barcode region and a molecular barcode with a relatively low G content as described herein and / or unique oligonucleotides that include a spacer 3' to the molecular barcode and 5' to the uniform region provided amplification with lower error rates and therefore higher accuracy than compositions comprising common polynucleotide species that do not include such features.

[0142] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are intended to be illustrative and not limiting, with the true scope and spirit being indicated by the following claims.

[0143] Those skilled in the art will recognize that in these and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in differing orders. Moreover, the outlined steps and operations are provided merely as examples, and some of the steps and operations may be optional and may be combined into fewer steps and operations and expanded to include additional steps and operations without departing from the essence of the embodiments of the present disclosure.

[0144] In connection with the use of substantially any plural and / or singular term described herein, those skilled in the art can convert from plural to singular and / or from singular to plural, where appropriate in the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0145] In general, those skilled in the art will understand that the terms used in this specification, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Furthermore, those skilled in the art will understand that where a specific number of introduced claim recitations is intended, such intent is expressly recited in the claim, and that no such intent exists in the absence of such recitations. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, even if such phrases are used, the introduction of a claim recitation with the indefinite article "a" or "an" should not be interpreted as meaning that any particular claim containing such an introduced claim recitation is limited to only the embodiment containing one such recitation, even if the same claim contains the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"). The same applies when a claim recitation is introduced with a definite article. In addition, those skilled in the art will understand that even if a specific number of introduced claim recitations is explicitly recited, such recitations should be interpreted as meaning at least the number recited (e.g., a bare recitation without other modifiers such as "two recitations" means at least two recitations or more than two recitations).Furthermore, when a condition similar to "at least one of A, B, and C" is used, it is generally intended that such a configuration be understood by one of ordinary skill in the art (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, a system having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). When a condition similar to "at least one of A, B, or C, etc.," is used, it is generally intended that such a configuration be understood by one of ordinary skill in the art (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, a system having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). Moreover, those skilled in the art will appreciate that virtually any disjunctive word and / or phrase expressing two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0146] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, one of skill in the art will recognize that the disclosure is thereby described in terms of any individual member or subgroup of members of the Markush group.

[0147] As will be understood by one of ordinary skill in the art, for all purposes, e.g., with respect to the provision of the specification, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any recited range is readily identifiable as being fully descriptive and that the range can be divided into at least two, three, four, five, ten, etc. For example, and without limitation, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third. Similarly, as will be understood by one of ordinary skill in the art, all expressions such as "up to," "at least," and the like, include the recited numbers and refer to ranges that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one of ordinary skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells means a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells means a group having 1, 2, 3, 4, or 5 cells, and so forth.

[0148] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims. Yet another aspect of the present invention may be as follows. [1] A composition comprising at least 1000 unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region comprising a molecular barcode comprising at least 7 nucleotides; the unique oligonucleotide species contain distinct nucleic acid sequences in their barcode regions; and (a) the composition consists essentially of unique oligonucleotide species, each molecular barcode of which has a G content of less than 50%, or (b) the molecular barcodes of all unique oligonucleotide species in said composition collectively have a G content of 12.5% ​​or less; The composition, [2] The composition described in [1], wherein the barcode region further comprises a sample barcode comprising at least 3 nucleotides. [3] The composition described in [1] or [2], wherein each unique oligonucleotide species further comprises a uniform region 3' to the barcode region. [4] The composition described in [3], wherein the uniform region further comprises a target-specific region 3' to the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid. [5] The composition described in any one of [1] to [4] above, wherein the composition consists essentially of unique oligonucleotide species, each molecular barcode of which has a G content of less than 50%. [6] The composition described in any one of [1] to [5], wherein the molecular barcodes of all unique oligonucleotide species in the composition have a G content of 12.5% ​​or less overall. [7] The composition described in any one of [1] to [6], wherein the unique oligonucleotide species are arranged in at least two spatially separated pools, each pool containing at least 100 unique oligonucleotides of the unique oligonucleotide species, the unique oligonucleotides of the same pool containing the same sample barcode sequence, and different unique oligonucleotides of the same pool containing different molecular barcode sequences. [8] The composition described in any one of [2] to [7] above, wherein the sample barcode of each unique oligonucleotide species has a G content of 50% or less. [9] The composition described in any one of [1] to [8] above, wherein the barcode region of each unique oligonucleotide species has a G content of 50% or less.

