Particles associated with oligonucleotides

Abstract

Disclosed herein are methods, compositions, and kits for barcoding nucleic acids. For example, a particle for barcoding is provided, wherein the particle comprises: a first plurality of oligonucleotide barcodes, each of which comprises a target binding region; and a second plurality of oligonucleotides, each of which comprises a target-specific target binding region.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent No. 62 / 607,812, filed on December 19, 2017, the contents of which are incorporated herein by reference in their entirety.

[0003] References to sequence listings

[0004] This application is submitted together with a sequence listing in electronic format. The sequence listing is provided as a file entitled Sequence_Listing_BDCRI_030WO.txt, created on December 18, 2018, and is 2.1 kilobytes in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety. Background of the Invention Technical Field

[0006] The present disclosure relates generally to the field of molecular barcoding of targets, and more particularly to target capture. Background Art

[0007] Barcoding methods and techniques such as using beads coupled to nucleic acid barcodes are useful for cell analysis, particularly using, for example, reverse transcription, polymerase chain reaction (PCR) amplification, and next generation sequencing (NGS) to decipher gene expression profiles to determine the state of cells. However, the sensitivity of these methods and techniques can be improved. Summary of the invention

[0008] The disclosure herein includes embodiments of synthetic particles. In some embodiments, the synthetic particles comprise: a first plurality of oligonucleotide barcodes; and a second plurality of oligonucleotide barcodes, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cellular marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region.

[0009] In some embodiments, the target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. In some embodiments, the target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a poly(dT) region. An oligonucleotide barcode of the second plurality of oligonucleotide barcodes may comprise a poly(dT) region.

[0010] In some embodiments, the ratio of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes is in the range of from 1:10 to 10:1. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a sample marker sequence. At least one of the second plurality of oligonucleotide barcodes is shorter than at least one of the first plurality of oligonucleotide barcodes. The target binding region and the target-specific target binding region may have up to 50% sequence identity. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may have different lengths. The sequences of at least two of the second plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may be the same. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain the same sequence.

[0011] In some embodiments, the synthetic particle comprises a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region and a target-specific target binding region. The synthetic particle may comprise a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise a target binding region and a target-specific target binding region.

[0012] In some embodiments, at least one of the first plurality of spacer oligonucleotides is shorter than at least one of the second plurality of spacer oligonucleotides.At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the first plurality of spacer oligonucleotides.At least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may have different lengths.The sequence of at least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may be the same.At least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may comprise the same sequence.

[0013] In some embodiments, at least one of the first plurality of oligonucleotide barcodes is shorter than at least one of the second plurality of oligonucleotide barcodes. At least one of the second plurality of oligonucleotide barcodes may be shorter than at least one of the first plurality of oligonucleotide barcodes. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may have different lengths. The sequences of at least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may be the same. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may comprise the same sequence.

[0014] In some embodiments, at least 100 of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes contain different molecular marker sequences. The target binding region may contain a target-specific sequence, an oligo (dT) sequence, a random polymer, or any combination thereof. The two target-specific target binding regions of two oligonucleotide barcodes in the second plurality of oligonucleotide barcodes may contain different target-specific sequences. Each cell marker sequence may contain at least 6 nucleotides. Each molecular marker sequence may contain at least 6 nucleotides. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a spatial marker sequence, and wherein the spatial marker sequences differ from each other by at least one nucleotide. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a universal sequence, wherein the universal sequence is the same.

[0015] In some embodiments, at least one of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes is fixed on a synthetic particle. At least one of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may be partially fixed on a synthetic particle. At least one of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may be encapsulated in a synthetic particle. At least one of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may be partially encapsulated in a synthetic particle. The synthetic particle may be destructible. The synthetic particle may comprise beads. The beads may comprise magnetic beads. The synthetic particle may comprise destructible hydrogel particles, such as destructible hydrogel beads.

[0016] In some embodiments, each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may comprise a linker functional group, the synthetic particle may comprise a solid support functional group, and the support functional group and the linker functional group may be associated with each other. The linker functional group and the support functional group may be individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, one or more sulfides, and any combination thereof.

[0017] The disclosure herein includes embodiments of kits or compositions. In some embodiments, the kit or composition comprises: a plurality of synthetic particles, wherein each synthetic particle comprises: a first plurality of oligonucleotide barcodes; and a second plurality of oligonucleotide barcodes, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cell marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region; and optionally, instructions for using the plurality of synthetic particles. In some embodiments, the kit comprises a buffer, one or more reagents for a reverse transcription reaction, one or more reagents for an amplification reaction, or a combination thereof.

[0018] In some embodiments, the plurality of synthetic particles comprises at least 94 synthetic particles. The kit may comprise a plurality of partitions, each partition comprising a synthetic particle from the plurality of synthetic particles. The plurality of partitions may comprise microwells of a microwell array. The plurality of partitions may comprise a plurality of droplets.

[0019] In some embodiments, the target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. The target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes may comprise a poly(dT) region. The oligonucleotide barcode of the second plurality of oligonucleotide barcodes may comprise a poly(dT) region.

[0020] In some embodiments, the ratio of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes is in the range of from 1:10 to 10:1. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a sample marker sequence. At least one of the second plurality of oligonucleotide barcodes may be shorter than at least one of the first plurality of oligonucleotide barcodes. The target binding region and the sequence-specific target binding region may have up to 50% sequence identity. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may have different lengths. The sequences of at least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may be the same. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain the same sequence.

[0021] In some embodiments, each synthetic particle comprises a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region and a target-specific target binding region. Each synthetic particle may comprise a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise a target binding region and a target-specific target binding region.

[0022] In some embodiments, at least one of the first plurality of spacer oligonucleotides is shorter than at least one of the second plurality of spacer oligonucleotides.At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the first plurality of spacer oligonucleotides.At least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may have different lengths.The sequence of at least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may be the same.At least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may comprise the same sequence.

[0023] In some embodiments, at least one of the first plurality of oligonucleotide barcodes is shorter than at least one of the second plurality of oligonucleotide barcodes. At least one of the second plurality of oligonucleotide barcodes may be shorter than at least one of the first plurality of oligonucleotide barcodes. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may have different lengths. The sequences of at least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may be the same. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may comprise the same sequence.

[0024] In some embodiments, at least 100 of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes contain different molecular marker sequences. The target binding region may contain a target-specific sequence, an oligo (dT) sequence, a random polymer, or any combination thereof. The two target-specific target binding regions of two oligonucleotide barcodes in the second plurality of oligonucleotide barcodes may contain different target-specific sequences. Each cell marker sequence may contain at least 6 nucleotides. Each molecular marker sequence may contain at least 6 nucleotides. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a spatial marker sequence, and wherein the spatial marker sequences differ from each other by at least one nucleotide. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a universal sequence, wherein the universal sequence is the same.

[0025] In some embodiments, at least one of the plurality of synthetic particle oligonucleotides is immobilized on a synthetic particle. At least one of the first plurality of oligonucleotide barcodes may be partially immobilized on a synthetic particle. At least one of the first plurality of oligonucleotide barcodes may be encapsulated in a synthetic particle. At least one of the first plurality of oligonucleotide barcodes may be partially encapsulated in a synthetic particle. The synthetic particle may be destructible. The synthetic particle may comprise beads. The beads may comprise magnetic beads. The synthetic particle may comprise destructible hydrogel particles, such as hydrogel beads.

[0026] In some embodiments, each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can be individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, one or more sulfides, and any combination thereof.

[0027] The disclosure herein includes embodiments of methods for determining the number of targets in a sample. In some embodiments, the method includes: barcoding copies of a first plurality of targets using a first plurality of oligonucleotide barcodes associated with a synthetic particle to produce a first plurality of barcoded copies of the target; barcoding copies of a second plurality of targets in the sample using a second plurality of oligonucleotide barcodes associated with the synthetic particle to produce a second plurality of barcoded copies of the target, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cellular marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region; and evaluating the number of each of the first plurality of targets and / or the second plurality of targets using the molecular marker sequences of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes.

[0028] In some embodiments, the target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. The target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes may comprise a poly(dT) region. The oligonucleotide barcode of the second plurality of oligonucleotide barcodes may comprise a poly(dT) region.

[0029] In some embodiments, the first plurality of targets comprises mRNA molecules of the cell, and wherein the second plurality of targets comprises DNA molecules of the cell. The first plurality of targets may comprise mRNA molecules of the cell, and the second plurality of targets may comprise RNA molecules of viruses, bacteria and / or fungi that infect the cell.

[0030] In some embodiments, barcoding copies of a first plurality of targets in a sample using a first plurality of oligonucleotide barcodes associated with a synthetic particle comprises hybridizing the first plurality of targets to the first plurality of oligonucleotide barcodes on the synthetic particle, wherein barcoding copies of the second plurality of targets in the sample using a second plurality of oligonucleotide barcodes associated with the synthetic particle comprises hybridizing the second plurality of targets to the second plurality of oligonucleotide barcodes on the synthetic particle. Barcoding copies of the first plurality of targets may comprise reverse transcribing the first plurality of targets to produce barcoded cDNAs of the first plurality of targets, and wherein barcoding copies of the second plurality of targets comprises reverse transcribing the second plurality of targets to produce barcoded cDNAs of the second plurality of targets. The method may comprise amplifying the barcoded cDNAs of the first plurality of targets and / or amplifying the barcoded cDNAs of the second plurality of targets using second strand synthesis to produce first double-stranded barcoded targets and second double-stranded barcoded targets, respectively. The method can include amplifying a first double-stranded barcoded target and a second double-stranded barcoded target to produce a first labeled target amplicon and a second labeled target amplicon, respectively.

[0031] The disclosure herein includes embodiments of methods for capturing targets in a sample. In some embodiments, the method includes: hybridizing a first plurality of copies of a target using a first plurality of oligonucleotide barcodes associated with a synthetic particle to produce a first plurality of copies of a barcoded target; hybridizing a second plurality of copies of a target using a second plurality of oligonucleotide barcodes associated with a synthetic particle to produce a second plurality of copies of a barcoded target, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cell marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region.

[0032] In some embodiments, the second plurality of copies of barcoded targets comprises copies of at least 50% of the targets in the plurality of targets. The second plurality of copies of barcoded targets may comprise copies of at least 75% of the targets in the second plurality of targets. The second plurality of copies of barcoded targets may comprise copies of at least 95% of the targets in the second plurality of targets. The second plurality of copies of barcoded targets may comprise copies of at least 50% of each target in the second plurality of targets. The second plurality of copies of barcoded targets may comprise copies of at least 75% of each target in the second plurality of targets. The second plurality of copies of barcoded targets may comprise copies of at least 95% of each target in the second plurality of targets.

[0033] In some embodiments, the first plurality of targets and the second plurality of targets comprise mRNA, and the method comprises: reverse transcribing the first plurality of targets to produce a first barcoded cDNA of the first plurality of targets; and reverse transcribing the second plurality of targets to produce a second barcoded cDNA of the second plurality of targets. The method may include amplifying the first barcoded cDNA and the second barcoded cDNA using second strand synthesis to produce a first double-stranded barcoded target and a second double-stranded barcoded target, respectively. The method may include amplifying the first barcoded target and the second barcoded target to produce a first labeled target amplicon and a second labeled target amplicon, respectively.

[0034] In some embodiments, the first plurality of targets and the second plurality of targets are from a single cell, and the method comprises associating the synthetic particle with the single cell. The single cell may comprise a rare cell, a tumor cell, a cell from a human, a cell from a tissue, a cell from a tumor, a cell infected with a viral polynucleotide, or any combination thereof.

[0035] In some embodiments, the method includes lysing the single cell after associating the synthetic particle with the single cell. Lysing the single cell may include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof. The synthetic particle and the single cell may be in the same well. The synthetic particle and the single cell may be in the same droplet.

[0036] In some embodiments, the first plurality of targets and / or the second plurality of targets include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNAs each containing a poly (A) tail, RNAs that do not contain any poly (A) tail, viral RNA, and any combination thereof.

[0037] In some embodiments, the ratio of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes is in the range of from 1:10 to 10:1. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a sample marker sequence. At least one of the second plurality of oligonucleotide barcodes may be shorter than at least one of the first plurality of oligonucleotide barcodes. The target binding region and the target-specific target binding region may have up to 50% sequence identity. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may have different lengths. The sequences of at least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes are the same. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain the same sequence.

[0038] In some embodiments, the synthetic particle comprises a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region and a target-specific target binding region. The synthetic particle may comprise a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise a target binding region and a target-specific target binding region.

[0039] In some embodiments, at least one of the first plurality of spacer oligonucleotides is shorter than at least one of the second plurality of spacer oligonucleotides.At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the first plurality of spacer oligonucleotides.At least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may have different lengths.The sequence of at least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may be the same.At least two of the first plurality of spacer oligonucleotides and / or the second plurality of spacer oligonucleotides may comprise the same sequence.

[0040] In some embodiments, at least one of the first plurality of oligonucleotide barcodes is shorter than at least one of the second plurality of oligonucleotide barcodes. At least one of the second plurality of oligonucleotide barcodes may be shorter than at least one of the first plurality of oligonucleotide barcodes. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may have different lengths. The sequences of at least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may be the same. At least two of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may comprise the same sequence.

[0041] In some embodiments, at least 100 of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes contain different molecular marker sequences. The target binding region may contain a target-specific sequence, an oligo (dT) sequence, a random polymer, or any combination thereof. The two target binding regions of two oligonucleotide barcodes in the second plurality of oligonucleotide barcodes may contain target-specific sequences. Each cell marker sequence may contain at least 6 nucleotides. Each molecular marker sequence may contain at least 6 nucleotides. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a spatial marker sequence, and wherein the spatial marker sequences differ from each other by at least one nucleotide. Each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes may contain a universal sequence, wherein the universal sequence is the same.

[0042] In some embodiments, at least one of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes is immobilized on a synthetic particle. At least one of the first plurality of oligonucleotide barcodes may be partially immobilized on a synthetic particle. At least one of the first plurality of oligonucleotide barcodes may be encapsulated in a synthetic particle. At least one of the first plurality of oligonucleotide barcodes may be partially encapsulated in a synthetic particle. The synthetic particle may be destructible. The synthetic particle may comprise beads. The beads may comprise magnetic beads. The synthetic particle may comprise destructible hydrogel particles, such as hydrogel beads.

[0043] In some embodiments, each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can be individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof.

[0044] The disclosure herein includes embodiments of synthetic particles. In some embodiments, the synthetic particles include: a plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes includes the same cell marker sequence, molecular marker sequence and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes include different molecular marker sequences; and a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not include a target binding region. The ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides may be in the range of from 1:10 to 10:1. Each of the first plurality of spacer oligonucleotides may include a cell marker sequence or a molecular marker sequence.

[0045] In some embodiments, at least one of the first plurality of spacer oligonucleotides may be shorter than at least one of the plurality of oligonucleotide barcodes. Each of the first plurality of spacer oligonucleotides may include a region corresponding to the target binding region, and wherein the region corresponding to the target binding region and the target binding region have different sequences. The region corresponding to the target binding region and the target binding region may have up to 50% sequence identity. At least two of the first plurality of spacer oligonucleotides may have different lengths. The sequences of at least two of the first plurality of spacer oligonucleotides may be the same. At least two of the first plurality of spacer oligonucleotides may include the same sequence.

[0046] In some embodiments, the synthetic particle comprises a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise the target binding region. At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the first plurality of spacer oligonucleotides. At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the plurality of oligonucleotide barcodes. At least two of the second plurality of spacer oligonucleotides may have different lengths. The sequences of at least two of the second plurality of spacer oligonucleotides may be the same. At least two of the second plurality of spacer oligonucleotides comprise the same sequence.

[0047] In some embodiments, at least 100 of the multiple oligonucleotide barcodes contain different molecular marker sequences. The target binding region may contain a gene-specific sequence, an oligo (dT) sequence, a random polymer, or any combination thereof. The two target binding regions of two oligonucleotide barcodes in the multiple oligonucleotide barcodes may contain different gene-specific sequences. Each cell marker sequence of the multiple oligonucleotide barcodes may contain at least 6 nucleotides. Each molecular marker sequence of the multiple oligonucleotide barcodes may contain at least 6 nucleotides. Each of the multiple oligonucleotide barcodes may contain a spatial marker sequence, and wherein the spatial marker sequences of the multiple oligonucleotide barcodes differ from each other by at least one nucleotide. Each of the multiple oligonucleotide barcodes may contain a universal marker sequence, wherein the universal marker sequences of the multiple oligonucleotide barcodes are the same.

[0048] In some embodiments, at least one of the multiple oligonucleotide barcodes is fixed on, partially fixed on, encapsulated in, partially encapsulated in, or any combination thereof. The synthetic particles may be destructible. The synthetic particles may include beads. The beads may include agarose gel beads, streptavidin protein beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo (dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particles may include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methyl styrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, and any combination thereof. The synthetic particles may include destructible hydrogel particles. Each of the multiple oligonucleotide barcodes may include a linker functional group, the synthetic particles may include a solid support functional group, and / or the support functional group and the linker functional group may be associated with each other. The linker functional group and the support functional group may be individually selected from the group consisting of the following: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof. In some embodiments, the multiple synthetic particles include at least 94 synthetic particles. The kit may include a microwell array, wherein each microwell in the microwell array includes the synthetic particles in the multiple synthetic particles. The kit may include a buffer, a box, one or more reagents for reverse transcription reaction, one or more reagents for amplification reaction, or a combination thereof. The ratio of the multiple oligonucleotide barcodes and the first multiple spacer oligonucleotides may be in the range of from 1:10 to 10:1. Each of the first multiple spacer oligonucleotides may include a cell marker sequence or a molecular marker sequence.

[0049] Provided herein are embodiments comprising a kit. In some embodiments, the kit comprises: a plurality of synthetic particles, wherein each synthetic particle comprises: a plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes comprises the same cell marker sequence, molecular marker sequence and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences; and a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region. The kit may comprise: instructions for using the plurality of oligonucleotide barcodes.

[0050] In some embodiments, at least one of the first plurality of spacer oligonucleotides is shorter than at least one of the plurality of oligonucleotide barcodes. Each of the first plurality of spacer oligonucleotides may include a region corresponding to the target binding region. The region corresponding to the target binding region and the target binding region may have different sequences. The region corresponding to the target binding region and the target binding region may have up to 50% sequence identity. At least two of the first plurality of spacer oligonucleotides may have different lengths. The sequences of at least two of the first plurality of spacer oligonucleotides may be the same. At least two of the first plurality of spacer oligonucleotides may include the same sequence.

[0051] In some embodiments, the synthetic particle comprises a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise the target binding region. At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the first plurality of spacer oligonucleotides. At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the plurality of oligonucleotide barcodes. At least two of the second plurality of spacer oligonucleotides may have different lengths. The sequences of at least two of the second plurality of spacer oligonucleotides may be the same. At least two of the second plurality of spacer oligonucleotides may comprise the same sequence.

[0052] In some embodiments, at least 100 of the multiple oligonucleotide barcodes contain different molecular marker sequences. The target binding region may contain a gene-specific sequence, an oligo (dT) sequence, a random polymer, or any combination thereof. The two target binding regions of two oligonucleotide barcodes in the multiple oligonucleotide barcodes may contain different gene-specific sequences. Each cell marker sequence of the multiple oligonucleotide barcodes may contain at least 6 nucleotides. Each molecular marker sequence of the multiple oligonucleotide barcodes may contain at least 6 nucleotides. Each of the multiple oligonucleotide barcodes may contain a spatial marker sequence, and wherein the spatial marker sequences of the multiple oligonucleotide barcodes differ from each other by at least one nucleotide. Each of the multiple oligonucleotide barcodes may contain a universal marker sequence, wherein the universal marker sequences of the multiple oligonucleotide barcodes are the same.

[0053] In some embodiments, at least one of the multiple oligonucleotide barcodes is fixed on, partially fixed on, encapsulated in, partially encapsulated in, or any combination thereof. The synthetic particles may be destructible. The synthetic particles may include beads. The beads may include agarose gel beads, streptavidin protein beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo (dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particles may include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methyl styrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, and any combination thereof. The synthetic particles may include destructible hydrogel particles. Each of the plurality of oligonucleotide barcodes may comprise a linker functional group, the synthetic particle may comprise a solid support functional group, and / or the support functional group and the linker functional group may be associated with each other. The linker functional group and the support functional group may be individually selected from the group consisting of C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof.

[0054] Embodiments disclosed herein include methods for determining the number of targets in a sample. In some embodiments, the method includes barcoding copies of multiple targets in a sample using a plurality of oligonucleotide barcodes associated with a synthetic particle to produce a plurality of barcoded copies of the target, wherein each of the plurality of oligonucleotide barcodes comprises the same cell marker sequence, molecular marker sequence, and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein the synthetic particle comprises a first plurality of spacer oligonucleotides, and wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region; and using the molecular marker sequences of the plurality of oligonucleotide barcodes, assessing the number of each of the plurality of targets in the sample. In some embodiments, barcoding copies of the plurality of targets in a sample using the plurality of oligonucleotide barcodes associated with the synthetic particle may include hybridizing the plurality of targets with the plurality of oligonucleotide barcodes on the synthetic particle. Barcoding copies of the plurality of targets may include reverse transcribing the plurality of targets to produce barcoded cDNAs of the plurality of targets. The method may include amplifying the barcoded cDNA using second strand synthesis to produce a barcoded target, wherein the barcoded target is double stranded. The method may include amplifying the barcoded target to produce a labeled target amplicon.

[0055] Disclosed herein are embodiments of methods for capturing targets in a sample. In some embodiments, the method comprises hybridizing copies of a plurality of targets in a sample using a plurality of oligonucleotide barcodes associated with a synthetic particle to produce a plurality of barcoded copies of the target, wherein each of the plurality of oligonucleotide barcodes comprises the same cell marker sequence, molecular marker sequence, and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein the synthetic particle comprises a first plurality of spacer oligonucleotides, and wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region. In some embodiments, the plurality of barcoded copies of the target may comprise at least 50%, 75%, 95%, or any combination thereof of the copies of the target in the plurality of targets in the sample. The plurality of barcoded copies of the target may comprise at least 50%, 75%, 95%, or any combination thereof of the copies of each of the plurality of targets in the sample. The method may comprise reverse transcribing the plurality of targets to produce barcoded cDNAs of the plurality of targets. The method may include amplifying the barcoded cDNA using second strand synthesis to produce a barcoded target, wherein the barcoded target is double stranded. The method may include amplifying the barcoded target to produce a labeled target amplicon.

[0056] In some embodiments, the sample comprises a single cell, and the method comprises associating the synthetic particle with the single cell in the sample. The single cell may comprise a rare cell, a tumor cell, a cell from a human, a cell from a tissue, a cell from a tumor, a cell infected by a viral polynucleotide, and any combination thereof. The method may comprise: after associating the synthetic particle with the single cell, lysing the single cell. The method may comprise: lysing the single cell comprises heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof. The synthetic particle and the single cell may be in the same well. The synthetic particle and the single cell may be in the same droplet. The multiple targets may comprise ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA each containing a poly (A) tail, and any combination thereof.

[0057] In some embodiments, the ratio of the plurality of oligonucleotide barcodes and the first plurality of spacer oligonucleotides is in the range of from 1:10 to 10:1. Each of the first plurality of spacer oligonucleotides may comprise a cell marker sequence or a molecular marker sequence. At least one of the first plurality of spacer oligonucleotides may be shorter than at least one of the plurality of oligonucleotide barcodes. Each of the first plurality of spacer oligonucleotides may comprise a region corresponding to the target binding region, and wherein the region corresponding to the target binding region and the target binding region have different sequences. The region corresponding to the target binding region and the target binding region may have up to 50% sequence identity. At least two of the first plurality of spacer oligonucleotides may have different lengths. The sequences of at least two of the first plurality of spacer oligonucleotides may be the same. At least two of the first plurality of spacer oligonucleotides may comprise the same sequence.