[10] The composition described in any one of [1] to [9], wherein the molecular barcodes of the unique oligonucleotide species have an overall G content of less than 12.5%.

[11] The composition described in any one of [1] to

[10] above, wherein the barcode region of the unique oligonucleotide species has an overall G content of 12.5% ​​or less.

[12] The composition according to any one of [1] to

[11] , wherein at least 95% of the unique oligonucleotide species have no Gs in the molecular barcode adjacent to any other G.

[13] The composition described in any one of [1] to

[12] above, wherein the composition consists essentially of unique oligonucleotide species, and none of the Gs in the molecular barcodes are adjacent to other Gs.

[14] The composition according to any one of [1] to

[13] , wherein at least 95% of the molecular barcodes of the unique oligonucleotide species contain a sequence in which H and N alternate a total of at least 6 times, each "H" being either A, C, or T, and each "N" being either A, G, C, or T.

[15] The composition described in any one of [1] to

[14] , wherein at least 95% of the molecular barcodes of the unique oligonucleotide species comprise the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T.

[16] The composition described in any one of [1] to

[15] , wherein each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T.

[17] The composition described in any one of [1] to

[16] , wherein each molecular barcode of the unique oligonucleotide species comprises the sequence HHHHHHHH, and each "H" is any one of A, C, or T.

[18] The composition described in any one of [1] to

[17] , wherein each of the unique oligonucleotide species comprises a spacer on the 3' side of the barcode region and on the 5' side of the target specific region, the spacer comprising the sequence HNHNHNHN, each "H" being A, C, or T, and each "N" being A, G, C, or T.

[19] The composition described in any one of [1] to

[17] , wherein each of the unique oligonucleotide species comprises a spacer on the 3' side of the barcode region and on the 5' side of the target specific region, the spacer comprising the sequence HHHHHHHH, and each "H" is any one of A, C, or T.

[20] The composition described in any one of [4] to

[19] above, wherein the target-specific region comprises an oligo dT sequence.

[21] The composition described in any one of [1] to

[20] , wherein the molecular barcode of each unique oligonucleotide species is located on the 3' side of the sample barcode.

[22] The composition described in any one of [1] to

[20] , wherein the sample barcode of each unique oligonucleotide species is located on the 3' side of the molecular barcode.

[23] The composition according to any one of [1] to

[22] above, wherein each oligonucleotide species has a length of at least 24 nucleotides.

[24] The composition according to any one of [1] to

[22] above, wherein each oligonucleotide species has a length of 24 to 140 nucleotides.

[25] The composition described in any one of [1] to

[24] above, wherein the composition comprises at least 6,500 unique oligonucleotide species.

[26] The composition described in any one of [1] to

[24] above, wherein the composition comprises at least 65,000 unique oligonucleotide species.

[27] The composition described in any one of [1] to

[26] above, wherein the composition comprises at least two oligonucleotides of the same unique oligonucleotide species.

[28] The composition described in any one of [1] to

[27] above, wherein no two oligonucleotides in the composition are the same unique oligonucleotide species.

[29] The composition described in any one of [7] to

[28] above, wherein the composition comprises at least 48 pools.

[30] The composition described in any one of [7] to

[29] , wherein the unique oligonucleotide species of each pool are immobilized on a substrate such that the sample barcodes, but not the molecular barcodes of the oligonucleotide species immobilized on each substrate, are identical.

[31] The composition described in

[30] , wherein the substrate comprises discrete surface regions such that the surface may comprise two or more substrates.

[32] The composition described in

[30] above, wherein the substrate comprises beads.

[33] The composition described in any one of

[27] to

[29] , wherein each of the unique oligonucleotide species further comprises an adapter configured to fix the unique oligonucleotide to the substrate, and the barcode region is on the 3' side of the adapter.

[34] A method for specifically barcoding multiple nucleic acids from two or more samples, each sample containing nucleic acid, comprising: contacting each sample with a pool comprising at least 100 unique oligonucleotide species under conditions in which each sample is contacted in a spatially separated manner from the other samples, wherein each unique oligonucleotide species comprises a barcode region comprising a molecular barcode comprising at least 7 nucleotides; the unique polynucleotide species of each pool contain the same sample barcode and a different molecular barcode; and (a) the unique oligonucleotide species contacted with the sample consist essentially of unique oligonucleotide species, each molecular barcode having a G content of less than 50%, or (b) the molecular barcodes of all unique oligonucleotide species collectively have a G content of 12.5% ​​or less; and hybridizing target specific regions of at least some of the oligonucleotides of the unique oligonucleotide species to at least some of the nucleic acids of the sample; extending the hybridized oligonucleotides to generate strands comprising oligonucleotides of the unique oligonucleotide species and sequences complementary to the target, wherein the strands comprise the same sample barcode and different molecular barcodes for each sample, and the molecular barcodes are different for different samples; The method includes:

[35] The method according to

[34] , wherein the barcode region further comprises a sample barcode comprising at least 3 nucleotides.

[36] The method according to

[34] or

[35] , wherein each unique oligonucleotide species further comprises a uniform region 3' to the barcode region.

[37] The method according to

[36] , wherein the uniform region further comprises a target-specific region 3' to the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid.

[38] The method according to any one of

[34] to

[37] , wherein the unique oligonucleotide species contacted with the sample essentially consists of unique oligonucleotide species, each molecular barcode having a G content of less than 50%.

[39] The method according to any one of

[34] to

[38] , wherein the molecular barcodes of all of the unique oligonucleotide species have a G content of 12.5% ​​or less overall.

[40] The method according to any one of

[34] to

[39] , further comprising confirming the nucleic acid sequence of a strand containing the unique oligonucleotide species and a sequence complementary to the target.

[41] The method according to any one of

[34] to

[40] , wherein at least 100 unique oligonucleotide species of each pool are immobilized to a substrate such that the unique oligonucleotide species immobilized to a given substrate contain the same sample barcode and such that different unique oligonucleotide species immobilized to the substrate contain different molecular barcodes.

[42] The method according to any one of

[34] to

[41] above, wherein each sample barcode has a G content of 50% or less.

[43] The method according to any one of

[34] to

[42] , wherein the molecular barcodes of the unique oligonucleotide species have an overall G content of less than 12.5%.

[44] The method according to any one of

[34] to

[43] , wherein the barcode region of the unique oligonucleotide species has an overall G content of 12.5% ​​or less.

[45] The method according to any one of

[34] to

[44] , wherein at least 95% of the unique oligonucleotide species have no Gs in the molecular barcode adjacent to any other Gs.

[46] The method according to any one of

[34] to

[44] , wherein each pool essentially consists of unique oligonucleotide species in which none of the Gs in the molecular barcodes are adjacent to any other G.

[47] The method according to any one of

[34] to

[46] , wherein at least 95% of the molecular barcodes of the unique oligonucleotide species contain a sequence in which H and N alternate a total of at least 6 times, each "H" being one of A, C, or T, and each "N" being one of A, G, C, or T.

[48] ​​The method according to any one of

[34] to

[47] , wherein at least 95% of the molecular barcodes of the unique oligonucleotide species comprise the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T.

[49] The method according to any one of

[34] to

[47] , wherein each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T.

[50] The method according to any one of

[34] to

[49] , wherein each molecular barcode of the unique oligonucleotide comprises the sequence HHHHHHHH, where each "H" is any one of A, C, or T.

[51] The method according to any one of

[34] to

[50] , wherein each unique oligonucleotide comprises a spacer on the 3' side of the barcode region and on the 5' side of the target-specific region, the spacer comprising the sequence HNHNHNHN, each "H" being any one of A, C, or T, and each "N" being any one of A, G, C, or T.

[52] The method according to any one of

[34] to

[50] , wherein each unique oligonucleotide comprises a spacer on the 3' side of the barcode region and on the 5' side of the target-specific region, the spacer comprising the sequence HHHHHHHH, where each "H" is any one of A, C, or T.

[53] The method according to any one of

[34] to

[52] above, wherein at least one pool contains at least two oligonucleotides of the same unique oligonucleotide species.

[54] The method according to any one of

[34] to

[52] above, wherein none of the pools contains two oligonucleotides of the same unique oligonucleotide species.

[55] The method according to any one of

[34] to

[54] , wherein the target-specific region comprises an oligo dT sequence.

[56] The method according to any one of

[34] to

[55] , wherein for each unique oligonucleotide species, the molecular barcode is located on the 3' side of the sample barcode.

[57] The method according to any one of

[34] to

[55] , wherein for each unique oligonucleotide species, the sample barcode is located on the 3' side of the molecular barcode.

[58] The method according to any one of

[34] to

[57] above, wherein each unique oligonucleotide species has a length of at least 24 nucleotides.