[0058] In some embodiments, the synthetic particle may include a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not include a target binding region. At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the first plurality of spacer oligonucleotides. At least one of the second plurality of spacer oligonucleotides may be shorter than at least one of the plurality of oligonucleotide barcodes. At least two of the second plurality of spacer oligonucleotides may have different lengths. The sequences of at least two of the second plurality of spacer oligonucleotides may be the same. At least two of the second plurality of spacer oligonucleotides may include the same sequence.

[0059] In some embodiments, at least 100 of the multiple oligonucleotide barcodes contain different molecular marker sequences. The target binding region may contain a gene-specific sequence, an oligo (dT) sequence, a random polymer, or any combination thereof. The two target binding regions of two oligonucleotide barcodes in the multiple oligonucleotide barcodes may contain different gene-specific sequences. Each cell marker sequence of the multiple oligonucleotide barcodes may contain at least 6 nucleotides. Each molecular marker sequence of the multiple oligonucleotide barcodes may contain at least 6 nucleotides. Each of the multiple oligonucleotide barcodes may contain a spatial marker sequence, and wherein the spatial marker sequences of the multiple oligonucleotide barcodes differ from each other by at least one nucleotide. Each of the multiple oligonucleotide barcodes may contain a universal marker sequence, wherein the universal marker sequences of the multiple oligonucleotide barcodes are the same.

[0060] In some embodiments, at least one of the multiple oligonucleotide barcodes is fixed on, partially fixed on, encapsulated in, partially encapsulated in, or any combination thereof. The synthetic particles may be destructible. The synthetic particles may include beads. The beads may include agarose gel beads, streptavidin protein beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo (dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particles may include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methyl styrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, and any combination thereof. The synthetic particles may include destructible hydrogel particles.

[0061] In some embodiments, each of the plurality of oligonucleotide barcodes comprises a linker functional group, the synthetic particle comprises a solid support functional group, and / or the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can be individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Non-limiting exemplary barcodes are illustrated (eg, random barcodes).

[0063] Figure 2 A non-limiting exemplary workflow for performing barcoding and digital counting (eg, performing random barcoding and digital counting) is shown.

[0064] Figure 3 is a schematic diagram showing a non-limiting exemplary process for generating an indexed library from a plurality of targets (eg, barcoded targets randomly barcoded targets).

[0065] Figure 4A are non-limiting exemplary synthetic particles associated with a plurality of oligonucleotide barcodes. Figure 4B is a non-limiting exemplary synthetic particle associated with a plurality of oligonucleotide barcodes and a plurality of spacer oligonucleotides. Figure 4C1-4C2 are other non-limiting exemplary synthetic particles associated with a plurality of oligonucleotide barcodes and a plurality of spacer oligonucleotides. Figure 4D1-4D2 are non-limiting exemplary synthetic particles associated with a plurality of oligonucleotide barcodes having target-specific target binding regions. Figure 4D1-4D2 The synthetic particles shown in the figure may include Figure 4B and 4C1 -One or more of the plurality of spacer oligonucleotides described in -4C2 (not shown).

[0066] Figures 5A-5C The following non-limiting exemplary comparison is shown: Oligonucleotide barcodes having a poly(dT) region capable of hybridizing to the poly(A) tail of an mRNA molecule ( Figure 5A ); an oligonucleotide barcode having a target-specific target binding region capable of binding to one or more targets of interest ( Figure 5B ); oligonucleotide barcodes with a poly(dT) region and a target-specific target binding region ( Figure 5C ).

[0067] Figure 6 is a bar graph showing non-limiting exemplary improvements in RNA capture using beads, each bead comprising a plurality of oligonucleotide barcodes having an oligo(dT) tail capable of capturing RNA molecules and a plurality of spacer oligonucleotides.

[0068] Figure 7 is a bar graph showing a non-limiting exemplary comparison of T cell receptor (TCR) capture efficiency using beads with TCR-specific oligonucleotide barcodes. DETAILED DESCRIPTION

[0069] Reference is made to the accompanying drawings which form a part of this document in the following detailed description. In the accompanying drawings, similar symbols generally identify similar parts unless the context indicates otherwise. The illustrative embodiments described in the specific embodiments, drawings, and claims are not meant to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It is readily understood that the aspects of the present disclosure as generally described herein and illustrated in the drawings may be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein and form a part of the present disclosure.

[0070] All patents, published patent applications, other publications, and sequences from GenBank, and other databases mentioned herein with respect to the relevant art are incorporated by reference in their entirety.

[0071] Determining the quantity of nucleic acid or target (for example messenger ribonucleotide (mRNA) molecule) is clinically important for determining the gene expressed in cells at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (for example, mRNA molecules) is also very challenging, especially when the number of molecules is very small. A method for determining the absolute number of molecules in a sample is digital polymerase chain reaction (PCR). Ideally, PCR produces the same copy of molecules in each cycle. However, PCR may have a disadvantage that each molecule is replicated with a random probability, and this probability varies according to PCR cycles and gene sequences, which results in amplification bias and inaccurate gene expression measurements.

[0072] Barcodes (e.g., random barcodes) with unique molecular labels (ML, also called molecular index (MI)) can be used to count the number of molecules. Barcodes with molecular labels unique to each cell can be used to count the number of molecules in each cell. Non-limiting exemplary assays for barcoding include Precise TM Assay (Cellular Research, Inc. (Palo Alto, CA)), Resolve TM Assay (Cell Research, Inc. (Palo Alto, CA)) or Rhapsody TM Assay (Becton, Dickinson and Company (Franklin Lakes, NJ)). However, these methods and techniques can introduce errors that, if not corrected, can lead to an overestimation of cell counts.

[0073] Rhapsody TM The assay can utilize a non-exhaustive pool with a large number of (e.g., 6561 to 65536) barcodes (e.g., random barcodes), unique molecular markers on poly (T) oligonucleotides to hybridize with all poly (A)-mRNAs in the sample during the RT step. In addition to molecular markers, cell markers of barcodes can be used to identify each single cell in each hole of a microplate. The barcode may include a universal PCR initiation site. During RT, the target gene molecule reacts randomly with the barcode. Each target molecule can hybridize with a barcode (e.g., a random barcode) to produce a barcoded complementary ribonucleotide (cDNA) molecule (e.g., a randomly barcoded cDNA molecule). After labeling, the barcoded cDNA molecules from the microwells of the microplate can be merged into a single tube for PCR amplification and sequencing. The raw sequencing data can be analyzed to obtain the number of barcodes with unique molecular markers.

[0074] The disclosure herein includes embodiments of synthetic particles. In some embodiments, the synthetic particles comprise: a first plurality of oligonucleotide barcodes; and a second plurality of oligonucleotide barcodes, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cell marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region. In some embodiments, the target binding region of the oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. In some embodiments, the target binding region of the oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a poly(dT) region. The oligonucleotide barcode of the second plurality of oligonucleotide barcodes may comprise a poly(dT) region.

[0075] The disclosure herein includes embodiments of kits or compositions. In some embodiments, the kit or composition comprises: a plurality of synthetic particles, wherein each synthetic particle comprises: a first plurality of oligonucleotide barcodes; and a second plurality of oligonucleotide barcodes, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cell marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region; and optionally, instructions for using the plurality of synthetic particles. In some embodiments, the kit comprises a buffer, one or more reagents for a reverse transcription reaction, one or more reagents for an amplification reaction, or a combination thereof. In some embodiments, the plurality of synthetic particles comprises at least 94 synthetic particles. The kit may comprise a plurality of partitions, each partition comprising synthetic particles in the plurality of synthetic particles. The plurality of partitions may comprise microwells of a microwell array. The plurality of partitions may comprise a plurality of droplets. In some embodiments, the target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. The target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes may comprise a poly(dT) region. The oligonucleotide barcode of the second plurality of oligonucleotide barcodes may comprise a poly(dT) region.

[0076] The disclosure herein includes embodiments of methods for determining the number of targets in a sample. In some embodiments, the method includes: barcoding copies of a first plurality of targets using a first plurality of oligonucleotide barcodes associated with a synthetic particle to produce a first plurality of barcoded copies of the target; barcoding copies of a second plurality of targets in the sample using a second plurality of oligonucleotide barcodes associated with the synthetic particle to produce a second plurality of barcoded copies of the target, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cellular marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region; and evaluating the number of each of the first plurality of targets and / or the second plurality of targets using the molecular marker sequences of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes. In some embodiments, the target binding region of the oligonucleotide barcodes of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. The target binding region of the oligonucleotide barcodes of the first plurality of oligonucleotide barcodes may comprise a poly (dT) region. The oligonucleotide barcodes of the second plurality of oligonucleotide barcodes may comprise a poly (dT) region. In some embodiments, the first plurality of targets comprise mRNA molecules of cells, and wherein the second plurality of targets comprise DNA molecules of cells. The first plurality of targets may comprise mRNA molecules of cells, and the second plurality of targets may comprise RNA molecules of viruses, bacteria and / or fungi that infect cells.

[0077] Embodiments of particles associated with multiple oligonucleotides are disclosed herein. In some embodiments, the particles are synthetic particles. In some embodiments, the synthetic particles include: multiple oligonucleotide barcodes, wherein each of the multiple oligonucleotide barcodes includes the same cell marker, molecular marker, and target binding region, and wherein at least 10 of the multiple oligonucleotide barcodes include different molecular marker sequences; and a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not include a target binding region. The ratio of the multiple oligonucleotide barcodes to the first plurality of spacer oligonucleotides can be in the range of from 1:10 to 10:1. Each of the first plurality of spacer oligonucleotides can include a cell marker or a molecular marker.

[0078] Also provided herein are embodiments comprising a kit comprising one or more synthetic particles of the present disclosure. In some embodiments, the kit comprises: a plurality of synthetic particles, wherein each synthetic particle comprises: a plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes comprises the same cell marker, molecular marker, and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences; and a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region. The kit may comprise: instructions for using the plurality of oligonucleotide barcodes.

[0079] Embodiments disclosed herein include methods for determining the number of targets in a sample. In some embodiments, the method includes: barcoding a plurality of copies of a target in a sample using a plurality of oligonucleotide barcodes associated with a synthetic particle to generate a plurality of barcoded copies of a target, wherein each of the plurality of oligonucleotide barcodes comprises the same cellular marker, molecular marker, and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein the synthetic particle comprises a first plurality of spacer oligonucleotides, and wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region; and using the molecular markers of the plurality of oligonucleotide barcodes, assessing the number of each of the plurality of targets in the sample.

[0080] Disclosed herein are embodiments of methods for capturing targets in a sample. In some embodiments, the method comprises: hybridizing a plurality of copies of a target in a sample using a plurality of oligonucleotide barcodes associated with a synthetic particle to produce a plurality of barcoded copies of the target, wherein each of the plurality of oligonucleotide barcodes comprises the same cellular marker, molecular marker, and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein the synthetic particle comprises a first plurality of spacer oligonucleotides, and wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region.

[0081] Some definitions

[0082] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, New York 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, New York 1989). For the purpose of the present disclosure, the following terms are defined as follows.

[0083] As used herein, the term "adapter" may mean a sequence that promotes the amplification or sequencing of an associated nucleic acid. The associated nucleic acid may include a target nucleic acid. The associated nucleic acid may include one or more of a spatial marker, a target marker, a sample marker, an index marker, a barcode, a random barcode, or a molecular marker. The adaptor may be linear. The adaptor may be a pre-adenylated adaptor. The adaptor may be double-stranded or single-stranded. One or more adaptors may be located at the 5' or 3' end of the nucleic acid. When the adaptor includes a known sequence at the 5' and 3' ends, the known sequence may be the same or different sequences. The adaptor located at the 5' and / or 3' ends of the polynucleotide can hybridize with one or more oligonucleotides fixed on the surface. In certain embodiments, the adaptor may include a universal sequence. The universal sequence may be a region of a nucleotide sequence common to two or more nucleic acid molecules. Two or more nucleic acid molecules may have regions of different sequences. Therefore, for example, the 5' adaptor may include the same and / or universal nucleic acid sequence, and the 3' adaptor may include the same and / or universal sequence. The universal sequence that can be present in different members of multiple nucleic acid molecules can allow the use of a single universal primer complementary to the universal sequence to replicate or amplify multiple different sequences. Similarly, at least one, two (e.g., a pair) or more universal sequences that can be present in different members of the set of nucleic acid molecules can allow the use of at least one, two (e.g., a pair) or more single universal primers complementary to the universal sequence to replicate or amplify multiple different sequences. Therefore, universal primers include sequences that can hybridize with such universal sequences. The molecule carrying the target nucleic acid sequence can be modified to attach a universal adapter (e.g., a non-target nucleic acid sequence) to one or both ends of different target nucleic acid sequences. One or more universal primers attached to the target nucleic acid can provide a site for universal primer hybridization. One or more universal primers attached to the target nucleic acid can be identical or different from each other.

[0084] As used herein, the term "association" or "associated with..." may mean that two or more species can be identified as being co-located at a certain point in time. Association may mean that two or more species are or have been in similar containers. Association may be an informatics association, in which, for example, digital information about two or more species is stored and can be used to determine that one or more of the species are co-located at a certain point in time. Association may be a direct or indirect physical association. In some embodiments, two or more associated species are "tethered", "attached" or "fixed" to each other or to a common solid or semi-solid surface. Association may refer to a covalent or non-covalent means for attaching a marker to a solid or semi-solid support (e.g., a synthetic particle or bead). Association may be a covalent bond between a target and a marker.

[0085] As used herein, the term "complementarity" can refer to the ability of accurate pairing between two nucleotides. For example, if the nucleotides at a given position of a nucleic acid can be hydrogen-bonded with the nucleotides of another nucleic acid, the two nucleic acids are considered to be complementary to each other at the position. The complementarity between two single-stranded nucleic acid molecules can be "partial", wherein only some of the nucleotides are combined, or when there is complete complementarity between the single-stranded molecules, this complementarity can be complete. If the first nucleotide sequence is complementary to the second nucleotide sequence, it can be considered that the first nucleotide sequence is the "complement" of the second sequence. If the first nucleotide sequence is complementary to the sequence opposite to the second sequence (that is, the nucleotide order is opposite), it can be considered that the first nucleotide sequence is the "reverse complement" of the second sequence. As used herein, the terms "complement", "complementary" and "reverse complement" can be used interchangeably. It can be understood from this disclosure that if a molecule can hybridize with another molecule, it can be the complement of the hybridized molecule.

[0086] As used herein, the term "digital counting" may refer to a method for estimating the number of target molecules in a sample. Digital counting may include the step of determining the number of unique markers that have been associated with the target in the sample. This stochastic method transforms the problem of counting molecules from one of localization and identification of identical molecules to a series of yes / no digital questions about the detection of a set of predefined markers.

[0087] As used herein, the term (one or more) "marks" can refer to a nucleic acid code associated with a target in a sample. The mark can be, for example, a nucleic acid marker. The mark can be a fully or partially amplifiable mark. The mark can be a fully or partially sequenceable mark. The mark can be a part of a natural nucleic acid that can be identified as being different. The mark can be a known sequence. The mark can include a junction of a nucleic acid sequence, such as a junction of a natural and non-natural sequence. As used herein, the term "mark" can be used interchangeably with the term "index", "label" or "mark-label". Marks can convey information. For example, in various embodiments, a mark can be used to determine the identity of a sample, the source of a sample, the identity of a cell and / or a target.

[0088] As used herein, the term "non-depleting reservoir" may refer to a pool of random barcodes consisting of many different markers. A non-depleting reservoir may include a large number of different random barcodes, such that when the non-depleting reservoir is associated with a target pool, each target may be associated with a unique random barcode. The uniqueness of each labeled target molecule can be determined by the statistics of random selection and depends on the number of copies of the same target molecule in the set compared to the diverse markers. The size of the resulting set of labeled target molecules can be determined by the stochastic nature of the barcoding process, and then analysis of the number of random barcodes detected allows calculation of the number of target molecules present in the original set or sample. When the ratio of the number of copies of the target molecules present to the number of unique random barcodes is low, the labeled target molecules are highly unique (i.e., the probability of labeling more than one target molecule with a given marker is very low).

[0089] As used herein, the term "nucleic acid" refers to a polynucleotide sequence, or a fragment thereof. Nucleic acid may include nucleotides. Nucleic acid may be exogenous or endogenous to a cell. Nucleic acid may be present in a cell-free environment. Nucleic acid may be a gene or a fragment thereof. Nucleic acid may be DNA. Nucleic acid may be RNA. Nucleic acid may include one or more analogs (e.g., altered backbones, sugars, or nucleobases). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, foreign nucleic acids, morpholinos, locked nucleic acids, diol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugar), thiols containing nucleotides, biotin-linked nucleotides, fluorescent analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braided glycosides, and wyoside. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" may be used interchangeably.

[0090] Nucleic acids may include one or more modifications (e.g., base modifications, backbone modifications) to provide new or enhanced features (e.g., improved stability) to the nucleic acid. Nucleic acids may include nucleic acid affinity tags. Nucleosides may be base-sugar combinations. The base portion of a nucleoside may be a heterocyclic base. The two most common categories of such heterocyclic bases are purine and pyrimidine. Nucleotides may be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides including furanose, the phosphate group may be linked to the 2', 3' or 5' hydroxyl portion of the sugar. In forming nucleic acids, the phosphate group may covalently link adjacent nucleosides to each other to form a linear polymer compound. In turn, the respective ends of this linear polymer compound may further join to form a cyclic compound; however, linear compounds are generally suitable. In addition, linear compounds may have internal nucleotide base complementarity, and may therefore be folded in a manner that produces a fully or partially double-stranded compound. In nucleic acids, the phosphate group may generally be referred to as the internucleoside backbone that forms the nucleic acid. The linkage or backbone may be a 3' to 5' phosphodiester bond.

[0091] Nucleic acids can include modified backbones and / or modified internucleoside bonds. Modified backbones can include those that retain phosphorus atoms in the backbone and those that do not have phosphorus atoms in the backbone. Suitable modified nucleic acid backbones containing phosphorus atoms can contain, for example, phosphorothioates; chiral phosphorothioates; phosphorodithioates; phosphotriesters; aminoalkylphosphotriesters; methylphosphonates and other alkylphosphonates, such as 3'-alkylenephosphonates, 5'-alkylenephosphonates; chiral phosphonates; phosphites; phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates; phosphorodiamidates; thiocarbonylphosphoramidates; thiocarbonylalkylphosphonates; thiocarbonylalkylphosphotriesters; selenophosphates; and boranophosphates with normal 3'-5' bonds, analogs of 2'-5' connections and those with reverse polarity, wherein one or more internucleotide bonds are 3' to 3', 5' to 5' or 2' to 2' bonds.

[0092] Nucleic acid can include a polynucleotide backbone formed by a short-chain alkyl or cycloalkyl nucleoside bond, a mixed heteroatom, and an alkyl or cycloalkyl nucleoside bond or one or more short-chain heteroatomic or heterocyclic nucleoside bonds. These can include those having the following structures: a morpholino bond (partially formed from the sugar moiety of a nucleoside); a siloxane backbone; a sulfide, sulfoxide, and sulfone backbone; a formyl acetyl and thioformyl acetyl backbone; a methylene formyl acetyl and thioformyl acetyl backbone; a ribose acetyl backbone; an alkene-containing backbone; a sulfamic acid salt backbone; a methylene imino and methylene hydrazine backbone; a sulfonate and sulfonamide backbone; an amide backbone; and other backbones with mixed N, O, S, and CH2 component parts.

[0093] Nucleic acid can include nucleic acid mimics.Term " mimics " can be intended to include polynucleotides in which only furanose ring or furanose ring and internucleotide bond are replaced by non-furanose groups, and only replacing furanose ring can be called sugar substitute.Heterocyclic base part or modified heterocyclic base part can be kept in order to hybridize with appropriate target nucleic acid.A kind of such nucleic acid can be peptide nucleic acid (PNA).In PNA, the sugar backbone of polynucleotide can be replaced by backbone (particularly aminoethylglycine backbone) containing amides.Nucleotide can be kept and directly or indirectly attached to the nitrogen-nitrogen atom of the amide part of backbone.The backbone in PNA compound can include two or more aminoethylglycine units connected, which give PNA the backbone containing amides.Heterocyclic base part can be directly or indirectly attached to the nitrogen-nitrogen atom of the amide part of backbone.

[0094] Nucleic acid can include morpholino backbone structure.For example, nucleic acid can include 6-membered morpholino rings replacing ribose rings.In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside bonds can replace phosphodiester bonds.

[0095] Nucleic acid can include morpholino units (i.e. morpholino nucleic acids) having connections with heterocyclic bases attached to the morpholino ring. Linking groups can connect morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds can have less undesirable interactions with cell proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds in the morpholino category can be connected using different linking groups. Another class of polynucleotide mimics can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose rings that are usually present in nucleic acid molecules can be replaced by cyclohexenyl rings. Phosphoramidite monomers protected by CeNA DMT can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. CeNA monomers are incorporated into nucleic acid chains to increase the stability of DNA / RNA hybrids. CeNA oligoadenylic acid can form complexes with nucleic acid complements having stability similar to that of natural complexes. Additional modifications may include locked nucleic acids (LNA), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C, 4'-C-oxymethylene bond, thereby forming a bicyclic sugar moiety. The bond may be a methylene group (-CH2-) bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNA and LNA analogs may exhibit very high duplex thermal stability (Tm = +3°C to +10°C) with complementary nucleic acids, stability to 3'-exonucleoside degradation, and good solubility properties.

[0096] Nucleic acids can also include modifications or substitutions of nucleobases (often referred to simply as "bases"). As used herein, "unmodified" or "natural" nucleobases can include purine bases (e.g., adenine (A) and guanine (G)), and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases can 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 other alkynyl derivatives of cytosine and pyrimidine bases, 6-azouracil , cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy 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-deazaadenine. The modified nucleobases may include tricyclic pyrimidines 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), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido (5,4-(b) (1,4) benzoxazin-2 (3H) -one), phenothiazine cytidine ( 1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as substituted phenoxazine cytidine (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 (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0097] As used herein, the term "sample" may refer to a composition that includes a target. Suitable samples for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms.

[0098] As used herein, the term "sampling device" or "device" may refer to a device that can take a portion of a sample and / or place the portion on a substrate. The sampling device may refer to, for example, a fluorescence activated cell sorter (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a cascade and / or an ultramicrotome.

[0099] As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which a plurality of random barcodes may be attached. A solid support may include any type of solid, porous or hollow sphere, ball, seat, cylinder or other similar configuration, which is composed of plastic, ceramic, metal or polymer material (e.g., hydrogel), on which nucleic acids may be fixed (e.g., covalently or non-covalently). A solid support may include discrete particles that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as a cube, rectangle, cone, cylinder, cone, ellipse or disc, etc. A plurality of solid supports spaced apart in an array may not include a substrate. A solid support may be a particle, such as a synthetic particle or a bead.

[0100] A solid support may refer to a "substrate". A substrate may be a solid support. A substrate may refer to a continuous solid or semisolid surface on which the methods of the present disclosure may be performed. For example, a substrate may refer to an array, a box, a chip, a device, and a slide.

[0101] As used herein, the term "spatial marker" may refer to a marker that may be associated with a location in space.