[59] The method according to any one of

[34] to

[58] above, wherein each unique oligonucleotide species has a length of 24 to 140 nucleotides.

[60] The method according to any one of

[34] to

[59] , wherein each pool contains at least 6,500 unique oligonucleotide species.

[61] The method according to any one of

[34] to

[59] , wherein each pool contains at least 65,000 unique oligonucleotide species.

[62] The method according to any one of

[34] to

[61] , wherein at least 48 unique samples are each contacted with a unique pool.

[63] The method according to any one of

[34] to

[62] , wherein at least 99% of the samples each contain one cell or less.

[64] The method according to any one of

[34] to

[63] , wherein the unique oligonucleotide species of each pool are immobilized on a substrate such that the sample barcodes, but not the molecular barcodes, of the unique oligonucleotide species immobilized on each of the multiple substrates are identical.

[65] The method according to

[64] , wherein the substrate comprises spatially separated surface regions such that the substrates of different pools comprise different spatially separated surface regions.

[66] The method according to

[64] , wherein the substrate comprises beads.

[67] The method according to any one of

[64] to

[66] , wherein each of the unique oligonucleotide species further comprises an adapter configured to fix the unique oligonucleotide to the substrate, and the barcode region is on the 3' side of the adapter.

[68] A method for making a composition comprising a unique oligonucleotide, comprising: providing a plurality of different sample barcodes each comprising at least three nucleotides; providing a plurality of distinct molecular barcodes, each comprising at least 7 nucleotides; synthesizing a plurality of unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region that comprises a sample barcode and a molecular barcode; (a) the plurality of unique oligonucleotide species consists essentially of unique oligonucleotide species, each molecular barcode having a G content of less than 50%, or (b) the molecular barcodes of all of said plurality of unique oligonucleotide species collectively have a G content of 12.5% ​​or less; and disposing said unique oligonucleotide species in spatially separated pools, each pool comprising a plurality of unique oligonucleotide species, such that unique oligonucleotide species of the same pool comprise the same sample barcode sequence and such that different unique oligonucleotide species of the same pool comprise different molecular barcode sequences; each pool containing at least 1000 unique oligonucleotide species; The method includes:

[69] The method according to

[68] , wherein each unique oligonucleotide species further comprises a uniform region 3' to the barcode region.

[70] The method according to

[69] , wherein the uniform region further comprises a target-specific region 3' to the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid.

[71] The method according to any one of

[68] to

[70] , wherein the plurality of unique oligonucleotide species essentially consists of unique oligonucleotide species, each molecular barcode having a G content of less than 50%.

[72] The method of

[68] or

[69] , wherein the molecular barcodes of all of the multiple unique oligonucleotide species have an overall G content of 12.5% ​​or less.

[73] The method according to

[68] , wherein each molecular barcode has a G content of 50% or less.

[74] The method according to any one of

[68] to

[69] , wherein each sample barcode has a G content of 50% or less.

[75] The method described in any one of

[68] to

[72] , wherein the sample barcodes of the unique oligonucleotide species have an overall G content of 12.5% ​​or less.

[76] The method according to any one of

[68] to

[72] , wherein the molecular barcodes of the unique oligonucleotide species have an overall G content of less than 12.5%.

[77] The method described in any one of

[68] to

[76] , wherein the barcode region of the unique oligonucleotide species has an overall G content of 12.5% ​​or less.

[78] The method according to any one of

[68] to

[77] , wherein at least 95% of the unique oligonucleotide species have no Gs in the molecular barcode adjacent to any other G.

[79] The method according to any one of

[68] to

[77] , wherein the plurality of unique oligonucleotide species essentially consists of unique oligonucleotide species in which no G in the molecular barcode is adjacent to any other G.

[80] The method according to any one of

[68] to

[79] , wherein at least 95% of the molecular barcodes of the unique oligonucleotide species contain a sequence in which H and N alternate a total of at least 6 times, each "H" being one of A, C, or T, and each "N" being one of A, G, C, or T.

[81] The method according to any one of

[68] to

[80] , wherein at least 95% of the molecular barcodes of the unique oligonucleotides comprise the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T.

[82] The method according to any one of

[68] to

[81] , wherein each molecular barcode of the unique oligonucleotide comprises the sequence HNHNHNHN, where each "H" is A, C, or T, and each "N" is A, G, C, or T.