[0102] As used herein, the term "random barcode" may refer to a polynucleotide sequence comprising a tag. A random barcode may be a polynucleotide sequence that can be used for random barcoding. A random barcode may be used to quantify a target in a sample. A random barcode may be used to control errors that may occur after a tag is associated with a target. For example, a random barcode may be used to assess amplification or sequencing errors. A random barcode associated with a target may be referred to as a random barcode-target or a random barcode-tag-target.

[0103] As used herein, the term "gene-specific random barcode" may refer to a polynucleotide sequence comprising a tag and a gene-specific target binding region. A random barcode may be a polynucleotide sequence that can be used for random barcoding. Random barcodes can be used to quantify targets in a sample. Random barcodes can be used to control errors that may occur after a tag is associated with a target. For example, random barcodes can be used to assess amplification or sequencing errors. A random barcode associated with a target may be referred to as a random barcode-target or a random barcode-tag-target.

[0104] As used herein, the term "random barcoding" can refer to random labeling (e.g., barcoding) of nucleic acids. Random barcoding can utilize a recursive Poisson strategy to correlate and quantify labels associated with a target. As used herein, the term "random barcoding" can be used interchangeably with "gene-specific random barcoding".

[0105] As used herein, the term "target" may refer to a composition that can be associated with a random barcode. Exemplary suitable targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, etc. The target may be single-stranded or double-stranded. In some embodiments, the target may be a protein. In some embodiments, the target is a lipid.

[0106] As used herein, the term "reverse transcriptase" may refer to a group of enzymes with reverse transcriptase activity (i.e., catalyzing the synthesis of DNA from an RNA template). Typically, such enzymes include, but are not limited to, reverse transcriptases derived from retroviral reverse transcriptases, retrotransposon reverse transcriptases, reverse transcriptase plasmid reverse transcriptases, reverse transcriptases, bacterial reverse transcriptases, and type II introns, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, reverse transcriptase plasmid reverse transcriptases, reverse transcriptases, and type II intron reverse transcriptases. Examples of type II intron reverse transcriptases include Lactococcus lactis L1.LtrB intron reverse transcriptases, elongated thermophilic synechococcus (Thermosynechococcuse longatus) TeI4c intron reverse transcriptases, or geobacillus stearothermophilus GsI-IIC intron reverse transcriptases. Other classes of reverse transcriptases can include many types of non-retroviral reverse transcriptases (ie, retrovirals, group II introns, and diversity-generating retroelements, etc.).

[0107] Disclosed herein are systems and methods for identifying signal cell markers. In some embodiments, the method includes: (a) using a plurality of random barcodes to randomly barcode a plurality of targets in a cell sample to create a plurality of randomly barcoded targets, wherein each of the plurality of random barcodes comprises a cell marker and a molecular marker; (b) obtaining sequencing data of the plurality of randomly barcoded targets; (c) determining the number of molecular markers with different sequences associated with each of the cell markers of the plurality of random barcodes; (d) based on the number of molecular markers with different sequences associated with each of the cell markers, determining the rank of each of the cell markers of the plurality of random barcodes ; (e) generating a cumulative sum graph based on the number of molecular markers with different sequences associated with each of the cell markers determined in (c) and the rank of each of the cell markers determined in (d); (f) generating a second-order derivative graph of the cumulative sum graph; (g) determining a minimum value of the second-order derivative graph of the cumulative sum graph, wherein the minimum value of the second-order derivative graph corresponds to a cell marker threshold; and (h) identifying each of the cell markers as a signal cell marker or a noise cell marker based on the number of molecular markers with different sequences associated with each of the cell markers determined in (c) and the cell marker threshold determined in (g).

[0108] Barcode

[0109] Barcoding (such as random barcoding) has been described in the following literature: for example, Fu et al., Proc Natl Acad Sci USA, May 31, 2011, 108 (22): 9026-31; U.S. Patent Application Publication No. US 2011 / 0160078; Fan et al., Science, February 6, 2015, 347 (6222): 1258367; U.S. Patent Application Publication No. US2015 / 0299784; and PCT Application Publication No. WO2015 / 031691; the contents of each of these documents (including any supporting or supplementary information or materials) are incorporated herein by reference in their entirety. In some embodiments, the barcodes disclosed herein can be random barcodes, which can be polynucleotide sequences that can be used to randomly label (e.g., barcodes, tags) a target. A barcode can be referred to as a stochastic barcode if the ratio of the number of different barcode sequences of a stochastic barcode to the number of occurrences of any target to be labeled is at or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. A target can be, for example, an mRNA species that includes mRNA molecules having identical or nearly identical sequences. A barcode can be referred to as a stochastic barcode if the ratio of the number of different barcode sequences of a stochastic barcode to the number of occurrences of any target to be labeled is at least, or at most, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100: 1. The barcode sequences of a stochastic barcode can be referred to as molecular markers.

[0110] A barcode (e.g., a random barcode) may include one or more tags. Exemplary tags may include a universal tag, a cell tag, a barcode sequence (e.g., a molecular tag), a sample tag, a plate tag, a spatial tag, and / or a pre-spatial tag. Figure 1An exemplary barcode 104 with spatial markers is shown. The barcode 104 may include a 5' amine that can link the barcode to a solid support 108. The barcode may include universal markers, dimensional markers, spatial markers, cellular markers, and / or molecular markers. The order of the different markers (including but not limited to universal markers, dimensional markers, spatial markers, cellular markers, and molecular markers) in the barcode may be changed. For example, Figure 1 In the embodiment of the present invention, the universal label can be a 5'-end label, and the molecular label can be a 3'-end label. Spatial label, dimensional label, and cell label can be in any order. In certain embodiments, universal label, spatial label, dimensional label, cell label, and molecular label are in any order. The barcode can include a target binding region. The target binding region can interact with the target (e.g., target nucleic acid, RNA, mRNA, DNA) in the sample. For example, the target binding region can include an oligo (dT) sequence that can interact with the poly (A) tail of mRNA. In some cases, the label of the barcode (e.g., universal label, dimensional label, spatial label, cell label, and barcode sequence) can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.

[0111] Mark (for example cell marker) can comprise a group of unique nucleic acid subsequences of defined length, for example each seven nucleotides (equivalent to the number of bits used in some Hamming error correction codes), which can be designed to provide error correction capability. Can design the error correction subsequence group comprising seven nucleotide sequences, so that any paired combination of the sequence in the group shows the "genetic distance" (or number of mismatched bases) of definition, for example a group of error correction subsequence can be designed to show the genetic distance of three nucleotides. In this case, the examination of the error correction sequence in the sequence data group of the target nucleic acid molecule through labeling (described more fully below) can allow detection or correction of amplification or sequencing errors. In certain embodiments, the length of the nucleic acid subsequence for generating error correction code can vary, for example, they can be, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 or the nucleotide length of the number or range between any two of these values. In certain embodiments, nucleic acid subsequences of other lengths can be used to generate error correction code.

[0112] The barcode may include a target binding region. The target binding region may interact with a target in a sample. The target may be or include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA each containing a poly (A) tail, or any combination thereof. In some embodiments, multiple targets may include deoxyribonucleic acid (DNA).

[0113] In some embodiments, the target binding region may include an oligo (dT) sequence that can interact with the poly (A) tail of the mRNA. One or more labels of the barcode (e.g., universal labels, dimensional labels, spatial labels, cell labels, and barcode sequences (e.g., molecular labels)) can be separated from another or two of the remaining labels of the barcode by one or more spacers. The length of the spacer can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, no label is separated by a spacer in the label of the barcode.

[0114] General Tags

[0115] The barcode may include one or more universal tags. In some embodiments, for all barcodes in the barcode group (attached to a given solid support), one or more universal tags may be the same. In some embodiments, for all barcodes attached to multiple particles (e.g., synthetic particles, such as beads), one or more universal tags may be the same. In some embodiments, the universal tag may include a nucleic acid sequence that can hybridize with a sequencing primer. A sequencing primer may be used to sequence the barcode including the universal tag. A sequencing primer (e.g., a universal sequencing primer) may include a sequencing primer associated with a high-throughput sequencing platform. In some embodiments, the universal tag may include a nucleic acid sequence that can hybridize with a PCR primer. In some embodiments, the universal tag may include a nucleic acid sequence that can hybridize with a sequencing primer and a PCR primer. The nucleic acid sequence of the universal tag that can hybridize with a sequencing or PCR primer may be referred to as a primer binding site. The universal tag may include a sequence that can be used to initiate transcription of the barcode. The universal tag may include a sequence that can be used to extend the barcode or the region within the barcode. The length of the universal marker can be, or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. For example, the universal marker can include at least about 10 nucleotides. The length of the universal marker can be at least, or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. In some embodiments, a cleavable linker or modified nucleotide can be part of the universal marker sequence to enable the barcode to be cleaved from the support.

[0116] Dimension tagging

[0117] The barcode may include one or more dimensional markers. In some embodiments, the dimensional marker may include a nucleic acid sequence that provides information about the dimension in which the marker (e.g., random marker) occurs. For example, the dimensional marker may provide information about the time when the target is randomly barcoded. The dimensional marker may be associated with the time of barcoding (e.g., random barcoding) in the sample. The dimensional marker may be activated at the time of the marker. Different dimensional markers may be activated at different times. The dimensional marker provides information about the order in which the target, target group, and / or sample are randomly barcoded. For example, a cell population may be randomly barcoded in the G0 phase of the cell cycle. In the G1 phase of the cell cycle, the cell may be pulsed again with a barcode (e.g., a random barcode). In the S phase of the cell cycle, the cell may be pulsed again with a barcode, and so on. The barcode at each pulse (e.g., each stage of the cell cycle) may contain different dimensional markers. In this way, the dimensional marker provides information about which targets are marked at which stage of the cell cycle. Dimensional markers can interrogate many different biological stages. Exemplary biological events may include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., cell, cell population) may be randomly labeled before and / or after treatment with a drug and / or therapy. Changes in the copy number of different targets may indicate the response of the sample to the drug and / or therapy.

[0118] The dimension marker can be activatable. The activatable dimension marker can be activated at a specific time point. The activatable marker can be, for example, constitutively activated (for example, not closed). The activatable dimension marker can be, for example, reversibly activated (for example, the activatable dimension marker can be turned on and off). The dimension marker can be, for example, reversibly activated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. The dimension marker can be reversibly activated, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. In some embodiments, the dimension marker can be activated by fluorescence, light, chemical events (for example, cutting, connection of another molecule, addition of modification (for example, pegylation, sumoization, acetylation, methylation, deacetylation, demethylation), photochemical events (for example, light locking) and introduction of non-natural nucleotides.

[0119] In some embodiments, the dimensional markings may be the same for all barcodes (e.g., random barcodes) attached to a given solid support (e.g., synthetic particles, such as beads), but may be different for different solid supports (e.g., synthetic particles). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the barcodes on the same solid support may contain the same dimensional markings. In some embodiments, at least 60% of the barcodes on the same solid support may include the same dimensional markings. In some embodiments, at least 95% of the barcodes on the same solid support may include the same dimensional markings.

[0120] Multiple solid supports (e.g., synthetic particles) can represent up to 10 6 The length of the dimension mark can be at least or at most 1,2,3,4,5,10,15,20,25,30,35,40,45,50 nucleotides or the nucleotides of the number or range between any two of these values. The length of the dimension mark can be at least or at most 1,2,3,4,5,10,15,20,25,30,35,40,45,50,100,200 or 300 nucleotides. The dimension mark can include the nucleotides between about 5 to about 200. The dimension mark can include the nucleotides between about 10 to about 150. The dimension mark can include the nucleotides of the length between about 20 to about 125.

[0121] Space Marking

[0122] The barcode may include one or more spatial markers. In some embodiments, the spatial marker may include a nucleic acid sequence that provides information about the spatial orientation of a target molecule associated with the barcode. The spatial marker may be associated with a coordinate in the sample. The coordinate may be a fixed coordinate. For example, the coordinate may be fixed with reference to a substrate. The spatial marker may be referenced to a two-dimensional or three-dimensional grid. The coordinate may be referenced to a landmark fixed coordinate. Landmarks may be identifiable in space. Landmarks may be structures that may be imaged. Landmarks may be biological structures, such as anatomical landmarks. Landmarks may be cell landmarks, such as organelles. Landmarks may be non-natural landmarks, such as structures with identifiable markers (such as color codes, barcodes, magnetism, fluorescence, radioactivity, or unique size or shape). The spatial marker may be associated with a physical partition (e.g., a hole, container, microsphere, tube, microcapsule, or droplet). In some embodiments, multiple spatial markers are used together to encode one or more positions in space.

[0123] The spatial markers may be the same for all barcodes attached to a given solid support (e.g., synthetic particles such as beads), but may be different for different solid supports (e.g., synthetic particles). In some embodiments, the percentage of barcodes on the same solid support including the same spatial markers may be, or about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of barcodes on the same solid support including the same spatial markers may be at least, or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, at least 60% of the barcodes on the same solid support may include the same spatial marker. In some embodiments, at least 95% of the barcodes on the same solid support may include the same spatial marker.

[0124] Multiple solid supports (e.g., synthetic particles such as beads) can represent up to 10 6 The length of spatial marker can be or about 1,2,3,4,5,10,15,20,25,30,35,40,45,50 nucleotides or the nucleotides of the number or scope between any two of these values. The length of spatial marker can be at least or at most 1,2,3,4,5,10,15,20,25,30,35,40,45,50,100,200 or 300 nucleotides. Spatial marker can include the nucleotide between about 5 to about 200. Spatial marker can include the nucleotide between about 10 to about 150. Spatial marker can include the nucleotide of length between about 20 to about 125.

[0125] Cell labeling

[0126] The barcode may include one or more cell markers. In some embodiments, the cell marker may include a nucleic acid sequence that provides information for determining which target nucleic acid is from which cell. In some embodiments, the cell marker is the same for all barcodes attached to a given solid support (e.g., a synthetic particle, such as a bead), but is different for different solid supports (e.g., a synthetic particle). In some embodiments, the percentage of barcodes on the same solid support including the same cell marker may be, or about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of barcodes on the same solid support including the same cell marker may be, or about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least 60% of the barcodes on the same solid support may include the same cell marker. As another example, at least 95% of the barcodes on the same solid support may include the same cell marker.

[0127] Multiple solid supports (e.g., synthetic particles) can represent up to 10 6 The length of cell labeling can be at or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides or the nucleotides of the number or scope between any two of these values. The length of cell labeling can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or 300 nucleotides. For example, cell labeling can include about 5 to about 200 nucleotides. As another example, cell labeling can include about 10 to about 150 nucleotides. Also as another example, cell labeling can include a length of about 20 to about 125 nucleotides.

[0128] Barcode sequence

[0129] The barcode may include one or more barcode sequences. In some embodiments, the barcode sequence may include a nucleic acid sequence that provides identification information for a particular type of target nucleic acid species that hybridizes to the barcode. The barcode sequence may include a nucleic acid sequence that provides a counter (e.g., provides a rough approximation) for a particular occurrence of a target nucleic acid species that hybridizes to a barcode (e.g., a target binding region).

[0130] In some embodiments, a plurality of barcode sequences are attached to a given solid support (e.g., a synthetic particle such as a bead). In some embodiments, there can be, or about 10 2 , 103 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 The plurality of barcodes may include at least 10, or at most 10, unique molecular marker sequences, or a number or range between any two of these values. For example, the plurality of barcodes may include about 6561 barcode sequences having different sequences. As another example, the plurality of barcodes may include about 65536 barcode sequences having different sequences. In some embodiments, there may be at least 10, or at most 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 The unique molecular marker sequence can be attached to a given solid support (e.g., a synthetic particle).

[0131] The barcode can be at or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values. The barcode can be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0132] Molecular markers

[0133] The barcode may include one or more molecular markers. The molecular marker may include a barcode sequence. In some embodiments, the molecular marker may include a nucleic acid sequence that provides identification information for a specific type of target nucleic acid species that hybridizes to the random barcode. The molecular marker may include a nucleic acid sequence that provides a counter for a specific occurrence of a target nucleic acid species that hybridizes to a random barcode (e.g., a target binding region).

[0134] In some embodiments, a plurality of molecular markers are attached to a given solid support (e.g., a synthetic particle such as a bead). In some embodiments, there can be, or about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9The plurality of random barcodes may include at least, or at most, 10 molecular marker sequences. For example, the plurality of random barcodes may include about 6561 molecular markers with different sequences. As another example, the plurality of random barcodes may include about 65536 molecular markers with different sequences. In some embodiments, there may be at least, or at most 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 A random barcode with a unique molecular marker sequence can be attached to a given solid support (e.g., a synthetic particle).

[0135] For stochastic barcoding using multiple stochastic barcodes, the ratio of the number of different molecular marker sequences to the number of occurrences of any target can be at or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. The target can be an mRNA species that includes mRNA molecules having the same or nearly identical sequence. In some embodiments, the ratio of the number of different molecular marker sequences to the number of occurrences of any target is at least, or at most, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

[0136] The length of a molecular marker can be or is about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values. The length of a molecular marker can be at least, or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0137] Target binding region

[0138] The barcode may include one or more target binding regions, such as capture probes. In certain embodiments, the target binding region may hybridize with the target of interest. In certain embodiments, the target binding region may include a nucleic acid sequence that specifically hybridizes with a target (e.g., a target nucleic acid, such as a cell nucleic acid to be analyzed). For example, the target binding region may hybridize with a target nucleic acid on a specific gene sequence. In certain embodiments, the target binding region may include a nucleic acid sequence that can be attached (e.g., hybridized) to a specific position of a specific target nucleic acid. In certain embodiments, the target binding region may include a nucleic acid sequence that can specifically hybridize with a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode may then be connected to any nucleic acid molecule comprising a sequence complementary to a restriction site overhang.

[0139] In some embodiments, the target binding region may include a non-specific target nucleic acid sequence. Non-specific target nucleic acid sequences may refer to sequences that are independent of a specific sequence of a target nucleic acid and that can be combined with multiple target nucleic acids. For example, the target binding region may include a random polymer sequence or an oligomer (dT) sequence hybridized with a poly (A) tail on an mRNA molecule. The random polymer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer or a higher polymer sequence of any length. In some embodiments, for all barcodes attached to a given synthetic particle (e.g., a bead), the target binding region is identical. In some embodiments, for multiple barcodes attached to a given synthetic particle, the target binding region may include two or more different target binding sequences. The length of the target binding region may be or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. The target binding region may be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length.

[0140] In some embodiments, the target binding region can include an oligo(dT) that can hybridize to an mRNA that includes a polyadenylated end. The target binding region can be gene specific. For example, the target binding region can be configured to hybridize to a specific region of the target. The length of the target binding region can be or is about 1, 2, 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 nucleotides, or a number or range of nucleotides between any two of these values. The target binding region can be at least, or at most, 1, 2, 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, or 30 nucleotides in length. The target binding region can be about 5 to 30 nucleotides in length. When a barcode includes a gene-specific target binding region, the barcode can be referred to as a gene-specific barcode.

[0141] In some embodiments, the barcode does not include a target binding region. In some embodiments, the barcode includes a region corresponding to a target binding region having a sequence that has low binding affinity (e.g., does not bind) to some, most, substantially all, or all mRNA molecules in one or more cells of the sample. For example, a barcode may include a region corresponding to a target binding region having a sequence that does not bind to an mRNA molecule of interest. If the target binding region of the first barcode includes an oligo(dT) sequence that can hybridize with the poly(A) tail of an mRNA molecule, the corresponding region of the second barcode may include, for example, a sequence that is dissimilar or substantially similar to the poly(dT) sequence. If the target binding region of the first barcode includes a target binding region having a sequence that can specifically hybridize with a specific gene sequence, the corresponding region of the second barcode may include, for example, a sequence that is dissimilar or substantially similar to the target binding region.

[0142] Directional characteristics

[0143] The barcode may include one or more directional properties that can be used to orient (e.g., align) the barcode. The barcode may include a portion for isoelectric focusing. Different barcodes may contain different isoelectric focusing points. When these barcodes are introduced into a sample, the sample may undergo isoelectric focusing so that the barcode is positioned in a known manner. In this way, the directional properties may be used to develop known mappings of barcodes in a sample. Exemplary directional properties may include electrophoretic mobility (e.g., based on the size of the barcode), isoelectric point, spin, conductivity, and / or self-assembly. For example, a barcode has a directional property of self-assembly and can self-assemble into a specific orientation (e.g., a nucleic acid nanostructure) when activated.

[0144] Affinity characteristics

[0145] The barcode may include one or more affinity properties. For example, the spatial marker may include an affinity property. The affinity property may include a chemical and / or biological part that may promote the binding of the barcode to another entity (e.g., a cell receptor). For example, the affinity property may include an antibody, for example, an antibody specific for a specific part (e.g., a receptor) on a sample. In some embodiments, the antibody may guide the barcode to a specific cell type or molecule. Targets at and / or near a specific cell type or molecule may be randomly labeled. In some embodiments, in addition to the spatially labeled nucleotide sequence, the affinity property may provide spatial information because the antibody may guide the barcode to a specific location. The antibody may be a therapeutic antibody, such as a monoclonal antibody or a polyclonal antibody. The antibody may be humanized or chimeric. The antibody may be a naked antibody or a fusion antibody.

[0146] Antibodies can be full-length (ie, naturally occurring or formed by normal immunoglobulin gene fragment recombination processes) immunoglobulin molecules (eg, IgG antibodies) or immunologically active (ie, specifically binding) portions of immunoglobulin molecules (such as antibody fragments).

[0147] The antibody fragment can be, for example, a part of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. In some embodiments, the antibody fragment can bind to the same antigen recognized by the full-length antibody. The antibody fragment can include a separated fragment consisting of the variable region of the antibody, such as a "Fv" fragment consisting of the variable region of the heavy chain and the light chain, and a recombinant single-chain polypeptide molecule ("scFv protein") in which the light chain and the heavy chain variable region are connected by a peptide linker. Exemplary antibodies can include, but are not limited to, cancer cell antibodies, viral antibodies, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

[0148] Universal adapter primer

[0149] The barcode can include one or more universal adapter primers. For example, a gene-specific barcode (such as a gene-specific random barcode) can include a universal adapter primer. A universal adapter primer can refer to a nucleotide sequence that is universal across all barcodes. A universal adapter primer can be used to construct a gene-specific barcode. The length of a universal adapter primer can be, or is about 1, 2, 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 nucleotides, or a number or range of nucleotides between any two of these values. The length of the universal adapter primer can be at least, or at most, 1, 2, 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, or 30 nucleotides. The length of the universal adapter primer can be 5 to 30 nucleotides.

[0150] Connectors

[0151] When the barcode includes more than one type of marker (e.g., more than one cell marker or more than one barcode sequence, such as a molecular marker), the markers can be interspersed with adapter marker sequences. The length of the adapter marker sequence can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides. The length of the adapter marker sequence can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides. In some cases, the length of the adapter marker sequence is 12 nucleotides. The adapter marker sequence can be used to facilitate the synthesis of the barcode. The adapter marker can include an error correction (e.g., Hamming) code.

[0152] Solid support

[0153] In some embodiments, the barcodes disclosed herein (such as random barcodes) can be associated with a solid support. For example, the solid support can be a particle or a synthetic particle. In some embodiments, some or all barcode sequences (such as molecular markers of random barcodes (e.g., first barcode sequences)) of a plurality of barcodes on a solid support have a difference of at least one nucleotide. The cell markers of the barcodes on the same solid support can be the same. The cell markers of the barcodes on different solid supports can have a difference of at least one nucleotide. For example, the first cell marker of the first plurality of barcodes on the first solid support can have the same sequence, and the second cell marker of the second plurality of barcodes on the second solid support can have the same sequence. The first cell marker of the first plurality of barcodes on the first solid support and the second cell marker of the second plurality of barcodes on the second solid support can have a difference of at least one nucleotide. The cell marker can be, for example, about 5 to 20 nucleotides long. The barcode sequence can be, for example, about 5 to 20 nucleotides long. The synthetic particle can be, for example, a bead.

[0154] The synthetic particles can be, for example, silica beads, controlled pore glass beads, magnetic beads, Dynabeads, cross-linked dextran / agarose beads, beaded cellulose, polystyrene beads, or any combination thereof. The synthetic particles can comprise materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogels, paramagnetic substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, or any combination thereof.

[0155] In some embodiments, the synthetic particles can be polymer particles (e.g., deformable particles or gel particles) functionalized with barcodes or random barcodes (such as gel beads from 10X Genomics (San Francisco, California)). In some implementations, the gel particles can include one or more polymer-based gels. For example, the gel particles can be produced by encapsulating one or more polymer precursors into droplets. The gel particles can be produced after exposing the polymer precursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)).

[0156] In some embodiments, the particles can be degradable. For example, the polymer particles or beads can dissolve, melt or degrade, for example, under desired conditions. The desired conditions can include environmental conditions. The desired conditions can cause the polymer particles to dissolve, melt or degrade in a controlled manner. Due to chemical stimulation, physical stimulation, biological stimulation, thermal stimulation, magnetic stimulation, electrical stimulation, light stimulation or any combination thereof, the gel particles can dissolve, melt or degrade.

[0157] Reagents (such as oligonucleotide barcodes) can, for example, be coupled / fixed to the inner surface of synthetic particles (the accessible interior of the diffusion of the oligonucleotide barcode and / or the material for generating the oligonucleotide barcode) and / or the outer surface of the gel particles or any other microcapsules described herein. The particles can be, for example, gel beads. The association (e.g., coupling / fixation) can be via any form of chemical bonds (e.g., covalent bonds, ionic bonds) or physical phenomena (e.g., van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, the association (e.g., coupling / fixation) of the reagent with the particle or any other solid support described herein (e.g., microcapsule) can be reversible, for example, via an unstable portion (e.g., via a chemical crosslinker, including a chemical crosslinker described herein). After applying a stimulus, the unstable portion can be cut and release the immobilized reagent. In some embodiments, the unstable portion is a disulfide bond. For example, in the case where the oligonucleotide barcode is fixed to the gel particles via a disulfide bond, exposing the disulfide bond to a reducing agent can cut the disulfide bond and release the oligonucleotide barcode from the particle. The labile moiety can be included as part of a gel particle, bead, or microcapsule, as part of a chemical linker that connects the agent to the gel bead or microcapsule, and / or as part of the agent. In some embodiments, at least one barcode of the plurality of barcodes can be immobilized on a particle, partially immobilized on a particle, encapsulated in a particle, partially encapsulated in a particle, or any combination thereof.

[0158] In some embodiments, the particles (e.g., gel beads) can comprise a variety of different polymers, including but not limited to: polymers, thermosensitive polymers, photosensitive polymers, magnetic polymers, pH sensitive polymers, salt sensitive polymers, chemical sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins and / or plastics. The polymer may include, but is not limited to, the following materials: such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrene sulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(bisallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrrole) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methyl methacrylate) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(o-phthalaldehyde) (PTHF), poly(hexylviologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), poly(lactic acid-polyglycolic acid) (PLGA).

[0159] Many chemical stimuli can be used to trigger the destruction, dissolution, or degradation of particles. Examples of these chemical changes may include, but are not limited to, pH-mediated changes in the particle wall, chemical cleavage of cross-linked bonds to decompose the particle wall, triggered depolymerization of the particle wall, and particle wall conversion reactions. Batch changes can also be used to trigger the destruction of particles.

[0160] Batch or physical changes to microcapsules or particles by various stimuli also provide many advantages in designing capsules to release agents. Batch or physical changes occur on a macroscopic scale, where particle rupture is the result of mechanical-physical forces caused by the stimulus. These processes may include, but are not limited to, pressure-induced rupture, particle wall melting, or changes in the porosity of the particle wall.

[0161] Biostimulation can also be used to trigger the destruction, dissolution, or degradation of particles. Generally, biological triggers are similar to chemical triggers, but many examples use biomolecules, or molecules common in living systems, such as enzymes, peptides, sugars, fatty acids, nucleic acids, etc. For example, particles may contain polymers with peptide crosslinks that are sensitive to cleavage by specific proteases. More specifically, one example may include microcapsules containing GFLGK peptide crosslinks. Upon addition of a biological trigger (such as the protease cathepsin B), the peptide crosslinks of the shell pores are cut and the contents of the particles are released. In other cases, the protease can be heat activated. In another example, the particle includes a shell wall containing cellulose. The addition of the hydrolase chitosan is used as a biotrigger for cellulose bond cleavage, shell wall depolymerization, and internal content release.

[0162] It is also possible to induce the particles to release their contents after applying a thermal stimulus. Changes in temperature can result in various changes in the particles. Changes in heat can cause the particles to melt, causing the particle wall to disintegrate. In other cases, heat can increase the internal pressure of the components inside the particles, causing the particles to rupture or explode. In still other cases, heat can cause the particles to become dehydrated and contracted. Heat can also act on the thermosensitive polymers in the particle wall, causing the destruction of the particles.

[0163] Including magnetic nanoparticles in the particle wall of the microcapsule can allow the triggered rupture of the particles and guide the particles into an array. The device disclosed herein can contain magnetic particles for any purpose. In one example, Fe3O4 nanoparticles are incorporated into particles containing polyelectrolytes to trigger rupture in the presence of an oscillating magnetic field stimulus.

[0164] Particles may also be destroyed, dissolved, or degraded as a result of electrical stimulation. Similar to the magnetic particles described in the previous section, electrosensitive particles can allow for triggered rupture of particles as well as other functions such as alignment in an electric field, conductivity, or redox reactions. In one example, particles containing electrosensitive materials are aligned in an electric field so that the release of an internal agent can be controlled. In other examples, the electric field can induce a redox reaction within the particle wall itself, which can increase porosity.

[0165] Particles can also be destroyed with light stimulation. Many light triggers are possible and can include systems using various molecules such as nanoparticles and chromophores that can absorb photons of a specific wavelength range. For example, metal oxide coatings can be used as capsule triggers. UV irradiation of polyelectrolyte capsules coated with SiO2 can lead to the disintegration of the particle wall. In yet another example, light-switchable materials such as azobenzene groups can be incorporated into the particle wall. After application of UV or visible light, chemicals such as these undergo reversible cis-trans isomerization after absorbing photons. In this regard, the incorporation of photon switching causes the particle wall to disintegrate or become more porous after the application of a light trigger.

[0166] For example, in Figure 2 In the non-limiting example of barcoding (e.g., random barcoding) shown in FIG, after introducing cells (such as single cells) into a plurality of microwells of a microwell array at block 208, particles can be introduced into a plurality of microwells of a microwell array at block 212. Each microwell can contain a particle. The particle can contain a plurality of barcodes. The barcode can contain a 5' amine region attached to the particle. The barcode can include a universal tag, a barcode sequence (e.g., a molecular tag), a target binding region, or any combination thereof.

[0167] The barcodes disclosed herein can be associated (e.g., attached) to a solid support (e.g., a particle or a bead). Each barcode associated with a solid support can include a barcode sequence selected from the group consisting of at least 100 or 1000 barcode sequences with a unique sequence. In some embodiments, different barcodes associated with a solid support can include barcode sequences of different sequences. In some embodiments, the percentage of barcodes associated with a solid support includes the same cell marker. For example, the percentage can be or is about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. As another example, the percentage can be at least, or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, the barcodes associated with a solid support can have the same cell marker. The barcodes associated with different solid supports can have different cell markers selected from the group consisting of at least 100 or 1000 cell markers having unique sequences.

[0168] The barcodes disclosed herein can be associated (e.g., attached) to a solid support (e.g., a particle, such as a bead). In some embodiments, a solid support including a plurality of synthetic particles associated with a plurality of barcodes can be used to randomly barcode a plurality of targets in a sample. In some embodiments, a solid support may include a plurality of synthetic particles associated with a plurality of barcodes. The spatial markings of a plurality of barcodes on different solid supports may have a difference of at least one nucleotide. A solid support may, for example, include a plurality of barcodes in two or three dimensions. Synthetic particles may be beads. Particles may be silica beads, controlled pore glass beads, magnetic beads, Dynabeads, cross-linked dextran / agarose beads, beaded cellulose, polystyrene beads, or any combination thereof. A solid support may include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, a solid support may be free floating. In some embodiments, a solid support may be embedded in a semi-solid or solid array. A barcode may not be associated with a solid support. A barcode may be a single nucleotide. A barcode may be associated with a substrate.

[0169] As used herein, the terms "tethered," "attached," and "fixed" are used interchangeably and may refer to covalent or non-covalent means for attaching a barcode to a solid support. Any of a variety of different solid supports may be used as a solid support for attaching pre-synthesized barcodes or for in situ solid phase synthesis of barcodes.

[0170] In some embodiments, the solid support is a particle, such as a bead. The particle may comprise one or more types of solid, porous or hollow spheres, balls, sockets, cylinders or other similar configurations, on which nucleic acids may be fixed (e.g., covalently or non-covalently). The particle may be, for example, made of plastic, ceramic, metal, polymeric material, or any combination thereof. The particle may be, or include, spherical (e.g., microspheres) or discrete particles having a non-spherical or irregular shape, such as a cube, rectangle, cone, cylinder, conical, elliptical or disc-shaped, etc. In some embodiments, the shape of the particle may be non-spherical.

[0171] The particles can comprise a variety of materials, including but not limited to paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (Fe3O4; magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramics, plastics, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, cross-linked agarose, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof.

[0172] In some embodiments, the particle (eg, the particle to which the label is attached) is a hydrogel bead. In some embodiments, the particle comprises a hydrogel.

[0173] Some embodiments disclosed herein include one or more particles (e.g., beads). Each of the particles may include multiple oligonucleotides (e.g., barcodes). Each of the multiple oligonucleotides may include a barcode sequence (e.g., a molecular marker), a cell marker, and a target binding region (e.g., an oligo (dT) sequence, a gene-specific sequence, a random polymer, or a combination thereof). The cell marker sequence of each of the multiple oligonucleotides may be the same. The cell marker sequences of the oligonucleotides on different particles may be different, so that the oligonucleotides on different particles may be identified. In different implementations, the number of different cell marker sequences may be different. In some embodiments, the number of cell marker sequences can be or is about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 10 ... 6 , 10 7 , 10 8 , 10 9, a number or range between any two of these values, or more. In some embodiments, the number of cell marker sequences can be at least, or at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 10 ... 6 , 10 7 , 10 8 , or 10 9 In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more of the plurality of particles include oligonucleotides having the same cell sequence. In some embodiments, the plurality of particles including oligonucleotides having the same cell sequence may be at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, no particles in the plurality of particles have the same cell marker sequence.

[0174] The plurality of oligonucleotides on each particle can comprise different barcode sequences (e.g., molecular markers). In some embodiments, the number of barcode sequences can be or is about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 10 ... 6 , 10 7 , 10 8 , 10 9, or a number or range between any two of these values. In some embodiments, the number of barcode sequences can be at least, or at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 10 ... 6 , 10 7 , 10 8 , or 10 9 For example, at least 100 of the plurality of oligonucleotides comprise a different barcode sequence. As another example, in a single particle, at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, a number or range between any two of these values, or more of the plurality of oligonucleotides comprise a different barcode sequence. Some embodiments provide a plurality of particles comprising a barcode. In some embodiments, the ratio of the occurrence (or copies or number) of the target to be labeled and the different barcode sequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or higher. In some embodiments, each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both. The particle can be, for example, a nanoparticle or a microparticle.

[0175] The size of the particles can vary. For example, the diameter of the particles can range from 0.1 microns to 50 microns. In some embodiments, the diameter of the particles can be, or is approximately, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 microns, or a number or range between any two of these values.

[0176] The diameter of the particle can be related to the diameter of the hole of the substrate. In some embodiments, the diameter of the particle can be longer or shorter, or about longer or shorter by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values ​​compared to the diameter of the hole. The diameter of the particle can be related to the diameter of the cell (e.g., a single cell trapped by the hole of the substrate). In some embodiments, the diameter of the particle can be longer or shorter by at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the diameter of the hole. The diameter of the particle can be related to the diameter of the cell (e.g., a single cell trapped by the hole of the substrate). In some embodiments, the diameter of the particle can be longer or shorter, or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% longer or shorter, or a number or range between any two of these values, compared to the diameter of the cell. In some embodiments, the diameter of the particle can be longer or shorter by at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% compared to the diameter of the cell.

[0177] The particle can be attached to and / or embedded in a substrate. The particle can be attached and / or embedded in a gel, a hydrogel, a polymer and / or a matrix. Using a spatial marker present in the particle that can serve as a barcode for a position address, the spatial position of the particle in a matrix (e.g., a gel, a matrix, a support, or a polymer) can be identified.

[0178] Examples of particles may include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, microbeads, antibody-conjugated beads (e.g., anti-immunoglobulin microbeads), protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and BcMag TM Carboxyl-terminated magnetic beads.

[0179] The particles can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make them fluorescent in one or more fluorescent light channels. The particles can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The particles can be identifiable. For example, the particles can be imaged using a camera. The particles can have a detectable code associated with the particles. For example, the particles can include a bar code. The size of the particles can vary, for example due to swelling in organic or inorganic solutions. The particles can be hydrophobic. The particles can be hydrophilic. The particles can be biocompatible.

[0180] The solid support (e.g., particle) can be visualized. The solid support can include a visualization label (e.g., a fluorescent dye). The solid support (e.g., particle) can be etched with a logo (e.g., a number). The logo can be visualized by imaging the particle.

[0181] The solid support may include insoluble, semi-soluble or insoluble materials. When the solid support includes a linker, a scaffold, a building block or other reactive moieties attached thereto, it may be referred to as "functionalized", whereas when the solid support lacks such a reactive moiety attached thereto, it may be referred to as "non-functionalized". The solid support may be utilized unconstrained in solution, such as in a microtiter well format; in a flow-through format, such as in a column; or in a dipstick.

[0182] Solid supports can include membranes, paper, plastics, coated surfaces, flat surfaces, glass, slides, chips, or any combination thereof. Solid supports can be in the form of resins, gels, microspheres, or other geometric configurations. Solid supports can include silica chips; microparticles; nanoparticles; flat plates; arrays; capillaries; flat supports, such as glass fiber filters, glass surfaces, metal surfaces (steel, gold, silver, aluminum, silicon, and copper), glass supports, plastic supports, silicon supports, chips, filters, membranes, microplates, slides; plastic materials include porous plates or membranes (e.g., formed by polyethylene, polypropylene, polyamide, polyvinylidene fluoride); and / or wafers; combs; needles or needles (e.g., needle arrays suitable for combinatorial synthesis or analysis); or particles (e.g., synthetic particles, such as beads) in an array of recesses or nanoliter holes on a flat surface (e.g., a wafer (e.g., a silicon wafer), a wafer with recesses with or without a filter bottom).

[0183] The solid support may include a polymer matrix (e.g., a gel, a hydrogel). The polymer matrix may be capable of penetrating intracellular spaces (e.g., around organelles). The polymer matrix may be capable of being pumped throughout the circulatory system.

[0184] The solid support can be a biomolecule. For example, the solid support can be or can include nucleic acids, proteins, antibodies, histones, cell compartments, lipids, carbohydrates, etc. The solid support as a biomolecule can be amplified, translated, transcribed, degraded and / or modified (for example, pegylated, sumoylated, acetylated, methylated). In addition to being attached to the spatial markers of the biomolecule, the solid support as a biomolecule can provide spatial and temporal information. For example, a biomolecule can include a first conformation when unmodified, but can be changed to a second conformation when modified. These different conformations can expose the barcode of the present disclosure (for example, a random barcode) to the target. For example, a biomolecule can include a barcode that is inaccessible due to the folding of the biomolecule. When modifying a biomolecule (for example, acetylated), these biomolecules can change conformations to expose these barcodes. The time setting of modification can provide another time dimension for the barcoding method of the present disclosure.

[0185] In some embodiments, biomolecules comprising the barcode reagents disclosed herein can be located in the cytoplasm of a cell. Upon activation, the biomolecules can move to the nucleus where they can be barcoded. In this way, modification of the biomolecules can encode additional spatio-temporal information of the target identified by the barcode.

[0186] Substrate and microwell array

[0187] As used herein, a substrate may refer to a solid support. A substrate may refer to a solid support that may include a barcode and a random barcode of the present disclosure. For example, a substrate may include a plurality of micropores. For example, a substrate may be a well array including two or more micropores. In some embodiments, a micropore may include a small reaction chamber with a determined volume. In some embodiments, a micropore may trap one or more cells. In some embodiments, a micropore may only trap one cell. In some embodiments, a micropore may trap one or more solid supports. In some embodiments, a micropore may only trap one solid support. In some embodiments, a micropore traps a single cell and a single solid support (e.g., a particle). A micropore may include a combined barcode reagent of the present disclosure.

[0188] Synthesis of barcodes on solid supports

[0189] Barcodes (e.g., random barcodes) can be synthesized on a solid support (e.g., a particle, such as a synthetic particle or bead). Pre-synthesized barcodes (e.g., comprising a 5' amine that can be attached to a solid support) can be attached to a solid support (e.g., a particle) by any of a variety of immobilization techniques (involving functional group pairs on the solid support and the barcode). The barcode can comprise a functional group. The solid support (e.g., a particle) can comprise a functional group. The barcode functional group and the solid support functional group can comprise, for example, biotin, streptavidin, one or more primary amines, one or more carboxyl groups, one or more hydroxyl groups, one or more aldehydes, one or more ketones, and any combination thereof. The barcode (e.g., random barcode) can be tethered to a solid support by coupling the 5' amino group on the barcode (e.g., using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) to the carboxyl group of the functionalized solid support. Residual uncoupled barcodes can be removed from the reaction mixture by performing multiple rinsing steps. In some embodiments, the barcode and solid support are indirectly attached via a linker molecule (e.g., a short functionalized hydrocarbon molecule or polyethylene oxide molecule) using similar attachment chemistry. The linker can be a cleavable linker, such as an acid-labile linker or a photocleavable linker.

[0190] Barcodes (e.g., random barcodes) can be synthesized on a solid support (e.g., particles) using any of a variety of solid phase oligonucleotide synthesis techniques (e.g., phosphodiester synthesis, phosphotriester synthesis, phosphite triester synthesis, and phosphoramidite synthesis). Single nucleotides can be coupled to a growing tethered oligonucleotide in a stepwise manner. Short presynthesized sequences (or blocks) of several oligonucleotides can be coupled to a growing tethered barcode.

[0191] Barcodes (e.g., random barcodes) can be synthesized using one or more rounds of split-pool synthesis by interspersed stepwise or block coupling reactions, in which the total pool of synthesized particles is divided into multiple individual smaller pools, which are then each subjected to a different coupling reaction, followed by recombining and mixing the individual pools to randomize the growing barcode sequence across the entire pool of particles. Split-pool synthesis is an example of a combinatorial synthesis process in which the maximum number of chemical compounds is synthesized using the minimum number of chemical coupling steps. The potential diversity of the resulting compound library is determined by the number of unique building blocks (e.g., nucleotides) that can be used for each coupling step and the number of coupling steps used to generate the library. For example, a split-pool synthesis using 4 different nucleotides in each step including 10 rounds of coupling will produce 4 10= 1,048,576 unique nucleotide sequences. In some embodiments, the split-pool synthesis can be performed using an enzymatic method (e.g., polymerase extension or ligation reaction) rather than chemical coupling. For example, in each round of the split-pool polymerase extension reaction, the 3' end of the barcode tethered to the particle in a given pool can be hybridized to the 5' end of a set of semi-random primers, such as 5'-(M) k -(X) i -(N) j -3' structure primer, where (X) i is a random nucleotide sequence of i nucleotides in length (the primer set includes (X) i all possible combinations of j is a specific nucleotide (or series of j nucleotides), and (M) k is a specific nucleotide (or series of k nucleotides) where a different deoxyribonucleotide triphosphate (dNTP) is added to each pool and incorporated into the tethered oligonucleotide by the polymerase.

[0192] Barcoding method

[0193] The present disclosure provides methods for assessing the number of different targets in a sample. In some embodiments, barcoding a plurality of targets comprises hybridizing a plurality of barcodes to a plurality of targets to create barcoded targets (e.g., randomly barcoded targets). Barcoding a plurality of targets may comprise generating an index library of barcoded targets. Generating an index library of barcoded targets may be performed with a solid support comprising a plurality of barcodes (e.g., random barcodes).

[0194] Bring the sample and barcode into contact

[0195] The present disclosure provides a method for contacting a sample (e.g., a cell) with a substrate of the present disclosure. A sample including, for example, a cell, an organ, or a tissue slice can be contacted with a barcode (e.g., a random barcode). For example, the cell can be contacted by gravity flow, wherein the cell can be precipitated and a monolayer of cells can be produced. The sample can be a tissue slice. The slice can be placed on a substrate. The sample can be one-dimensional (e.g., forming a plane). The sample (e.g., a cell) can be coated on a substrate, for example, by growing / culturing the cell on a substrate.

[0196] When the barcode is close to the target, the target can hybridize with the barcode. The barcodes can be contacted at a non-exhaustive ratio so that each different target can be associated with a different barcode of the present disclosure. To ensure effective association between the target and the barcode, the target and the barcode can be cross-linked.

[0197] Cell lysis

[0198] After the distribution of cells and barcodes, the cells can be lysed to release the target molecules. Cell lysis can be accomplished by any of a variety of means, such as by chemical or biochemical means, by osmotic shock, or by thermal lysis, mechanical lysis or optical lysis. Cells can be lysed by adding a cell lysis buffer including a detergent (e.g., SDS, lithium dodecyl sulfate, Triton X-100, Tween-20 or NP-40), an organic solvent (e.g., methanol or acetone) or a digestive enzyme (e.g., proteinase K, pepsin or trypsin) or any combination thereof. In order to increase the association of the target and the barcode, the diffusion rate of the target molecule can be changed by, for example, lowering the temperature of the lysate and / or increasing the viscosity of the lysate.

[0199] In certain embodiments, filter paper can be used for sample cracking. The filter paper can be impregnated with lysis buffer on the top of the filter paper. The filter paper can be applied to the sample under pressure, and the pressure can promote the cracking of the sample, as well as the hybridization of the target of the sample with the substrate.