[83] The method described in any one of

[68] to

[82] , wherein each molecular barcode of the unique oligonucleotide comprises the sequence HHHHHHHH, where each "H" is any one of A, C, or T.

[84] The method according to any one of

[68] to

[83] , wherein each unique oligonucleotide species comprises a spacer on the 3' side of the barcode region and on the 5' side of the target specific region, the spacer comprising the sequence HNHNHNHN, each "H" being any one of A, C, or T, and each "N" being any one of A, G, C, or T.

[85] The method described in any one of

[68] to

[84] , wherein each unique oligonucleotide species comprises a spacer on the 3' side of the barcode region and on the 5' side of the target specific region, the spacer comprising the sequence HHHHHHHH, where each "H" is any one of A, C, or T.

[86] The method according to any one of

[70] to

[85] , wherein the target-specific region comprises an oligo dT sequence.

[87] The method described in any one of

[68] to

[86] , wherein the molecular barcode of each unique oligonucleotide species is located on the 3' side of the sample barcode.

[88] The method described in any one of

[68] to

[86] , wherein the sample barcode of each unique oligonucleotide species is on the 3' side of the molecular barcode.

[89] The method according to any one of

[68] to

[88] above, wherein each unique oligonucleotide species has a length of at least 24 nucleotides.

[90] The method according to any one of

[68] to

[89] above, wherein each unique oligonucleotide species has a length of 24 to 140 nucleotides.

[91] The method according to any one of

[68] to

[90] above, wherein each pool contains at least 6,500 unique oligonucleotide species.

[92] The method according to any one of

[68] to

[91] above, wherein each pool contains at least 65,000 unique oligonucleotide species.

[93] The method according to any one of

[68] to

[92] above, wherein at least 48 pools are produced.

[94] The method according to any one of

[68] to

[93] , further comprising immobilizing unique oligonucleotide species of each pool to a substrate such that the sample barcodes, but not the molecular barcodes, of the oligonucleotide species immobilized on each of the plurality of substrates are identical.

[95] The method of

[94] , wherein the substrate comprises discrete surface regions.

[96] The method according to

[94] , wherein the substrate comprises beads.

[97] The method according to any one of

[68] to

[96] , wherein the unique oligonucleotide species are arranged in a spatially separated pool simultaneously with the synthesis.

[98] The method according to any one of

[68] to

[96] , wherein the unique oligonucleotide species are arranged in spatially separated pools after the synthesis.

[99] An oligonucleotide comprising a barcode region on the 3' side of an adapter region, wherein the barcode region comprises a molecular barcode comprising at least 7 nucleotides, and the molecular barcode has a G content of 50% or less.

[100] The oligonucleotide according to

[99] , further comprising a sample barcode comprising at least three nucleotides.

[101] The oligonucleotide described in

[99] or

[100] , further comprising a uniform region on the 3' side of the barcode region.

[102] The oligonucleotide according to

[101] , wherein the uniform region comprises a target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid.

[103] The oligonucleotide described in any one of

[99] to

[102] above, further comprising an adapter region on the 5' side of the barcode region.

[104] The composition described in any one of [3] to

[19] or

[21] to

[33] , wherein the uniform region includes a target-specific region including a sequence flanking an immune cell receptor or an immunoglobulin variable region coding sequence.

[105] The composition described in

[104] , wherein the variable region coding sequence of the immune cell receptor is selected from the group consisting of a variable region coding sequence of a T cell receptor, a variable region coding sequence of a B cell receptor, and a combination thereof.

[106] A kit comprising the composition according to

[104] or

[105] , further comprising a primer configured to hybridize opposite the variable region to the target specific region and to hybridize to the complementary strand of the strand to which the target specific region hybridizes, thereby amplifying the variable region together with the target specific region.

[107] The kit described in

[106] , wherein the primers and target specific regions are configured to amplify at least 1 kb of nucleic acid comprising the variable region.

[108] The method described in any one of

[36] to

[54] ,

[56] to

[67] , 69 to 85, or 87 to 98, wherein the uniform region comprises a target-specific region comprising a sequence flanking an immune cell receptor or an immunoglobulin variable region coding sequence.

[109] The method described in

[108] , wherein the variable region coding sequence of the immune cell receptor is selected from the group consisting of a variable region coding sequence of a T cell receptor, a variable region coding sequence of a B cell receptor, and a combination thereof.

[110] The oligonucleotide according to any one of

[101] to

[103] , wherein the uniform region includes a target-specific region comprising a sequence flanking an immune cell receptor or an immunoglobulin variable region.