[0200] In some embodiments, the cleavage can be performed by mechanical cleavage, thermal cleavage, optical cleavage, and / or chemical cleavage. Chemical cleavage can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. The cleavage can be performed by adding a lysis buffer to the substrate. The lysis buffer can include Tris HCl. The lysis buffer can include at least about, or at most about 0.01, 0.05, 0.1, 0.5, or 1M Tris HCl or more or less. The lysis buffer can include about 0.01, 0.05, 0.1, 0.5, or 1M Tris HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher. The pH of the lysis buffer can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer can include a salt (e.g., LiCl). In the lysis buffer, the concentration of salt can be at least about 0.1M, 0.5M, or 1M, or higher. In the lysis buffer, the concentration of salt can be at most about 0.1M, 0.5M, or 1M, or higher. In certain embodiments, the concentration of salt in the lysis buffer is about 0.5M. The lysis buffer can include a detergent (e.g., SDS, lithium dodecyl sulfate, Triton X, tween, NP-40). In the lysis buffer, the concentration of detergent can be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. The concentration of detergent in the lysis buffer may be at most about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. In some embodiments, the concentration of detergent in the lysis buffer is about 1% lithium dodecyl sulfate. The time used in the lysis method may depend on the amount of detergent used. In some embodiments, the more detergent used, the shorter the time required for lysis. The lysis buffer may include a chelating agent (e.g., EDTA, EGTA). The concentration of chelating agent in the lysis buffer may be at least about 1, 5, 10, 15, 20, 25, or 30 mM or higher. The concentration of chelating agent in the lysis buffer may be at most about 1, 5, 10, 15, 20, 25, or 30 mM or higher. In some embodiments, the concentration of chelating agent in the lysis buffer is about 10 mM. The lysis buffer may include a reducing agent (e.g., β-mercaptoethanol, DTT). The concentration of the reducing agent in the lysis buffer may be at least about 1, 5, 10, 15, or 20 mM or higher. The concentration of the reducing agent in the lysis buffer may be at most about 1, 5, 10, 15, or 20 mM or higher.In some embodiments, the concentration of the reducing agent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer can include about 0.1 M Tris HCl, about pH 7.5, about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

[0201] Lysis can be performed at a temperature of about 4°C, 10°C, 15°C, 20°C, 25°C, or 30°C. Lysis can be performed for about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes or more minutes. Lysed cells can contain at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000 or more target nucleic acid molecules. Lysed cells can contain up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000 or more target nucleic acid molecules.

[0202] Attaching barcodes to target nucleic acid molecules

[0203] After cell lysis and nucleic acid molecules are released, nucleic acid molecules can be randomly associated with the barcode of the co-located solid support. Association can include hybridization of the target recognition region of the barcode with the complementary portion of the target nucleic acid molecule (for example, the oligo (dT) of the barcode can interact with the poly (A) tail of the target). The assay conditions (such as buffer pH, ionic strength, temperature, etc.) for hybridization can be selected to promote the formation of specific stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells can be associated with multiple probes on the substrate (for example, hybridized with probes on the substrate). When the probe includes oligo (dT), the mRNA molecule can be hybridized with the probe and reverse transcribed. The oligo (dT) portion of the oligonucleotide can be used as a primer for the first chain synthesis of the cDNA molecule. For example, Figure 2 In the non-limiting example of barcoding shown in (at box 216), an mRNA molecule can hybridize to a barcode on a particle. For example, a single-stranded nucleotide fragment can hybridize to a target binding region of a barcode.

[0204] Attachment can further include connecting the target recognition region of the barcode to a portion of the target nucleic acid molecule. For example, the target binding region can include a nucleic acid sequence that can be specifically hybridized with a restriction site overhang (e.g., an EcoRI sticky end overhang). The assay procedure can also include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to produce a restriction site overhang. The barcode can then be connected to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to connect the two fragments.

[0205] For example, in Figure 2 In the non-limiting example of barcoding described in (at block 220), the labeled targets (e.g., target-barcode molecules) from multiple cells (or multiple samples) can then be pooled, for example, into a tube. The labeled targets can be pooled by, for example, recovering the barcodes and / or particles to which the target-barcode molecules are attached.

[0206] Retrieval of a solid support-based collection of attached target-barcode molecules can be achieved by using magnetic particles and an externally applied magnetic field. Once the target-barcode molecules have been pooled, all further processing can be performed in a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and / or nucleic acid extension reactions. Further processing reactions can be performed within microwells, i.e., without first pooling labeled target nucleic acid molecules from multiple cells.

[0207] Reverse transcription

[0208] The present disclosure provides methods for producing target-barcode conjugates using reverse transcription (in Figure 2 224). The target-barcode conjugate may include a barcode and a complementary sequence to all or part of a target nucleic acid (i.e., a barcoded cDNA molecule, such as a randomly barcoded cDNA molecule). Reverse transcription of the associated RNA molecule may occur by adding a reverse transcription primer together with a reverse transcriptase. The reverse transcription primer may be an oligo dT primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The oligo (dT) primer may be, or may be, about 12 to 18 nucleotides in length and binds to an endogenous poly (A) tail at the 3' end of a mammalian mRNA. The random hexanucleotide primer may bind to the mRNA at multiple complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest.

[0209] In some embodiments, the reverse transcription of the RNA molecule of the mark can be carried out by adding a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo (dT) primer, a random hexanucleotide primer or a target-specific oligonucleotide primer. Typically, the length of the oligo (dT) primer is 12-18 nucleotides and is bound to the endogenous poly (A) + tail at the 3' end of the mammalian mRNA. Random hexanucleotide primers can be bound to mRNA at multiple complementary sites. Target-specific oligonucleotide primers typically selectively trigger the mRNA of interest.

[0210] Reverse transcription can occur repeatedly to produce multiple labeled cDNA molecules. The methods disclosed herein may include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 reverse transcription reactions. The methods may include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 reverse transcription reactions.

[0211] Amplification

[0212] One or more nucleic acid amplification reactions can be performed (e.g., Figure 2 The amplification reaction may be performed in a multiplexed manner, wherein multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction may be used to add sequencing adapters to the nucleic acid molecules. The amplification reaction may include amplifying at least a portion of the sample label (if present). The amplification reaction may include amplifying at least a portion of the cell label and / or barcode sequence (e.g., molecular marker). The amplification reaction may include amplifying at least a portion of the sample label, cell label, spatial label, barcode (e.g., molecular marker), target nucleic acid, or a combination thereof. The amplification reaction may include amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100% of the plurality of nucleic acids, or a number or range between any two of these values. The method may further include performing one or more cDNA synthesis reactions to generate one or more cDNA copies of a target-barcode molecule comprising a sample marker, a cell marker, a spatial marker, and / or a barcode sequence (e.g., a molecular marker).

[0213] In some embodiments, polymerase chain reaction (PCR) can be used for amplification. As used herein, PCR can refer to a reaction for amplifying a specific DNA sequence in vitro by primer extension of a complementary strand of DNA. As used herein, PCR can include derivative forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, and assembly PCR.

[0214] The amplification of labeled nucleic acid can include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), amplification based on nucleic acid sequences (NASBA), chain displacement amplification (SDA), real-time SDA, rolling circle amplification or circle-to-circle amplification. Other non-PCR-based amplification methods include multiple cycles of DNA synthesis and transcription of DNA synthesis driven by DNA-dependent RNA polymerase or RNA-guided amplification to amplify DNA or RNA targets, ligase chain reaction (LCR), and Qβ replicase (Qβ) methods, the use of palindromic probes, chain displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, primers hybridized to nucleic acid sequences and amplification methods of cutting the resulting duplex before extension reactions and amplification, chain displacement amplification, rolling circle amplification, and branch extension amplification (RAM) using nucleic acid polymerases lacking 5' exonuclease activity. In certain embodiments, amplification does not produce circularized transcripts.

[0215] In some embodiments, the method disclosed herein further includes performing a polymerase chain reaction on a labeled nucleic acid (e.g., labeled RNA, labeled DNA, labeled cDNA) to produce a labeled amplicon (e.g., a randomly labeled amplicon). The labeled amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized with a DNA molecule. One or both chains of the double-stranded molecule can include a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acid disclosed herein can include a synthetic or altered nucleic acid.

[0216] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include light unstable or triggerable nucleotides. Examples of non-natural nucleotides can include but are not limited to peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides can be added to one or more cycles of an amplified reaction. Adding non-natural nucleotides can also be used to identify the product of a specific cycle or time point in an amplified reaction.

[0217] Carrying out one or more amplification reactions can include using one or more primers.One or more primers can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more nucleotides.One or more primers can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more nucleotides.One or more primers can include less than 12-15 nucleotides.One or more primers can anneal to at least a portion of a plurality of labeled targets (e.g., randomly labeled targets).One or more primers can anneal to 3' ends or 5' ends of a plurality of labeled targets.One or more primers can anneal to the internal region of a plurality of labeled targets. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the plurality of labeled targets. The one or more primers can include a set of fixed primers. The one or more primers can include at least one or more custom primers. The one or more primers may include at least one or more control primers.The one or more primers may include at least one or more gene-specific primers.

[0218] One or more primers may include universal primers. Universal primers may anneal to universal primer binding sites. One or more custom primers may anneal to a first sample marker, a second sample marker, a spatial marker, a cell marker, a barcode sequence (e.g., a molecular marker), a target, or any combination thereof. One or more primers may include universal primers and custom primers. Custom primers may be designed to amplify one or more targets. The target may include a subset of total nucleic acids in one or more samples. The target may include a subset of total labeled targets in one or more samples. One or more primers may include at least 96 or more custom primers. One or more primers may include at least 960 or more custom primers. One or more primers may include at least 9600 or more custom primers. One or more custom primers may anneal to two or more different labeled nucleic acids. Two or more different labeled nucleic acids may correspond to one or more genes.

[0219] Any amplification scheme can be used in the methods of the present disclosure. For example, in one scheme, the first round of PCR can use gene-specific primers and primers for the universal Illumina sequencing primer 1 sequence to amplify molecules attached to particles (e.g., beads). The second round of PCR can use nested gene-specific primers flanking the Illumina sequencing primer 2 sequence and primers for the universal Illumina sequencing primer 1 sequence to amplify the first PCR product. The third round of PCR adds P5 and P7 and a sample index to allow the PCR product to enter the Illumina sequencing library. Sequencing using 150bp x 2 sequencing can reveal cell markers and barcode sequences (e.g., molecular markers) on read 1, genes on read 2, and sample indexes on index 1 reads.

[0220] In some embodiments, nucleic acid can be removed from substrate using chemical cleavage. For example, chemical groups or modified bases present in nucleic acid can be used to promote its removal from solid support. For example, enzymes can be used to remove nucleic acid from substrate. For example, nucleic acid can be removed from substrate by restriction endonuclease digestion. For example, nucleic acid containing dUTP or ddUTP can be removed from substrate using uracil-d-glycosylase (UDG) treatment. For example, nucleic acid can be removed from substrate using enzymes for nucleotide excision (e.g., base excision repair enzymes (e.g., apurinic / apyrimidinic (AP) endonucleases)). In some embodiments, nucleic acid can be removed from substrate using photocleavable groups and light. In some embodiments, nucleic acid can be removed from substrate using cleavable joints. For example, cleavable joints can include at least one of the following: biotin / avidin, biotin / streptavidin protein, biotin / neutravidin, Ig protein A, photolabile joints, acid or base labile joint groups, or aptamers.

[0221] When the probe is gene specific, the molecule can be hybridized with the probe and reverse transcribed and / or amplified. In some embodiments, after the nucleic acid has been synthesized (e.g., reverse transcribed), it can be amplified. Amplification can be performed in a multiplex manner, wherein multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adapters to nucleic acids.

[0222] In certain embodiments, for example, amplification can be carried out on substrates with bridging amplification.cDNA can be a homopolymer tail, using an oligo (dT) probe on a substrate to produce a compatible end for bridging amplification.In bridging amplification, a primer complementary to the 3' end of a template nucleic acid can be the first primer of each pair of primers covalently attached to a solid particle.When a sample containing a template nucleic acid contacts a particle and performs a single thermal cycle, the template molecule can be annealed to the first primer, and the first primer is extended forward by adding nucleotides to form a duplex molecule, which is composed of a template molecule and a newly formed DNA chain complementary to the template.In the heating step of the next cycle, the duplex molecule can be denatured, the template molecule is released from the particle, and the complementary DNA chain is attached to the particle by the first primer.In the annealing stage of the subsequent annealing and extension steps, the complementary chain can be hybridized with the second primer, which is complementary to the fragment of the complementary chain at the position removed from the first primer. The hybridization can cause complementary chains to form bridges between the first and second primers that are fixed to the first primer by a covalent bond, and form a second primer by hybridization. In the extension phase, by adding nucleotides in the same reaction mixture, the second primer can be extended in the opposite direction, thereby converting the bridge into a double-stranded bridge. Then start the next cycle, and the double-stranded bridge can be denatured to produce two single-stranded nucleic acid molecules, one end of each single-stranded nucleic acid molecule is attached to the particle surface through the first and second primers respectively, and the other end of each single-stranded nucleic acid molecule is unattached. In the annealing and extension steps of the second cycle, each chain can be hybridized on the same particle with other complementary primers that were not previously used, to form a new single-stranded bridge. Two previously unused primers that are now hybridized are extended so that two new bridges are converted into double-stranded bridges.

[0223] The amplification reaction can include amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% of a plurality of nucleic acids.

[0224] Amplification of labeled nucleic acid may include PCR-based methods or non-PCR-based methods. Amplification of labeled nucleic acid may include indexed amplification of labeled nucleic acid. Amplification of labeled nucleic acid may include linear amplification of labeled nucleic acid. Amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for amplifying a specific DNA sequence in vitro by simultaneous primer extension of complementary strands of DNA. PCR may encompass derivative forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, inhibition PCR, semi-inhibition PCR, and assembly PCR.

[0225] In some embodiments, the amplification of the labeled nucleic acid includes a non-PCR-based method. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), chain displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-to-circle amplification. Other non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-guided DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ), the use of palindromic probes, chain displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, primers hybridized to nucleic acid sequences and amplification methods in which the resulting duplex is cut before extension reactions and amplification, chain displacement amplification, rolling circle amplification, and branch extension amplification (RAM) using nucleic acid polymerases lacking 5' exonuclease activity.

[0226] In some embodiments, the method disclosed herein further comprises performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized with a DNA molecule. One or both chains of the double-stranded molecule can include a sample label or a molecular identifier marker. Alternatively, the amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acid of the present invention can include a synthetic or altered nucleic acid.

[0227] In some embodiments, the method comprises repeatedly amplifying the labeled nucleic acid to produce a plurality of amplicons. The method disclosed herein may comprise performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the method comprises performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0228] Amplification may further include adding one or more control nucleic acids to one or more samples comprising a plurality of nucleic acids. Amplification may further include adding one or more control nucleic acids to a plurality of nucleic acids. The control nucleic acids may include a control marker.

[0229] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include light unstable and / or triggerable nucleotides. Examples of non-natural nucleotides include but are not limited to peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA) and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides can be added to one or more cycles of an amplified reaction. Adding non-natural nucleotides can also be used to identify the product of a specific cycle or time point in an amplified reaction.

[0230] Carrying out one or more amplification reactions can include using one or more primers. One or more primers can include one or more oligonucleotides. One or more oligonucleotides can include at least about 7 to 9 nucleotides. One or more oligonucleotides can include less than 12 to 15 nucleotides. One or more primers can anneal to at least a portion of a plurality of labeled nucleic acids. One or more primers can anneal to the 3' end and / or the 5' end of a plurality of labeled nucleic acids. One or more primers can anneal to the internal region of a plurality of labeled nucleic acids. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3' end of the multiple labeled nucleic acids. One or more primers can include a set of fixed primers. One or more primers can include at least one or more custom primers. One or more primers may include at least one or more control primers. One or more primers may include at least one or more housekeeping gene primers. One or more primers may include universal primers. Universal primers may anneal to universal primer binding sites. One or more custom primers may anneal to the first sample label, the second sample label, a molecular identifier label, a nucleic acid or their products. One or more primers may include universal primers and custom primers. Custom primers may be designed to amplify one or more target nucleic acids. Target nucleic acids may include a subset of total nucleic acids in one or more samples. In certain embodiments, the primers are probes attached to the array of the present disclosure.

[0231] In some embodiments, barcoding (e.g., randomly barcoding) multiple targets in a sample further comprises generating an index library of barcoded fragments. The barcode sequences of different barcodes (e.g., molecular markers of different random barcodes) can be different from each other. Generating an index library of barcoded targets (e.g., randomly barcoded targets) comprises generating multiple index polynucleotides from multiple targets in a sample. For example, for an index library of barcoded targets comprising a first index target and a second index target, the marker region of the first index polynucleotide and the marker region of the second index polynucleotide can have, have about, have at least, or have a difference of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or a number or range of nucleotide differences between any two of these values. In some embodiments, generating an index library of barcoded targets includes contacting a plurality of targets (e.g., mRNA molecules) with a plurality of oligonucleotides comprising a poly(T) region and a tag region; and performing first strand synthesis using a reverse transcriptase to generate single-stranded labeled cDNA molecules (each comprising a cDNA region and a tag region), wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences. Generating an index library of barcoded targets may further include amplifying the single-stranded labeled cDNA molecules to generate double-stranded labeled cDNA molecules; and performing nested PCR on the double-stranded labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method may include generating adapter-tagged amplicons.

[0232] Random barcoding can use nucleic acid barcodes or tags to label individual nucleic acid (e.g., DNA or RNA) molecules. In some embodiments, it involves adding DNA barcodes or tags to cDNA molecules because they are generated from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adaptors can be added for sequencing using, for example, next generation sequencing (NGS). For example, in Figure 2 At block 232 of the sequencing, the sequencing results can be used to determine a cellular marker, a barcode sequence (eg, a molecular marker), and a sequence of nucleotide fragments of one or more copies of a target.

[0233] Figure 3is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of barcoded targets (e.g., randomly barcoded targets), such as mRNA. As shown in step 1, the reverse transcription process can encode each mRNA molecule with a unique barcode sequence (e.g., a molecular marker), a cell marker, and a universal PCR site. For example, by hybridizing (e.g., randomly hybridizing) a set of barcodes (e.g., random barcodes) 310 to a poly (A) tail region 308 of an RNA molecule 302, an RNA molecule 302 can be reverse transcribed to generate a labeled cDNA molecule 304 (including a cDNA region 306). Each barcode 310 can include a target binding region, such as a poly (dT) region 312, a barcode sequence or molecular marker 314, and a universal PCR region 316.

[0234] In some embodiments, the cell marker may include 3 to 20 nucleotides. In some embodiments, the barcode sequence (e.g., molecular marker) may include 3 to 20 nucleotides. In some embodiments, each of a plurality of random barcodes further includes one or more of a universal marker and a cell marker, wherein the universal marker is the same for a plurality of random barcodes on a solid support and the cell marker is the same for a plurality of random barcodes on a solid support. In some embodiments, the universal marker may include 3 to 20 nucleotides. In some embodiments, the cell marker includes 3 to 20 nucleotides.

[0235] In some embodiments, the labeling region 314 may include a barcode sequence or molecular label 318 and a cell label 320. In some embodiments, the labeling region 314 may include one or more of a universal label, a dimensional label, and a cell label. The length of the barcode sequence or molecular label 318 may be, may be about, may be at least, or may be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any of these values. The length of the cell label 320 may be, may be about, may be at least, or may be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any of these values. The length of the universal tag can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any of these values. The universal tag can be the same for multiple random barcodes on the solid support, and the cell tags are the same for multiple random barcodes on the solid support. The length of the dimensional tag can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any of these values.

[0236] In some embodiments, the marker region 314 can include, include about, include at least, or include at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 different markers, or a number or range between any of these values, such as barcode sequences or molecular markers 318 and cell markers 320. Each marker can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides in length, or a number or range between any of these values ​​of nucleotides. A set of barcodes or random barcodes 310 may contain, contain about, contain at least, or may be at most 10, 20, 40, 50, 70, 80, 90, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7, 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 The barcodes or random barcodes 310 may be a number or range of barcodes or random barcodes 310 between any of these values. And the group of barcodes or random barcodes 310 may, for example, each contain a unique tag region 314. The labeled cDNA molecules 304 may be purified to remove excess barcodes or random barcodes 310. Purification may include Ampure bead purification.

[0237] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled into 1 tube and PCR amplified using the first PCR primer pool and the first universal PCR primer. Pooling is possible because of the unique tag region 314. In particular, the labeled cDNA molecules 304 can be amplified to produce nested PCR labeled amplicons 322. Amplification can include multiplex PCR amplification. Amplification can include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, the multiplex PCR amplification can utilize, utilize about, utilize at least, or utilize at most 10, 20, 40, 50, 70, 80, 90, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 Multiple primers, or multiple primers with a number or range between any of these values. Amplification may include a first PCR primer pool 324 of custom primers 326A-C targeting a specific gene and a universal primer 328. Custom primers 326 can hybridize to a region within the cDNA portion 306' of the labeled cDNA molecule 304. Universal primers 328 can hybridize to universal PCR region 316 of the labeled cDNA molecule 304.

[0238] like Figure 3In step 3 of, the product from the PCR amplification in step 2 can be amplified with a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. For example, the amplicon 322 of the nested PCR marker can be further amplified by nested PCR. Nested PCR can include multiple PCR using a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328' in a single reaction volume. The nested PCR primer pool 328 can contain, contain about, contain at least, or contain at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 different nested PCR primers 330, or a number or range of different nested PCR primers 330 between any of these values. Nested PCR primer 332 may contain adapter 334 and hybridize to a region within cDNA portion 306″ of labeled amplicon 322. Universal primer 328′ may contain adapter 336 and hybridize to universal PCR region 316 of labeled amplicon 322. Thus, step 3 produces adapter-tagged amplicon 338. In some embodiments, nested PCR primer 332 and second universal PCR primer 328′ may not contain adapters 334 and 336. Instead, adapters 334 and 336 may be ligated to the product of nested PCR to produce adapter-tagged amplicon 338.

[0239] As shown in step 4, the PCR product from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, adapters 334 and 336 can be used to perform one or more additional assays on adapter-tagged amplicon 338. Adapters 334 and 336 can hybridize with primers 340 and 342. One or more primers 340 and 342 can be PCR amplification primers. One or more primers 340 and 342 can be sequencing primers. One or more adapters 334 and 336 can be used for further amplification of adapter-tagged amplicon 338. One or more adapters 334 and 336 can be used to sequence adapter-tagged amplicon 338. Primer 342 can contain a plate index 344 so that amplicons generated using the same set of barcodes or random barcodes 310 can be sequenced in one round of sequencing reactions using next generation sequencing (NGS).

[0240] Synthetic particles with spacer oligonucleotides

[0241] Disclosures herein include particles (e.g., synthetic particles), wherein the particles comprise: a plurality of oligonucleotide barcodes (e.g., capture oligonucleotide barcodes), wherein each of the plurality of oligonucleotide barcodes comprises the same cellular marker, molecular marker, and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences); and a first plurality of spacer oligonucleotides (or oligonucleotides having a target-specific target binding region), wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region (i.e., a nucleic acid sequence of a target binding region) present in the oligonucleotide barcode (e.g., capture oligonucleotide barcode). For example, the spacer oligonucleotide does not comprise a nucleic acid sequence having greater than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25% sequence identity with a target binding region present in the oligonucleotide barcode (e.g., capture oligonucleotide barcode). In some embodiments, the spacer oligonucleotide comprises a nucleic acid sequence having no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25% sequence identity with the target binding region present in the oligonucleotide barcode (e.g., capture oligonucleotide barcode). In some embodiments, the spacer oligonucleotide does not comprise a region having a length greater than, for example, 10-30 nucleotides long, and the region has more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25% sequence identity with the target binding region present in the oligonucleotide barcode (e.g., capture oligonucleotide barcode). In some embodiments, the spacer oligonucleotide does not hybridize with a sequence that specifically hybridizes to the target binding region under the desired nucleic acid hybridization conditions. In some embodiments, the spacer oligonucleotide specifically binds to a sequence that does not specifically hybridize to the target binding region under desired nucleic acid hybridization conditions.

[0242] Density of oligonucleotide barcodes

[0243] As described herein, the density of oligonucleotide barcodes (e.g., random barcodes) on particles (e.g., synthetic particles) used for barcoding (e.g., random barcoding) can affect the efficiency of target capture (e.g., the efficiency of capturing mRNA targets). Figure 4A400a) is a non-limiting exemplary synthetic particle associated with a plurality of oligonucleotide barcodes. For example, synthetic particle 400a can be associated with (e.g., coupled or conjugated to) oligonucleotide barcode 404 (barcode 404 having 16 oligonucleotides is shown). Oligonucleotide barcode 404 is also referred to herein as capture oligonucleotide. If the density of oligonucleotide barcodes 404 on synthetic particle 400a is higher, the efficiency of target 408 capture may be lower than if the density of oligonucleotide barcodes on the synthetic particle is lower (e.g., see Figure 4B 4C). Without being bound by any particular theory, if the density of oligonucleotide barcodes 404 on the synthetic particle 400a is too high, there may be too much steric hindrance between the oligonucleotide barcodes 404, so that if the density is lower, the synthetic particle 400a captures fewer target molecules 408. For example, if the density of the target binding region 412 (displayed poly (dT) tail) of the oligonucleotide barcode 404 is too high, there may be steric hindrance in the oligonucleotide barcode 404 (or steric hindrance in the target binding region) so that some target molecules 408 (displayed poly (A) tail 416 of the target molecule 408) do not hybridize with the oligonucleotide barcode 404. Therefore, in some embodiments, it may be advantageous to reduce the density of capture oligonucleotide barcodes in the particle, for example by associating a spacer oligonucleotide with the particle. As described herein, the form, type, length, density and position of the spacer oligonucleotide can vary. The number of spacer oligonucleotides associated with a particle can be greater than, less than, equal to, or substantially equal to the number of capture oligonucleotides associated with the same particle. Although the illustrated synthetic particle 400a is associated with a barcode 404 having 16 oligonucleotides, the number of oligonucleotide barcodes 404 is for illustration only and is not intended to be limiting.

[0244] Figure 4Bis a non-limiting exemplary synthetic particle associated with multiple oligonucleotide barcodes and multiple spacer oligonucleotides. As shown, the synthetic particle 400b can include a spacer oligonucleotide 420b (six spacer oligonucleotides 420b shown) and an oligonucleotide barcode 404. The oligonucleotide barcode 404 can include a target binding region 412 capable of capturing (e.g., hybridizing) a target molecule 408. The spacer oligonucleotide 420b can include a spacer region 424b, which corresponds to the target binding region 412 and cannot capture (e.g., hybridize) the target molecule 408. Including a spacer oligonucleotide 420b on the synthetic particle 400b can improve the capture of the target 408 (e.g., mRNA capture) and the subsequent quantification of the captured target molecule 408. For example, synthetic particles 400b can be coupled to both oligonucleotide barcodes 404 (e.g., barcodes each having a poly(dT) region that can bind to a copy or occurrence of an mRNA molecule or species via a poly(dA) tail 416) and spacer oligonucleotides 420b, which can improve mRNA capture and gene expression quantification. Although the illustrated synthetic particle 400b is associated with 10 oligonucleotide barcodes 404 and six spacer oligonucleotides 420b, the number of oligonucleotide barcodes 404 and spacer oligonucleotides is for illustration only and is not intended to be limiting.

[0245] The spacer oligonucleotides associated with the synthetic particle can be shorter or longer than the oligonucleotide barcode. For example, all spacer oligonucleotides associated with the synthetic particle can be shorter than the oligonucleotide barcode (e.g., see Fig.4C1 As another example, all spacer oligonucleotides associated with a synthetic particle may be longer than the oligonucleotide barcode (see, e.g., Figure 4C2 ). For example, some spacer oligonucleotides associated with the synthetic particles can be longer than the oligonucleotide barcode, and some spacer oligonucleotides associated with the synthetic particles can be shorter than the oligonucleotide barcode.

[0246] Fig.4C1 It is a non-limiting exemplary synthetic particle associated with multiple oligonucleotide barcodes and multiple spacer oligonucleotides, each spacer oligonucleotide is shorter than the oligonucleotide barcode. Compared to the oligonucleotide barcode 404, the spacer oligonucleotide 420c1 on the synthetic particle 400c1 can be shorter. Synthetic particles 400c1 associated with oligonucleotide barcodes 404 (or target binding regions 412 with the same sequence) can be first produced. An enzyme (e.g., an exonuclease (5' or 3') or an endonuclease) can be used to remove the target binding region 412 of some of the oligonucleotide barcodes 404. The shorter oligonucleotide barcodes obtained on the synthetic particles are referred to as spacer oligonucleotides 420c in this article.

[0247] In some embodiments, the Fig.4C1 Both the oligonucleotide barcode 404 and the spacer oligonucleotide 420c1 shown in FIG. 4 are associated with (eg, coupled to) the synthetic particle 400c1 , the density of the oligonucleotide barcode 404 is reduced, and then the density is reduced using an exonuclease or an endonuclease.

[0248] Figure 4C2It is a non-limiting exemplary synthetic particle associated with multiple oligonucleotide barcodes and multiple spacer oligonucleotides, each spacer oligonucleotide is longer than the oligonucleotide barcode. Compared to the oligonucleotide barcode 404, the spacer oligonucleotide 420c2 on the synthetic particle 400c2 can be longer. For example, a synthetic particle 400c2 associated with an oligonucleotide barcode 404 (or a target binding region 412 with the same sequence) can be first produced. An enzyme such as a polymerase or a ligase can be used to add one or more sequences to some of the oligonucleotide barcode 404 by extension or connection to produce a spacer oligonucleotide 420c2 longer than the oligonucleotide barcode 404. The same sequence or different sequences can be added to produce the spacer oligonucleotide 420c2 of the synthetic particle 400c2. In some embodiments, the number of different sequences added to generate spacer oligonucleotide 420c2 can be, or about 1, 2, 3, 4, 5, 6, 7, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any two of these values. In some embodiments, the number of different sequences added to generate spacer oligonucleotide 420c2 can be at least, or at most 1, 2, 3, 4, 5, 6, 7, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. The spacer oligonucleotides 420c2 of the synthetic particles 400c2 can have the same sequence or different sequences. In some embodiments, the spacer oligonucleotides 420c2 can have or have a sequence of about 1, 2, 3, 4, 5, 6, 7, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values. In some embodiments, the spacer oligonucleotide 420c2 can have at least, or have at most 1, 2, 3, 4, 5, 6, 7, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 different sequences. The spacer oligonucleotides 420c2 of the synthetic particles 400c2 can have the same length or different lengths.In some embodiments, the spacer oligonucleotide 420c2 can have or have about 1, 2, 3, 4, 5, 6, 7, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any two of these values ​​between the number or range of different lengths. In some embodiments, the spacer oligonucleotide 420c2 can have at least or have at most 1, 2, 3, 4, 5, 6, 7, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 different lengths. The resulting longer oligonucleotide barcode on the synthetic particle is referred to herein as spacer oligonucleotide 420c1.

[0249] In some embodiments, the Figure 4C2 Both the oligonucleotide barcode 404 and the spacer oligonucleotide 420c2 shown in FIG. 4 are associated with (eg, coupled to) the synthetic particle 400c2 , reducing the density of the oligonucleotide barcode 404 .

[0250] The density of the oligonucleotide barcodes can be changed by adjusting the association conditions used to associate the oligonucleotide barcodes with the synthetic particles (e.g., reducing the amount of cross-linking agent, reducing the amount of oligonucleotide barcodes during the coupling process). Accurately controlling the association (e.g., coupling) by changing the association conditions may require a large number of tests. In some embodiments, the density of the oligonucleotide barcodes on the synthetic particles can be first reduced by adjusting the association conditions, and further reduced using a nuclease exonuclease or endonuclease. In some embodiments, the density of the oligonucleotide barcodes can be reduced by associating the oligonucleotide barcodes and the spacer oligonucleotides with the synthetic particles under conditions adjusted to produce synthetic particles with a lower density of oligonucleotide barcodes, and further reduced using a nuclease exonuclease or endonuclease.

[0251] In some embodiments, spacer oligonucleotides (e.g., spacer oligonucleotides associated on synthetic particles) can have the same length or different lengths. In some embodiments, one or more spacer oligonucleotides can be shorter than one or more oligonucleotide barcodes. For example, a spacer oligonucleotide and an oligonucleotide barcode can be identical, except that the oligonucleotide barcode comprises a target binding region, and the spacer oligonucleotide does not comprise a target binding region. In some embodiments, one or more spacer oligonucleotides can have the same length as one or more oligonucleotide barcodes. For example, in some embodiments (except that the oligonucleotide barcode comprises a target binding region, and the spacer oligonucleotide comprises a region having the same length as the target binding region but having a different nucleotide sequence), the spacer oligonucleotide and the oligonucleotide barcode can be identical.

[0252] Oligonucleotide barcodes with target-specific target binding regions

[0253] The disclosure herein includes embodiments of synthetic particles. In some embodiments, the synthetic particles comprise: a first plurality of oligonucleotide barcodes; and a second plurality of oligonucleotide barcodes, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cell marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region. In some embodiments, the target binding region of the oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. In some embodiments, the target binding region of the oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a poly(dT) region. The oligonucleotide barcode of the second plurality of oligonucleotide barcodes may comprise a poly(dT) region.

[0254] The disclosure herein includes embodiments of methods for determining the number of targets in a sample. In some embodiments, the method includes: barcoding copies of a first plurality of targets using a first plurality of oligonucleotide barcodes associated with a synthetic particle to produce a first plurality of barcoded copies of the target; barcoding copies of a second plurality of targets in the sample using a second plurality of oligonucleotide barcodes associated with the synthetic particle to produce a second plurality of barcoded copies of the target, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cellular marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region; and evaluating the number of each of the first plurality of targets and / or the second plurality of targets using the molecular marker sequences of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes. In some embodiments, the target binding region of the oligonucleotide barcodes of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. The target binding region of the oligonucleotide barcodes of the first plurality of oligonucleotide barcodes may comprise a poly (dT) region. The oligonucleotide barcodes of the second plurality of oligonucleotide barcodes may comprise a poly (dT) region. In some embodiments, the first plurality of targets comprise mRNA molecules of cells, and wherein the second plurality of targets comprise DNA molecules of cells. The first plurality of targets may comprise mRNA molecules of cells, and the second plurality of targets may comprise RNA molecules of viruses, bacteria and / or fungi that infect cells.

[0255] Figure 4D1-4D2 is a non-limiting exemplary synthetic particle associated with multiple oligonucleotide barcodes having target-specific target binding regions. The target binding region of the oligonucleotide barcode on the synthetic particle can include or can be a target-specific target binding region and / or a non-target-specific target binding region (e.g., a poly (dT) region and a random polymer). The target binding region of the oligonucleotide barcode on the synthetic particle (e.g., a synthetic particle associated with a spacer oligonucleotide) can include or be a poly (dT) region 404, a random polymer, and / or a target-specific sequence 412d (e.g., a sequence capable of hybridizing with one or more target of interest). For example, oligonucleotide barcode 404d1 associated with synthetic particle 400d1 can include a target binding region 412d1 capable of specifically binding to many target of interest (e.g., four target of interest, such as CD4, IL2, CD8, and CR7) and an oligonucleotide barcode 404 ( Fig.4D1 ).

[0256] In some embodiments, the target of interest may include an oligonucleotide conjugated to a cellular component binding agent (e.g., a protein binding agent, such as an antibody or a protein binding aptamer), wherein the oligonucleotide comprises a unique identifier for the cellular component binding agent conjugated thereto. Cellular component binding agents associated with oligonucleotides (e.g., barcoded antibodies) and their uses (e.g., sample indexing, identification or tracking of cells and samples, and protein expression profiling) have been described in U.S. Patent Application Publication No. US 2018 / 0088112 and U.S. Patent Application Publication No. US 2018 / 0346970 (the contents of each of these documents are incorporated herein in their entirety by reference).

[0257] Sample indexing, identification or trackingIn some embodiments, performing sample indexing, identification, or tracking may include: contacting one or more cells from each of a plurality of samples with a sample indexing composition of a plurality of sample indexing compositions, wherein each of the one or more cells comprises one or more cellular component targets (e.g., protein targets), wherein at least one sample indexing composition of the plurality of sample indexing compositions comprises two or more cellular component binding reagents (e.g., antigen binding reagents or antibodies), wherein each of the two or more cellular component binding reagents is associated with a sample indexing oligonucleotide, wherein at least one of the two or more cellular component binding reagents is capable of specifically binding to at least one of the one or more cellular component targets, wherein the sample indexing oligonucleotide comprises a sample indexing sequence, and wherein the sample indexing sequences of at least two of the plurality of sample indexing compositions comprise different sequences; barcoding the sample indexing oligonucleotides using a plurality of barcodes (e.g., oligonucleotide barcodes associated with synthetic particles) to create a plurality of barcoded sample indexing oligonucleotides; acquiring sequencing data of the plurality of barcoded sample indexing oligonucleotides; and identifying the sample origin of at least one of the one or more cells based on the sample indexing sequence of at least one barcoded sample indexing oligonucleotide of the plurality of barcoded sample indexing oligonucleotides. In some embodiments, the sample index sequence is 25-60 nucleotides in length (e.g., 45 nucleotides in length), about 128 nucleotides in length, or at least 128 nucleotides in length. The sample index sequences of at least 10 sample index compositions in a plurality of sample index compositions may comprise different sequences. The sample index sequences of at least 10 sample index compositions in a plurality of sample index compositions may comprise different sequences. The sample index sequences of at least 10 sample index compositions in a plurality of sample index compositions comprise different sequences. In some embodiments, the sample index oligonucleotide is not homologous to the genomic sequences of cells of a plurality of samples. The sample index oligonucleotide may comprise a molecular marker sequence; the target binding region of the barcode may bind (e.g., hybridize) to a binding sequence (e.g., a poly(A) region or a poly(A) region); or a combination thereof. The sample index oligonucleotide may comprise a sequence complementary to a capture sequence (e.g., a target binding region) of at least one barcode in a plurality of barcodes. The target binding region of the barcode may comprise a capture sequence. For example, the target binding region may comprise a poly(dT) region. For example, the sequence of the sample index oligonucleotide that is complementary to the capture sequence of the barcode may comprise a poly(dA) tail. In some embodiments, the sample index oligonucleotide may comprise a molecular tag.

[0258] Expression profileIn some embodiments, performing protein expression profiling (or profiling of cellular component targets) may include: providing a sample comprising a plurality of protein targets (or other cellular component targets, such as carbohydrates, lipids, etc.); providing a plurality of compositions, each of which comprises a protein binding agent (or cellular component binding agent) conjugated to an oligonucleotide, wherein the oligonucleotide comprises a unique identifier for the protein binding agent conjugated thereto, and the protein binding agent (or cellular component binding agent) is capable of specifically binding to at least one of the plurality of protein targets (or cellular component targets); contacting the plurality of compositions with the sample for specific binding to the plurality of protein targets (or cellular component targets); heterosexual binding; removing unbound compositions; providing a plurality of oligonucleotide probes (e.g., oligonucleotide barcodes associated with synthetic particles), wherein each of the plurality of oligonucleotide probes comprises a target binding region and a barcode sequence (e.g., a molecular marker sequence), wherein the barcode sequence is from a multi-group of unique barcode sequences; contacting the plurality of oligonucleotide probes with the oligonucleotides of the plurality of compositions; extending the oligonucleotide probes hybridized with the oligonucleotides to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acids comprises a unique identifier and a barcode sequence; and determining the number of unique barcode sequences for each unique identifier, thereby determining the amount of each protein target in the sample. In some embodiments, the unique identifier comprises a nucleotide sequence of 25-45 nucleotides in length. In some embodiments, the unique identifier is selected from a multi-group of unique identifiers. In some embodiments, the multi-group of unique identifiers comprises at least 100 different unique identifiers. In some embodiments, the multi-group of unique identifiers comprises at least 1,000 different unique identifiers. In some embodiments, the multi-group of unique identifiers comprises at least 10,000 different unique identifiers. In some embodiments, the plurality of compositions comprises a plurality of antibodies, a plurality of aptamers, or a combination thereof. In some embodiments, the oligonucleotide is conjugated to a protein binding reagent via a joint. In some embodiments, the joint includes a chemical group. In some embodiments, the oligonucleotide includes a joint. In some embodiments, the chemical group is reversibly attached to a protein binding reagent. In some embodiments, the chemical group is selected from the group consisting of: UV photolyzable groups, streptavidin protein, biotin, amines, and any combination thereof. In some embodiments, the sample includes a single cell. In some embodiments, a plurality of protein targets are expressed on the surface of a single cell. In some embodiments, removing unbound compositions includes washing a single cell with a wash buffer. In some embodiments, these methods include cracking a single cell. In some embodiments, these methods include separating oligonucleotides from a protein binding reagent. In some embodiments, oligonucleotides are separated from a protein binding reagent by UV photolysis, chemical treatment (dithiothreitol), heating, enzyme treatment, or any combination thereof.In some embodiments, each of the oligonucleotide probes includes a cell marker, a binding site for a universal primer, or any combination thereof. In some embodiments, the target binding region includes poly (dT). In some embodiments, a plurality of oligonucleotide probes are fixed on a solid support. In some embodiments, the solid support is a bead. In some embodiments, the methods further include amplifying a plurality of labeled nucleic acids to produce a plurality of amplicons. In some embodiments, the amplification includes PCR amplification of at least a portion of the barcode sequence and at least a portion of the unique identifier. In some embodiments, the multi-group of unique barcode sequences includes at least 100 unique barcode sequences. In some embodiments, the multi-group of unique barcode sequences includes at least 1,000 unique barcode sequences. In some embodiments, the multi-group of unique barcode sequences includes at least 10,000 unique barcode sequences. A plurality of compositions may further include a second protein binding agent that is not conjugated to the oligonucleotide. The protein binding agent and the second protein binding agent may be the same. In some embodiments, the methods further include sequencing a plurality of amplicons. In some embodiments, sequencing includes sequencing at least a portion of the barcode sequence and at least a portion of the unique identifier. The method can be used to simultaneously quantify multiple protein targets and multiple nucleic acid target molecules in a sample.

[0259] In some embodiments, the oligonucleotide conjugated to the cellular component binding agent may have a poly (dA) region 416 that can hybridize with the poly (dT) region 412 of the oligonucleotide barcode 404. In some embodiments, the oligonucleotide barcode 404d1 may have a target-specific target binding region 412d that can specifically hybridize and capture the oligonucleotide associated with the cellular component binding agent. The oligonucleotide region associated with the cellular component binding agent that is bound or hybridized to the target-specific target binding region 412 may be at the 3' end of the oligonucleotide (corresponding to the poly (dA) region of the mRNA), at the 5' end of the oligonucleotide, or inside the oligonucleotide. In some embodiments, the oligonucleotide dissociates from the cellular component binding agent before being captured by the oligonucleotide barcode 404d1 having the target-specific target binding region 412d.

[0260] like Fig.4D1As shown, the poly(dT) region of the oligonucleotide barcode 404 can capture (e.g., hybridize) a target (e.g., an mRNA molecule) having a poly(A) tail 416. The poly(dT) region 412 of the oligonucleotide barcode 404 can capture a target having a poly(A) region or tail 416. The target-specific target binding region 412d of the oligonucleotide barcode 404d1 can capture (e.g., hybridize) a target of interest. For example, the target-specific target binding region 412d can include or can be a portion of a CD4 sequence that can hybridize with a non-poly(A) region 416d of a CD4 mRNA molecule (or a region 416d of a CD4 mRNA having non-A bases) and capture the CD4 mRNA molecule. As another example, the target-specific target binding region 412d can include or can be a sequence for capturing a target (e.g., a viral mRNA) that does not have a poly(A) tail. In some embodiments, synthetic particles 400d1 can be used to simultaneously determine the genotype and / or phenotype of cells infected by a virus and the genotype and / or phenotype of the virus. For example, synthetic particles 400d1 can include oligonucleotide barcodes 404 having poly(dT) regions 412 for capturing mRNA of infected cells and oligonucleotide barcodes 404d1 having target-specific target binding regions 412d for capturing viral RNA (e.g., viral mRNA). In some embodiments, beads 400d1 can be produced by associating (e.g., coupling) oligonucleotide barcodes 404 with poly(dT) regions 412 and oligonucleotide barcodes 404d1 with target-specific target binding regions 412d to synthetic particles 400d1.

[0261] refer to Figure 4D2 The oligonucleotide barcode 404d2 associated with the synthetic particle 400d2 may include a target binding region 412d1 that is capable of specifically binding to a number of targets of interest and a poly(dT) region 404( Figure 4D2 ).like Figure 4D2 As shown, synthetic particle 400d2 may also include an oligonucleotide barcode 404 having a non-target specific target binding region (e.g., poly (dT) region 400). Figure 4D2 As shown, the poly(dT) region 412 of the oligonucleotide barcode 404 can capture (e.g., hybridize) a target (e.g., an mRNA molecule) having a poly(A) tail 416. The poly(dT) region 412 of the oligonucleotide barcode 404 can capture a target having a poly(A) region or tail 416. The target-specific target binding region 412d of the oligonucleotide barcode 404d2 can capture (e.g., hybridize) a target of interest. Figure 4D2As shown, in some embodiments, the oligonucleotide barcode 404d2 having a target-specific target binding region 412d can include a poly (dT) region 412. In some embodiments, the synthetic particle 400d2 can be used to simultaneously determine the genotype and / or phenotype of a cell infected with a virus and the genotype and / or phenotype of the virus. For example, the synthetic particle 400d2 can include an oligonucleotide barcode 404 having a poly (dT) region 412 for capturing mRNA of an infected cell and an oligonucleotide barcode 404d2 having a target-specific target binding region 412d for capturing viral RNA (e.g., viral mRNA).

[0262] In some embodiments, beads 400d2 can be produced by associating (e.g., coupling) an oligonucleotide barcode 404 with a poly(dT) region 412 and an oligonucleotide barcode 404d2 with a target-specific target binding region 412d and a poly(dT) region 412 to a synthetic particle 400d. In some embodiments, synthetic particles 400d2 associated with oligonucleotide barcodes 404 having the same sequence (or poly(dT) region 412) can be first produced. An enzyme such as a polymerase or a ligase can be used to add a target-specific target binding region 412d to some of the oligonucleotide barcodes 404 by extension or ligation to produce an oligonucleotide barcode 404d2 having a target-specific target binding region 412d and a poly(dT) region 412. For example, a single-stranded DNA template having a poly (dA) sequence (e.g., at the 3' end of the template) and a partial sequence of a target of interest (e.g., at the 5' end of the template) can be hybridized with the poly (dT) region 412 of the oligonucleotide barcode 404, which can be extended by a polymerase to produce an oligonucleotide barcode 404d2 having a target-specific target binding region 412d and a poly (dT) region 412. As another example, a partially duplex DNA template can be used to produce an oligonucleotide barcode 404d2 having a target-specific target binding region 412 and a poly (dT) region 412. One strand of the partially duplex DNA template can include a poly (dA) sequence (e.g., at the 5' end of the strand) and a partial sequence of a target of interest (e.g., at the 3' end of the strand). The other strand of the partially duplex DNA template can include a target-specific target binding region. A partially duplex DNA template can be formed by hybridizing a partial sequence of a target of interest on one strand with a target-specific target binding region on another strand. The partially duplex DNA template can be hybridized with the oligonucleotide barcode 404 via hybridization of the poly(dT) region 412 of the oligonucleotide barcode 404 and the poly(dA) sequence of the partially duplex DNA template. After hybridization, the partially double-stranded DNA template can be ligated with the oligonucleotide barcode 404 having the poly(dT) region 412 using a ligase. The template strand having the poly(dA) sequence can be removed by denaturing the partially duplex DNA template ligated to the oligonucleotide barcode 404 to generate an oligonucleotide barcode 404 having a target-specific target binding region 412d and a poly(dT) region 412. The synthetic particles 400d1, 400d2 may include a spacer oligonucleotide (not shown).