[111] The oligonucleotide described in

[110] , wherein the variable region coding sequence of the immune cell receptor is selected from the group consisting of a variable region coding sequence of a T cell receptor, a variable region coding sequence of a B cell receptor, and a combination thereof.

Claims

1. A composition comprising a plurality of unique oligonucleotide species, each unique oligonucleotide species comprising: a barcode region comprising a molecular barcode comprising at least 7 nucleotides and a sample barcode comprising at least 3 nucleotides; a uniform region comprising a target specific region; a non-G spacer comprising at least 3 nucleotides; Including, the target specific region comprises at least 10 nucleotides complementary to a target nucleic acid, and the uniform region is 3' to the barcode region; the spacer is between the barcode region and the uniform region, and the spacer contains only T; the nucleic acid sequence of the molecular barcode of each unique oligonucleotide species is different from the nucleic acid sequence of the molecular barcode of any other unique oligonucleotide species; composition.

2. The composition of claim 1 , wherein the uniform region comprises a target-specific region comprising sequences flanking an immune cell receptor or an immunoglobulin variable region coding sequence.

3. 3. The composition of claim 2, wherein the immune cell receptor variable region coding sequence is selected from the group consisting of a T cell receptor variable region coding sequence, a B cell receptor variable region coding sequence, and a combination thereof.

4. The composition according to any one of claims 1 to 3, wherein the spacer is 3 to 10 nucleotides in length.

5. The composition of any one of claims 1 to 4, wherein the spacer is TTT or TTTTT.

6. 6. The composition of any one of claims 1 to 5, wherein for each unique oligonucleotide species, the molecular barcode is 3' to a sample barcode.

7. the unique oligonucleotide species are arranged in at least two spatially separated pools, each pool comprising at least 100 unique oligonucleotides of the unique oligonucleotide species; The unique oligonucleotides in the same pool contain the same sample barcode sequence, and The composition of any one of claims 1 to 6, wherein different unique oligonucleotides of the same pool comprise different molecular barcode sequences.

8. The composition of claim 7 , wherein the unique oligonucleotide species are arranged in at least 48 pools.

9. 9. The composition of claim 7 or 8, wherein the unique oligonucleotide species of each pool are immobilized to a substrate such that the sample barcode, but not the molecular barcode, is identical for each oligonucleotide species immobilized to each substrate.

10. 10. The composition of claim 9, wherein the substrate comprises discrete surface regions such that a surface may comprise two or more substrates.

11. The composition of claim 9 , wherein the substrate comprises a bead.

12. 12. The composition of any one of claims 9-11, wherein each of the unique oligonucleotide species further comprises an adapter configured to anchor the unique oligonucleotide to the substrate, and the barcode region is 3' to the adapter.

13. The composition of any one of claims 1 to 12, wherein each oligonucleotide species has a length of at least 24 nucleotides.

14. The composition of any one of claims 1 to 13, comprising at least 1000 unique oligonucleotide species.

15. The composition of any one of claims 1 to 14, comprising at least two oligonucleotides of the same unique oligonucleotide species.

16. The composition of any one of claims 1 to 14, wherein no two oligonucleotides of the composition are of the same unique oligonucleotide species.

17. 1. A method for specifically barcoding a plurality of nucleic acids from two or more samples, each of which contains nucleic acid, comprising: contacting each sample with a pool comprising the composition of any one of claims 1 to 16, wherein each sample is contacted with spatial separation from the other samples; hybridizing target specific regions of at least some of the oligonucleotides of said unique oligonucleotide species to at least some of the nucleic acids of said sample; extending the hybridized oligonucleotides to generate strands comprising oligonucleotides of the unique oligonucleotide species and sequences complementary to the target, wherein for each sample, the strands comprise the same sample barcode and a different molecular barcode, and for different samples, the sample barcodes are different; The method includes:

18. 4. A kit comprising the composition of claim 2 or 3, further comprising a primer configured to hybridize to a strand complementary to a strand hybridized by the target specific region, the strand hybridized by the target specific region comprising the variable region coding sequence, and the primer configured to amplify the variable region coding sequence along with a unique oligonucleotide species comprising the target specific region.

19. 20. The kit of claim 18, wherein the primers and unique oligonucleotide species comprising the target specific region are configured to amplify at least 1 kb of nucleic acid comprising the variable region coding sequence.

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