[0263] Figures 5A-5C A non-limiting exemplary comparison is shown of an oligonucleotide barcode 404 having a poly(dT) region 412 capable of hybridizing to the poly(A) tail of an mRNA molecule. Figure 5A); an oligonucleotide barcode 404d1 having a target-specific target binding region 412 capable of binding to one or more target genes; and an oligonucleotide barcode 404d2 having a poly(dT) region 412 and a target-specific target binding region 412d. Oligonucleotide barcodes 404, 404d1, 404d2 can be associated with a single synthetic particle. In some embodiments, instead of coupling (e.g., covalently coupling) a poly(dT) oligonucleotide (for capturing mRNA via a poly(A) tail) to a particle (such as a synthetic particle or bead), oligonucleotide barcodes having various target-specific sequences 412 (e.g., gene-specific) can be coupled to the particle. These oligonucleotides can have a similar structure to the oligonucleotide having a poly(dT) region 404. Oligonucleotide barcodes 404d1, 404d2 can have different sequences for capturing RNA (including RNA molecules that do not have a poly(A) tail). For example, depending on the target of the oligonucleotide 404d1, 404d2 coupled, the particle can allow the capture of a specific subset of RNA. Because a single cell has about 300,000 mRNA molecules, even if only a subset of these molecules will eventually be targeted for genome amplification, it is necessary to couple to each particle to capture the quantity of the oligonucleotide barcode with poly (dT) district of all mRNA molecules (e.g., greater than 300,000). Using target-specific oligonucleotide barcodes 404d1, 404d2, a more selective group of cDNA molecules can be produced, because only the mRNA molecules for subsequent amplification and sequencing are captured. In addition, the quantity of the molecule (e.g., for each gene or for all target genes) for subsequent amplification and sequencing may be much lower than 300,000 copies, so it is necessary to couple the less oligonucleotide barcodes 404d1, 404d2 and particles targeting the gene or all target genes. The particles can be coupled with oligonucleotide barcodes 404d1, 404d2 that are capable of specifically capturing mRNA molecules of genes that are expected to be expressed by one or more cell types (e.g., CD4+ helper T cells). Thus, only the mRNA molecules of these genes can be captured and further analyzed (e.g., expression quantification).

[0264] A first plurality of oligonucleotides (e.g., spacer oligonucleotides and target-specific oligonucleotide barcodes)

[0265] Embodiments of synthetic particles are disclosed herein. In some embodiments, the synthetic particles (e.g., synthetic particles 400b, 400c1, 400c2 of references 4B and 4C1-4C2) include: a plurality of oligonucleotide barcodes (e.g., Figure 4B and 4C1 -Oligonucleotide barcode 404 in 4C2, and Figure 4D1-4D2Each of the plurality of oligonucleotide barcodes 404, 404d1, 404d may contain the same cell marker, molecular marker, and target binding region (e.g., Figure 4B , 4C1 -4C2 and 4D1-4D2). At least, or at most 10, 100, 1000, 10000, 100000, or more of the plurality of oligonucleotide barcodes may comprise different molecular marker sequences. The synthetic particle may include a first plurality of spacer oligonucleotides (e.g., reference Figure 4B and 4C1 -spacer oligonucleotides 420b, 420c1, 420c2 as described in -4C2).

[0266] In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions, referred to herein as target-specific oligonucleotide barcodes) can be or is about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) can be at least or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 .

[0267] In various implementations, the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) can be different, ranging from 1:10 to9 Up to 10 9 As used herein, the ratio of two said plurality of oligonucleotides refers to the ratio between the number of oligonucleotides in the first plurality of oligonucleotides and the number of oligonucleotides in the second plurality of oligonucleotides. In some embodiments, the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having a target-specific target binding region) can be or can be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1:10 8 , 1:10 9 , or a number or range between any two of these values. In some embodiments, the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides can be at least or at most 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1:10 8 or 1:10 9In some embodiments, the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having a target-specific target binding region) can be or is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 4 :1,10 5 :1,10 6 :1,10 7 :1,10 8 :1,10 9 :1, or a number or range between any two of these values. In some embodiments, the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having a target-specific target binding region) can be at least or at most 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 4 :1,10 5 :1,10 6 :1,10 7 :1,10 8 :1, or 10 9 :1.

[0268] The spacer oligonucleotide (or oligonucleotide barcode with target-specific target binding region) can be shorter, of the same length, or longer than the oligonucleotide barcode. In different implementations, the number of spacer oligonucleotides (or oligonucleotide barcode with target-specific target binding region) that are shorter, of the same length, or longer than each oligonucleotide barcode (or at least one oligonucleotide barcode) can be different. In some embodiments, the number of spacer oligonucleotides (or oligonucleotide barcodes having a target-specific target binding region) that is shorter, of the same length, or longer than each oligonucleotide barcode (or at least one oligonucleotide barcode) can be or can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of spacer oligonucleotides (or oligonucleotide barcodes with target-specific target binding regions) that are shorter, have the same length or are longer than each oligonucleotide barcode (or at least one oligonucleotide barcode) can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 For example, at least one of the first plurality of spacer oligonucleotides may be shorter than at least one of the plurality of oligonucleotide barcodes.

[0269] In some embodiments, the number of shorter, same length or longer oligonucleotide barcodes compared to each (or at least one) spacer oligonucleotide (or oligonucleotide with a target-specific target binding region) can be or can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of shorter, same length or longer oligonucleotide barcodes can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 90 ... 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 .

[0270] Each of the first plurality of spacer oligonucleotides may not comprise a target binding region. For example, Figure 4B The spacer oligonucleotide 420b shown in FIG. 4 may include a spacer region 424b that corresponds to the target binding region and cannot be used to hybridize with the target of interest. As another example, compared to the oligonucleotide barcode 404 lacking the target binding region 412, in Fig.4C1 As another example, compared to oligonucleotide barcode 404, Figure 4C2 The spacer oligonucleotide 420c1 shown in FIG. 4 is longer. Each of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having a target-specific target binding region) may include a cell marker or a molecular marker. Each of the first plurality of spacer oligonucleotides may include a region corresponding to a target binding region (referred to herein as a spacer region, e.g., in Figure 4B The spacer region 424b in the embodiment.

[0271] The region 424b corresponding to the target binding region and the target binding region (or two target binding regions) may have different sequences. The spacer region 424b and the target binding region (or two target binding regions) may be different. In different implementations, the sequence identity between the spacer region 424b and the target binding region (or between two target binding regions such as the poly(dT) region 412 and the target-specific target binding region 412d) may be different (in the range of 1% to 99%). In some embodiments, the sequence identity between the spacer region 424b and the target binding region 412, 412d (or both target binding regions) can be or can be about 1%, 2%, 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%, 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%, 99%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%,In some embodiments, the sequence identity between the spacer region 424b and the target binding region 412, 412d (or between two target binding regions) can be at least, or at most 1%, 2%, 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%, 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%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 98%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, For example, region 424b corresponding to a target binding region and target binding regions 412, 412d (or two target binding regions) can have up to 50% sequence identity. In some embodiments, spacer region 424b and target binding regions 412, 412d (or two target binding regions) can differ from each other by or about 1, 2, 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, 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 , 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, 99, 100, or a number or range between any two of these values.In some embodiments, the spacer region 424b and the target binding region 412, 412d (or both target binding regions) can differ from each other by at least, or at most, 1, 2, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 4, 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, 99, or 100.

[0272] In different implementations, different lengths (e.g. Figure 4C1-4C2 In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) of the spacer oligonucleotides 420c1, 420c2 can be different. In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having different lengths can be or can be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having different lengths can be at least or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6, 10 7 , 10 8 , or 10 9. For example, at least two of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) can have different lengths. In some embodiments, the percentage of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having different lengths can be or can be about 1%, 2%, 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%, 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%, 99%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having different lengths can be at least or at most 1%, 2%, 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%, 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%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 111%, 112%, 113%, , 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%, 99% or 100%.

[0273] In different implementations, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having the same sequence can be different. In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having the same sequence can be or can be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having the same sequence can be at least, at most, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 . For example, the sequences of at least two of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) can be identical. In different implementations, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having the same subsequence can be different. In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having the same subsequence can be or can be approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 107 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) having the same subsequence can be at least, at most, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 For example, at least two of the first plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) can comprise the same sequence.

[0274] A second plurality of oligonucleotides (e.g., a spacer oligonucleotide and a target-specific oligonucleotide barcode)

[0275] In some embodiments, the synthetic particle comprises a second plurality of spacer oligonucleotides (or an oligonucleotide barcode with a target-specific target binding region). Each of the second plurality of spacer oligonucleotides may not comprise a target binding region. One or more of the first plurality of spacer oligonucleotides and one or more of the second plurality of spacer oligonucleotides may have the same length and different nucleotide sequences. One or more of the first plurality of spacer oligonucleotides and one or more of the second plurality of spacer oligonucleotides may have different lengths and similar or different nucleotide sequences. One or more of the first plurality of target-specific oligonucleotides and one or more of the second plurality of target-specific oligonucleotides may have the same length and different nucleotide sequences. One or more of the first plurality of target-specific oligonucleotides and one or more of the second plurality of target-specific oligonucleotides may have different lengths and similar or different nucleotide sequences.

[0276] In some embodiments, the number of the second plurality of spacer oligonucleotides (or oligonucleotide barcodes having target-specific target binding regions) can be or is about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 104 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of the second plurality of spacer oligonucleotides (or oligonucleotide barcodes having a target-specific target binding region) can be at least or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 .

[0277] The second spacer oligonucleotides of the second plurality of spacer oligonucleotides (or the second target-specific oligonucleotide barcodes of the second plurality of target-specific oligonucleotide barcodes) can have different lengths. The number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be shorter, can have the same length, or can be longer than at least one or each of the first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes). In some embodiments, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) that is shorter, has the same length, or is longer than at least one or each of the first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be or is about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9, or a number or range between any two of these values. In some embodiments, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) that is shorter, has the same length, or is longer than at least one or each of the first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be at least or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9For example, at least one of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be shorter than at least one of the first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes). In some embodiments, the percentage of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) that is shorter, has the same length, or is longer than at least one or each of the first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be or is about 1%, 2%, 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%, 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%, 99%, 100%, or a number or range between any two of these values.In some embodiments, the percentage of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) that is shorter, has the same length, or is longer than at least one or each of the first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be or is about 1%, 2%, 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%, 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%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, %, 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%, 99% or 100%.

[0278] In some embodiments, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) that is shorter, has the same length, or is longer than at least one or each of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be or is about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9, or a number or range between any two of these values. In some embodiments, the number of first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) that is shorter, has the same length, or is longer than at least one or each of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be at least or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9For example, at least one of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be shorter than at least one of the plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes). In some embodiments, the percentage of the first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) that are shorter, of the same length, or longer than at least one or each of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be or can be about 1%, 2%, 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%, 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%, 99%, 100%, or a number or range between any two of these values.In some embodiments, the percentage of the first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) that are shorter, have the same length, or are longer than at least one or each of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be or can be about 1%, 2%, 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%, 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%, 99%, %, 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%, 99%, or 100%.

[0279] In different implementations, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having different lengths can be different. In some embodiments, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having different lengths can be or can be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9, or a number or range between any two of these values. In some embodiments, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having different lengths can be at least or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9. For example, at least two of the second plurality of spacer oligonucleotides can have different lengths. In some embodiments, the percentage of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having different lengths can be or can be about 1%, 2%, 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%, 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%, 99%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having different lengths can be at least or at most 1%, 2%, 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%, 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%, 99%, , 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%, 99% or 100%.

[0280] In different implementations, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having the same sequence can be different. In some embodiments, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having the same sequence can be or can be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having the same sequence can be at least, at most, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 . For example, the sequences of at least two of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) can be identical. In different implementations, the number of second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having the same subsequence can be different. In some embodiments, the number of second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having the same subsequence can be or can be approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9, or a number or range between any two of these values. In some embodiments, the number of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) having the same subsequence can be at least, at most, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 For example, at least two of the second plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes) may comprise the same sequence.

[0281] Target binding region

[0282] like Figure 4B and 4C1-4C2, the synthetic particles 400b, 400c1, 400c2 can include spacer oligonucleotides 420b, 420c1, 420c2 and oligonucleotide barcodes 404. The oligonucleotide barcodes 404 can include a target binding region 412 capable of capturing (e.g., hybridizing) a target molecule 408. The spacer oligonucleotide 420b can include a spacer region 424b that corresponds to the target binding region 412 and is unable to capture (e.g., hybridize) the target molecule 408 (or a subset of target molecules). The spacer oligonucleotides 420c1, 420c2 can include or lack a region corresponding to the target binding region 412. Including the spacer oligonucleotides 420b, 420c1, 420c2 on the synthetic particles 400b, 400c1, 400c2 can improve the capture of the target 408 (e.g., mRNA capture) and the subsequent quantification of the captured target molecule 408. For example, synthetic particles 400b, 400c1, 400c2 can be coupled to oligonucleotide barcodes 404 (e.g., barcodes each having a poly (dT) region, which can be bound to copies or occurrences of mRNA molecules or species) and spacer oligonucleotides 420b, 420c1, 420c2, which can improve mRNA capture and gene expression quantification. In some embodiments, the target binding region can hybridize with the target of interest. In some embodiments, the target binding region can include a nucleic acid sequence that specifically hybridizes with a target (e.g., a target nucleic acid, such as a cell nucleic acid to be analyzed). For example, the target binding region can hybridize with a target nucleic acid on a specific gene sequence. In some embodiments, the target binding region can include a nucleic acid sequence that can be attached (e.g., hybridized) to a specific position of a specific target nucleic acid. In some embodiments, the target binding region can include a nucleic acid sequence that can be specifically hybridized with a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode can then be connected to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang.

[0283] In some embodiments, the target binding region may include a non-specific target nucleic acid sequence (also referred to herein as a non-target specific target binding region). A non-specific target nucleic acid sequence may refer to a sequence that is independent of a specific sequence of a target nucleic acid and that can bind to multiple target nucleic acids. For example, the target binding region may include a random polymer sequence, or an oligo (dT) sequence hybridized with a poly (A) tail on an mRNA molecule. A random polymer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or a higher polymer sequence of any length. In some embodiments, for all barcodes attached to a given synthetic particle (e.g., a bead), the target binding region is the same. In some embodiments, for multiple barcodes attached to a given synthetic particle, the target binding region may include two or more different target binding sequences. The length of the target binding region may be or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. The target binding region may be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length.

[0284] In some embodiments, the target binding region can comprise an oligo(dT) that can hybridize to an mRNA comprising a polyadenylated (poly(A)) end. The target binding region can be target-specific (e.g., gene-specific). For example, the target binding region can be configured to hybridize to a specific region of the target. The target binding region can be or is about 1, 2, 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 nucleotides in length, or a number or range of nucleotides between any two of these values. The target binding region can be at least, or at most, 1, 2, 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, or 30 nucleotides in length. The target binding region can be about 5 to 30 nucleotides in length. When a barcode includes a gene-specific target binding region, the barcode can be referred to as a gene-specific barcode.

[0285] The target binding region may comprise a gene-specific sequence, an oligo(dT) sequence, a random polymer, or any combination thereof. In different implementations, the number of target binding regions of the oligonucleotide barcodes of the plurality of oligonucleotide barcodes (having different gene-specific sequences) may be different. In some embodiments, the number of target binding regions of the oligonucleotide barcodes of the plurality of oligonucleotide barcodes (having different gene-specific sequences) may be or may be approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of target binding regions of the oligonucleotide barcodes of the plurality of oligonucleotide barcodes (having different gene-specific sequences) can be at least or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9In some embodiments, the percentage of the target binding region of the oligonucleotide barcodes of the plurality of oligonucleotide barcodes (having different gene-specific sequences) can be or can be about 1%, 2%, 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%, 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%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of the target binding region of an oligonucleotide barcode of a plurality of oligonucleotide barcodes (having different gene-specific sequences) can be at least or at most 1%, 2%, 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%, 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%, 99%, %, 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%, 99% or 100%. For example, the two target binding regions of two oligonucleotide barcodes in the plurality of oligonucleotide barcodes can comprise different gene-specific sequences.

[0286] In some embodiments, at least or at most 100, 1000, 10000, 100000, or more of the multiple oligonucleotide barcodes contain different molecular marker sequences. Each cell marker in the multiple oligonucleotide barcodes can contain at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Each molecular marker in the multiple oligonucleotide barcodes can contain at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Each of the multiple oligonucleotide barcodes can contain a spatial marker, and wherein the spatial markers of the multiple oligonucleotide barcodes differ from each other by at least one nucleotide. Each of the multiple oligonucleotide barcodes can contain a universal marker, wherein the universal markers of the multiple oligonucleotide barcodes are the same.

[0287] In some embodiments, at least one of the multiple oligonucleotide barcodes is fixed on, partially fixed on, encapsulated in, partially encapsulated in, or any combination thereof. The synthetic particles may be destructible. The synthetic particles may include particles. The particles may include agarose gel beads, streptavidin protein beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo (dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particles may include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methyl styrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, and any combination thereof. The synthetic particles may include destructible hydrogel particles. Each of the plurality of oligonucleotide barcodes may comprise a linker functional group, the synthetic particle may comprise a solid support functional group, and / or the support functional group and the linker functional group may be associated with each other. The linker functional group and the support functional group may be individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof. In some embodiments, the plurality of synthetic particles comprises at least 94 synthetic particles. The kit may comprise a microwell array, wherein each microwell in the microwell array comprises a synthetic particle in the plurality of synthetic particles. The kit may comprise a buffer, a box, one or more reagents for a reverse transcription reaction, one or more reagents for an amplification reaction, or a combination thereof.

[0288] The disclosure herein includes embodiments of kits or compositions. In some embodiments, the kit or composition comprises: a plurality of synthetic particles, wherein each synthetic particle comprises: a first plurality of oligonucleotide barcodes; and a second plurality of oligonucleotide barcodes, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cell marker sequence and comprises a molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region; and optionally, instructions for using the plurality of synthetic particles. In some embodiments, the kit comprises a buffer, one or more reagents for a reverse transcription reaction, one or more reagents for an amplification reaction, or a combination thereof. In some embodiments, the plurality of synthetic particles comprises at least 94 synthetic particles. The kit may comprise a plurality of partitions, each partition comprising synthetic particles in the plurality of synthetic particles. The plurality of partitions may comprise microwells of a microwell array. The plurality of partitions may comprise a plurality of droplets. In some embodiments, the target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a target-specific target binding region. The target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes may comprise a poly(dT) region. The oligonucleotide barcode of the second plurality of oligonucleotide barcodes may comprise a poly(dT) region.

[0289] The disclosure provided herein includes embodiments of a kit comprising one or more synthetic particles of the present disclosure. In some embodiments, the kit comprises: a plurality of synthetic particles, wherein each synthetic particle comprises: a plurality of oligonucleotide barcodes, wherein each of the plurality of oligonucleotide barcodes comprises the same cell marker, molecular marker, and target binding region, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences; and a first plurality of spacer oligonucleotides (or target-specific oligonucleotide barcodes), wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region. The kit may comprise: instructions for using the plurality of oligonucleotide barcodes.

[0290] Barcoding using synthetic particles with spacer oligonucleotides

[0291] Embodiments disclosed herein include methods for determining the amount of a target in a sample (see Figure 2 and 3). In some embodiments, the method comprises: barcoding copies of a plurality of targets in a sample using a plurality of oligonucleotide barcodes associated with a synthetic particle to produce a plurality of barcoded copies of the targets, wherein each of the plurality of oligonucleotide barcodes comprises the same cellular marker, molecular marker, and target binding region, and wherein at least 10, 100, 1000, 10000, or more of the plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein the synthetic particle comprises a first plurality of spacer oligonucleotides, and wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region; and assessing the quantity of each of the plurality of targets in the sample using the molecular markers of the plurality of oligonucleotide barcodes. In some embodiments, barcoding copies of the plurality of targets in a sample using the plurality of oligonucleotide barcodes associated with the synthetic particle may comprise: hybridizing the plurality of targets with the plurality of oligonucleotide barcodes on the synthetic particle. Barcoding copies of the plurality of targets may comprise: reverse transcribing the plurality of targets to produce barcoded cDNAs of the plurality of targets. The method may include amplifying the barcoded cDNA using second strand synthesis to produce a barcoded target, wherein the barcoded target is double stranded. The method may include amplifying the barcoded target to produce a labeled target amplicon.

[0292] Disclosed herein are embodiments of methods for capturing targets in a sample. In some embodiments, the method comprises hybridizing copies of multiple targets in a sample using multiple oligonucleotide barcodes associated with a synthetic particle to produce multiple barcoded copies of the target, wherein each of the multiple oligonucleotide barcodes comprises the same cell marker, molecular marker, and target binding region, and wherein at least 10, 100, 1000, 10000, or more of the multiple oligonucleotide barcodes comprise different molecular marker sequences, wherein the synthetic particle comprises a first plurality of spacer oligonucleotides, and wherein each of the first plurality of spacer oligonucleotides does not comprise a target binding region. The method may comprise reverse transcribing the multiple targets to produce barcoded cDNAs of the multiple targets. The method may comprise amplifying the barcoded cDNAs using second strand synthesis to produce barcoded targets, wherein the barcoded targets are double stranded. The method may comprise amplifying the barcoded targets to produce labeled target amplicons.

[0293] The plurality of barcoded target copies can comprise a percentage of the copies of a target among a plurality of targets in a sample, a number of targets (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more targets), or every target in a sample. Such a percentage is a measure or indication of capture efficiency. In some embodiments, to generate copies of multiple barcoded targets, the percentage of copies of a target, two targets, three targets, or each target in a plurality of targets in a sample that hybridizes to an oligonucleotide barcode can be or can be about 1%, 2%, 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%, 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%, 99%, 100%, or a number or range between any two of these values.In some embodiments, to generate copies of a plurality of barcoded targets, the percentage of copies of a target, two targets, three targets, or each target in a sample that hybridizes to an oligonucleotide barcode can be at least, or at most, 1%, 2%, 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%, 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%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 111%, 112%, 113%, 3%, 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%, 99%, or 100%.

[0294] In some embodiments, the sample comprises a single cell, and the method comprises associating the synthetic particle with the single cell in the sample. The single cell may comprise a rare cell, a tumor cell, a cell from a human, a cell from a tissue, a cell from a tumor, a cell infected by a viral polynucleotide, and any combination thereof. The method may comprise: after associating the synthetic particle with the single cell, lysing the single cell. The method may comprise: lysing the single cell comprises heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof. The synthetic particle and the single cell may be in the same well. The synthetic particle and the single cell may be in the same droplet. The multiple targets may comprise ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA each containing a poly (A) tail, and any combination thereof.

[0295] target

[0296] The target can be or include a DNA target, an RNA target, or a combination thereof. In certain embodiments, the target is an mRNA target with a poly (A) tail. For example, the target can be an mRNA encoding a heavy immunoglobulin (e.g., IgA, IgD, IgE1, IgE2, IgG, and IgM) and a light immunoglobulin (e.g., IgK and IgL). For example, a bead with an oligonucleotide barcode with a target binding region specific to heavy immunoglobulins and light immunoglobulins can be used to determine the pairing of heavy immunoglobulins and light immunoglobulins in immune cells (e.g., T cells, B cells). Such beads can be used to determine the pairing of heavy immunoglobulins and light immunoglobulins of antibodies, thereby determining an antibody library. In certain embodiments, the target is an RNA target (also referred to herein as a non-polyadenylated target) without a poly (A) tail, such as non-coding RNA, histone mRNA, and viral RNA (e.g., reovirus mRNA). For example, the target can be from two or more organisms, such as the RNA of a cell and the RNA of bacteria, fungi, or viruses. Cells can be infected with bacteria, fungi and / or viruses. For example, beads having oligonucleotide barcodes with poly(dT) regions (for target binding) and oligonucleotide barcodes with target binding regions for viral mRNA can be used to determine cell phenotypes and viral genotypes during infection. In some embodiments, the target is a DNA target (e.g., a genomic DNA fragment without a poly(A) tail).

[0297] In some embodiments, targets (e.g., mRNA transcripts of genes) include CD4, FOX01, CD45RO, MYC, IL1R2, PRF1, GZMK, LGALS1, IL17F, IL23R, LYNX1, PRDM1, SELL, SMAD4, ICOS, IKZF5, RORC, AHRR, CTLA4, ITGB7, ENTPD1, ​​CCR8, TSHR, TGFB2, IL12A, IL7R, HLA-DMA, CCR5, TIAF1, BCL6, BHLHE40, CXCR4, and CD307c. Targets may include CD3D, GSTP1, TCF7, CD3E, RNB6, RB1, MYB, CD3G, KRT8, CDH1, ERBB3, ERBB2, TCTN1, ESR1, CDKN1A, and TFF3. Targets may include ABCB1, ABCG2, ADAM23, AKT1, APC, AR, ATM, BAD, BCL2, BIRC5, BRCA1, BRCA2, C4A, CA12, CCNA1, CCND1, CCND2, CCNE1, CDH1, CDH13, CDK2, CD326, CDKN1A, CDKN1C, CDKN2A, CSF1, CST6, CTNNB1, CTSD, EGF, EGFR, EMAP-2, ERBB2, ERBB3, ESR1, ESR2, FOXA1, GATA3, GLI1, GPI, GRB7, GSTP1, HIC1, HPRT1, ID1, IGF1, IGF1R, IGFBP3, IL6, JUN, KRT18, KRT19, KRT5, KRT8, LAMP1, MA PK1, MAPK3, MAPK8, MGMT, MKI67, MLH1, MMP2, MMP9, MUC1, MYB, MYC, NME1, NOTCH1, NR3C1, PGR, PLAU, PRDM2, PSMB2, PSMB4, PTEN, PTGS2, PYCARD, RAB7A, RARA, RARB, RASSF1, RB1, REEP5, RN B6, SERPINE1, SFN, SFRP1, SLC39A6, SLIT2, SNAI2, SRC, TBC1D9, TCTN1, TFF3, TGFB1, THBS1, TP73, TWIST1, VEGFA, XBP1, CD3E, CD3G, CD3G, TCF7, ALCAM, CD25, ITGA6, THY1, PROM1, and CXCR4. Targets may include BRCA1, BRCA2, TP53, PTEN, MSH2, MLH1, MSH6, PMS2, EPCAM, APC, RB1, MEN1, RET, and VHL.

[0298] The targets may be associated with: blood and lymphatic diseases; cancer; digestive system; ear, nose and throat; eye diseases; female-specific diseases; male-specific diseases; glands and hormones; heart and blood vessels; immune system diseases; male-specific diseases; muscle and bone; neonatal diseases; nervous system; nutritional and metabolic diseases; respiratory diseases; and / or skin and connective tissue.

[0299] Samples and target cells

[0300] In some embodiments, the sample can be or include a clinical sample, a biological sample, an environmental sample, or a combination thereof. For example, the sample can include one or more biological fluids, tissues, and cells from a subject. In some embodiments, the sample can include blood, urine, cerebrospinal fluid, pleural fluid, amniotic fluid, semen, saliva, bone marrow, biopsy samples, or a combination thereof. In some embodiments, the target cell can include stem cells, cancer cells, blood cells, peripheral blood mononuclear cells, circulating tumor cells (CTCs), breast cancer cells, cells in a desired cell cycle stage, or a combination thereof.

[0301] As used herein, the term "target cell" refers to a cell being studied. Non-limiting examples of target cells in a sample include cells expressing a malignant phenotype; tumor cells, such as tumor cells that have been shed from a tumor into the blood or other body fluids or bone marrow; benign tumor cells; cancer cells; cancer cells in peripheral blood; thyroid cancer cells; breast cancer cells; circulating tumor cells ("CTCs"); leukemia cells; cancer stem cells; single cells from different cell cycle stages (G0 / G1, S, G2); sperm with X and Y chromosomes; stem cells; fetal or adult stem cells; pluripotent stem cells; nucleated red blood cells ("NRBCs") in thalassemia patients; fetal cells, such as fetal cells in maternal peripheral blood; fetal nucleated red blood cells ("FNRBCs") in maternal circulation; and cells characterized by CD71, CD8, CD34, or CD133. The sample may include cells from a mixed cancer cell sample.

[0302] Other examples of cells of interest include, but are not limited to, the following: circulating endothelial cells; cells infected with viruses, such as cells infected with HIV, cells transfected with a gene of interest; and abnormal subtypes of T cells or B cells present in the peripheral blood of subjects suffering from autoimmune or autoreactive disorders; activated lymphocytes; antigen presenting cells, such as monocytes and dendritic cells; cells infected with pathogenic or parasitic organisms (e.g., mycoplasmas), cells containing intracellular parasites; and cells or microorganisms in dilute fluids (such as urine).

[0303] Non-limiting examples of cell lines include: Jurkat cells (a T leukemia cell line); SKBR3 (an adenocarcinoma-derived breast cancer cell line known for overexpression of Her2 / neu); T47D (a ductal carcinoma-derived breast cancer cell line showing low to moderate expression of Her2 / neu); and HeLa.

[0304] In some embodiments, multiple targets may be included in one or more samples. The sample may include one or more cells, or nucleic acids from one or more cells. The sample may be a single cell or nucleic acids from a single cell. The one or more cells may be one or more cell types. At least one of the one or more cell types may be a brain cell, a heart cell, a cancer cell, a circulating tumor cell, an organ cell, an epithelial cell, a metastatic cell, a benign cell, a primary cell, a circulating cell, or any combination thereof.

[0305] The sample used in the method of the present disclosure may include one or more cells. The sample may refer to one or more cells. In certain embodiments, a plurality of cells may include one or more cell types. At least one of the one or more cell types may be a brain cell, a heart cell, a cancer cell, a circulating tumor cell, an organ cell, an epithelial cell, a metastatic cell, a benign cell, a primary cell, a circulating cell, or any combination thereof. In certain embodiments, the cell is a cancer cell removed from a cancerous tissue, such as breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, brain cancer, melanoma, and non-melanoma skin cancer. In certain embodiments, the cell is derived from cancer, but is collected from body fluids (e.g., circulating tumor cells). Non-limiting examples of cancer may include adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma, and fibrosarcoma. The sample may include tissue, monolayer cells, fixed cells, tissue sections, or any combination thereof. The sample may include biological samples, clinical samples, environmental samples, biological fluids, tissues or cells from a subject. The sample can be obtained from a human, mammal, dog, rat, mouse, fish, fly, worm, plant, fungus, bacteria, virus, vertebrate, or invertebrate.

[0306] In some embodiments, the cell is a cell that has been infected by a virus and contains a viral oligonucleotide. In some embodiments, viral infection can be caused by a virus such as a single-stranded (+ strand or "sense") DNA virus (e.g., parvovirus), or a double-stranded RNA virus (e.g., reovirus). The beads disclosed herein can be used to determine the cell phenotype of infected cells and the genotype of the virus that infects the cell. For example, beads having an oligonucleotide barcode with a poly (dT) region (for target binding) and an oligonucleotide barcode with a target binding region for viral mRNA can be used to simultaneously determine the cell phenotype and / or genotype and viral genotype and / or phenotype during infection. In some embodiments, the cell is a cell that has been infected by bacteria (or fungi) and contains a bacterial oligonucleotide (or fungal oligonucleotide). The beads disclosed herein can be used to simultaneously determine the genotype and / or phenotype of the phenotype infected cell and the genotype and / or phenotype of the infected organism. In some embodiments, the cell is a bacterium. These can include gram-positive bacteria or gram-negative bacteria. In some embodiments, the cell is a fungus. In some embodiments, the cell is a protozoan or other parasite. In some embodiments, the cell is a host cell of a parasite.The beads disclosed herein can be used to simultaneously determine the phenotype and / or genotype of the host cell and the genotype and / or phenotype of the parasite.

[0307] As used herein, the term "cell" may refer to one or more cells. In some embodiments, the cell is a normal cell, for example, a human cell at a different stage of development, or a human cell from a different organ or tissue type. In some embodiments, the cell is a non-human cell, for example, a mammalian cell of other types (e.g., mouse, rat, pig, dog, cow or horse). In some embodiments, the cell is an animal or plant cell of other types. In other embodiments, the cell may be any prokaryotic or eukaryotic cell.

[0308] In some embodiments, the cells are sorted prior to associating the cells with the beads. For example, the cells can be sorted by fluorescence activated cell sorting or magnetic activated cell sorting, or more generally by flow cytometry. The cells can be filtered by size. In some embodiments, the retentate comprises cells to be associated with the beads. In some embodiments, the flow through comprises cells to be associated with the beads.

[0309] A sample can refer to a plurality of cells. A sample can refer to a monolayer of cells. A sample can refer to a thin section (e.g., a tissue thin section). A sample can refer to a solid or semi-solid collection of cells that can be placed in one dimension on an array.

[0310] Sequencing

[0311] In some embodiments, estimating the number of different barcoded targets (e.g., randomly barcoded targets) can include determining the sequence of a labeled target, a spatial marker, a molecular marker, a sample marker, a cell marker, or any product thereof (e.g., a labeled amplicon, or a labeled cDNA molecule). The amplified target can be subjected to sequencing. Determining the sequence of a barcoded target (e.g., a randomly barcoded target) or any product thereof can include performing a sequencing reaction to determine the sequence of at least a portion of a sample marker, a spatial marker, a cell marker, a molecular marker, a labeled target (e.g., a randomly labeled target), its complement, the sequence of at least a portion of its reverse complement, or the sequence of any combination thereof.

[0312] Determination of the sequence of a barcoded target or a randomly barcoded target (e.g., an amplified nucleic acid, a labeled nucleic acid, a cDNA copy of a labeled nucleic acid, etc.) can be performed using various sequencing methods, including but not limited to: sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ), FISSEQ beads, wobble sequencing, multiplex sequencing, polycolony (POLONY) sequencing; nanogrid rolling circle sequencing (ROLONY), allele-specific oligo ligation assay (e.g., oligo ligation assay (OLA), single template molecule OLA using ligated linear probes and rolling circle amplification (RCA) readouts, ligated padlock probes, or single template molecule OLA using ligated circular padlock probes and rolling circle amplification (RCA) readouts), etc.

[0313] In some embodiments, determining the sequence of a barcoded target (e.g., a randomly barcoded target) or any product thereof comprises paired end sequencing, nanopore sequencing, high throughput sequencing, shotgun sequencing, dye terminator sequencing, multiplexed DNA sequencing, primer walking, Sanger dideoxy sequencing, Maxim-Gilbert sequencing, pyrosequencing, true single molecule sequencing, or any combination thereof. Alternatively, the sequence of a barcoded target or any product thereof can be determined by electron microscopy analysis or a chemically sensitive field effect transistor (chemFET) array.

[0314] High throughput sequencing methods can be used, such as cycle array sequencing using a platform such as Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrent, Complete Genomics, Pacific Bioscience, Helicos, or Polonator platform. In some embodiments, sequencing can include MiSeq sequencing. In some embodiments, sequencing can include HiSeq sequencing.

[0315] The labeled target (e.g., a randomly labeled target) may include nucleic acids representing about 0.01% to about 100% of the genomic genes of an organism. For example, a target complementary region comprising a plurality of polymers may be used to sequence about 0.01% of the genomic genes of an organism to about 100% of the genomic genes of an organism by capturing genes containing complementary sequences from the sample. In some embodiments, the barcoded target includes nucleic acids representing from about 0.01% of the transcripts of the transcriptome of an organism to about 100% of the transcripts of the transcriptome of an organism. For example, a target complementary region comprising a poly (T) tail may be used to sequence about 0.501% of the transcripts of the transcriptome of an organism to about 100% of the transcripts of the transcriptome of an organism by capturing mRNA from the sample.

[0316] Determining the sequence of the spatial and molecular markers of the plurality of barcodes (e.g., random barcodes) may include sequencing 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, 100%, or a number or range between any two of these values ​​of the plurality of barcodes. Determining the sequence of the markers (e.g., sample markers, spatial markers, and molecular markers) of the plurality of barcodes may include sequencing 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 1019 , 10 20 , 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a number or range between any two of these values. Sequencing some or all of the plurality of barcodes can include generating a sequence having, having about, having at least, or having a read length of at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 5000, 6000, 7000, 8000, 9000, 10000, or a number or range between any two of these values.

[0317] Sequencing can include sequencing at least or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides or base pairs of the barcoded targets. For example, sequencing can include generating sequencing data by polymerase chain reaction (PCR) amplification of a plurality of barcoded targets, wherein the sequences have a read length of 50, 75, or 100, or more nucleotides. Sequencing can include sequencing at least or at least about 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more nucleotides or base pairs of the barcoded targets. Sequencing can include sequencing at least or at least about 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 or more nucleotides or base pairs of the barcoded target.

[0318] Sequencing can include at least about 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more sequencing reads / run. In some embodiments, sequencing includes sequencing at least or at least about 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 or more sequencing reads / run. Sequencing can include less than or equal to about 1,600,000,000 sequencing reads / run. Sequencing can include less than or equal to about 200,000,000 reads / run.

[0319] Examples

[0320] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way.

[0321] Example 1

[0322] Improving RNA capture using beads with spacer oligonucleotides

[0323] This experiment shows that beads with spacer oligonucleotides can capture RNA molecules with higher efficiency than beads without spacer oligonucleotides.

[0324] For beads with spacer oligonucleotides, quantitative polymerase chain reaction (qPCR) experiments using bulk RNA showed improved capture efficiency ( Figure 6 ). Figure 6 : is a bar graph showing a non-limiting exemplary improvement in RNA capture using beads, each bead comprising multiple oligonucleotide barcodes having an oligo (dT) tail and multiple spacer oligonucleotides capable of capturing RNA molecules. The same amount of RNA was hybridized to 10,000 beads (with or without spacer oligonucleotides). After generating cDNA from the captured RNA molecules, qPCR was performed with primers that amplify GAPDH. Compared to beads without spacer oligonucleotides, beads with spacer oligonucleotides were about 0.3-0.5 Cqs lower (quantitative cycles, cycles in which fluorescence from amplification exceeds background fluorescence). This means that the amount of GAPDH captured by the spacer beads is about 125%-138% relative to GAPDH captured by beads with only dT.

[0325] Together, these data suggest that RNA capture efficiency can be improved by reducing the density of oligonucleotide barcodes using spacer oligonucleotides.

[0326] Example 2

[0327] Target-specific RNA capture

[0328] This example shows that beads with target-specific oligonucleotides can capture RNA molecules of interest with higher efficiency compared to beads with oligonucleotides with poly(dT) regions for RNA capture.

[0329] The target-specific oligonucleotide includes a target binding region with a sequence of 5'-ACAAGTCTGTCTG-3' (SEQ ID NO: 4) and 5'-CACACCCAAAAGGCCACACTGGT-3' (SEQ ID NO: 5), which is specific to the constant region of TCRα and TCRβ. For beads with target-specific oligonucleotides (specific to the constant region of TCRα and TCRβ), quantitative polymerase chain reaction (qPCR) experiments with bulk RNA show that compared with beads with oligonucleotides with poly (dT) sequences, the capture efficiency is improved (Figure 143). Figure 143 is a bar graph showing a non-limiting exemplary comparison of T cell receptor (TCR) capture efficiency using beads with TCR-specific oligonucleotide barcodes. The same amount of RNA is hybridized to 10,000 beads (beads with oligonucleotides with poly (dT) target binding regions or beads with oligonucleotides with poly (dT) target binding regions and target binding regions specific to the constant regions of TCRα and TCRβ). After generating cDNA from the captured RNA molecules, qPCR was performed using primers targeting the variable and constant regions of TCRα and the variable and constant regions of TCRβ to amplify TCRα and TCRβ. The amount of TCRα molecules captured by beads with target-specific oligonucleotide barcodes was about 260% relative to TCRα captured by beads with oligonucleotide barcodes with oligo (dT) regions for non-specific target binding. The amount of TCRβ molecules captured by beads with target-specific oligonucleotide barcodes was about 425% relative to TCRβ captured by beads with oligonucleotide barcodes with oligo (dT) regions for non-specific target binding.

[0330] Together, these data suggest that the use of oligonucleotide barcodes with target-specific binding regions can improve specific RNA capture efficiency.

[0331] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0332] With respect to substantially any plural and / or singular terms used herein, those skilled in the art may convert from the plural to the singular and / or from the singular to the plural where appropriate for the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural references unless the context clearly indicates otherwise. Any reference to "or" herein is intended to encompass "and / or" unless otherwise indicated.

[0333] Those skilled in the art will understand that, in general, the terms used herein, especially in the appended claims (e.g., the bodies 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 "includes but is not limited to", etc.). Those skilled in the art will further understand that if a specific number of an introduced claim statement is anticipated, such anticipation will be expressly stated in the claim, and in the absence of such a statement no such intention exists. For example, as an aid to understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim statements. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim recitation by the indefinite article "a" or "an" will limit any specific claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "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 to the use of definite articles to introduce claim recitations. In addition, even if a specific number of introduced claim recitations is explicitly stated, one skilled in the art will recognize that such statement should be interpreted to mean at least the stated number (e.g., merely stating "two recitations" without other modifiers means at least two recitations, or two or more recitations). Furthermore, in those cases where a convention similar to “at least one of A, B, and C, etc.” is used, generally such syntactic structure is intended in the sense that persons skilled in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but is not limited to systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to “at least one of A, B, or C, etc.” is used, generally such syntactic structure is intended in the sense that persons skilled in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but is not limited to systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will further appreciate that, in fact, any disjunctive words and / or phrases presenting two or more alternative terms, whether in the specification, claims or drawings, should be understood to contemplate the possibility of including one, either or both terms.

[0334] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0335] As will be understood by those skilled in the art, for any and all purposes, such as in providing written description, all scopes disclosed herein also include any and all possible sub-ranges and sub-range combinations thereof. Any listed scope can be easily identified as fully describing and enabling the same scope to be decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each scope discussed here can be easily decomposed into lower third, middle third and upper third, etc. As will be understood by those skilled in the art, all languages, such as "up to", "at least", "greater than", "less than", etc., include stated numbers, and refer to the scope that can be subsequently decomposed into the sub-ranges discussed above. Finally, as will be understood by those skilled in the art, scope includes each individual member. Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles. Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0336] 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 for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. Sequence Listing <110> Cellular Research, Inc. Lam, Gretchen Yinbon Shum, Eleen <120> Particles associated with oligonucleotides <130> BDCRI.030WO <150> 62 / 607,812 <151> 2017-12-19 <160> 5 <170> PatentIn Version 3.5 <210> 1 <211> 103 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> n <222> (78)..(85) <223> n = a, t, c, or g <220> <221> Features not yet classified <222> (78)..(85) <223> n is a, c, g, or t <400> 1 tctctctcta cacgacgctc ttccgatctg atggatgact ggcctgcgac tacacgaggt 60 agcggtgact tccaaggnnn nnnnnttttt tttttttttt ttt 103 <210> 2 <211> 103 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> n <222> (78)..(103) <223> n = a, t, c, or g <220> <221> Features not yet classified <222> (78)..(103) <223> n is a, c, g, or t <400> 2 tctctctcta cacgacgctc ttccgatctg atggatgact ggcctgcgac tacacgaggt 60 agcggtgact tccaaggnnn nnnnnnnnnn nnnnnnnnnn nnn 103 <210> 3 <211> 121 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> n <222> (75)..(85) <223> n = a, t, c, or g <220> <...

Claims

1. A synthetic particle comprising: a first plurality of oligonucleotide barcodes coupled to the synthetic particle; and a second plurality of oligonucleotide barcodes coupled to the synthetic particle, wherein each of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprises the same cellular marker sequence and molecular marker sequence, wherein at least 10 of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes comprise different molecular marker sequences, Each cell marker sequence comprises at least 6 nucleotides, each molecular marker sequence comprises at least 6 nucleotides, wherein each of the first plurality of oligonucleotide barcodes comprises a target binding region comprising a poly(dT) region or a random polymer, and wherein each of the second plurality of oligonucleotide barcodes comprises a target-specific target binding region comprising a target-specific sequence for hybridizing to a target nucleic acid, Wherein a ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is in a range from 1:10 to 10:

1. 2 . The synthetic particle of claim 1 , wherein the target binding region of an oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a poly(dT) region.

3. The synthetic particle of claim 2, wherein the oligonucleotide barcodes of the second plurality of oligonucleotide barcodes comprise: the poly(dT) region; and a target-specific target binding region 3' to the poly(dT) region. 4 . The synthetic particle of claim 1 , wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a sample marker sequence.

5. The synthetic particle of claim 1, wherein the target binding region and the target-specific target binding region have at most 50% sequence identity.

6. The synthetic particle of claim 1, wherein at least two of the first plurality of oligonucleotide barcodes have different lengths, and / or at least two of the second plurality of oligonucleotide barcodes have different lengths.

7. The synthetic particle of claim 1, wherein the sequences of at least two of the first plurality of oligonucleotide barcodes are identical, and / or the sequences of at least two of the second plurality of oligonucleotide barcodes are identical.

8. The synthetic particle of claim 1, wherein at least two of the first plurality of oligonucleotide barcodes comprise the same sequence, and / or at least two of the second plurality of oligonucleotide barcodes comprise the same sequence.

9. The synthetic particle of any one of claims 1-8, comprising a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not comprise the target binding region or the target-specific target binding region.

10. The synthetic particle of claim 9, wherein the synthetic particle comprises a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise the target binding region or the target-specific target binding region.

11. The synthetic particle of any one of claims 1-8, wherein at least one of the first plurality of oligonucleotide barcodes is shorter than at least one of the second plurality of oligonucleotide barcodes.

12. The synthetic particle of any one of claims 1-8, wherein at least one of the second plurality of oligonucleotide barcodes is shorter than at least one of the first plurality of oligonucleotide barcodes.

13. The synthetic particle of any one of claims 1-8, wherein at least 100 of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprise different molecular marker sequences.

14. The synthetic particle of any one of claims 1-8, wherein the two target-specific target binding regions of two oligonucleotide barcodes in the second plurality of oligonucleotide barcodes comprise different target-specific sequences.

15. The synthetic particle of any one of claims 1-8, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a spatial marker sequence, and wherein the spatial marker sequences differ from each other by at least one nucleotide.

16. The synthetic particle of any one of claims 1-8, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a universal sequence, wherein the universal sequences are the same.

17. The synthetic particle of any one of claims 1-8, wherein at least one of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

18. The synthetic particle of any one of claims 1-8, wherein at least a portion of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes are immobilized on the synthetic particle.

19. The synthetic particle of any one of claims 1-8, wherein at least one of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes is encapsulated in the synthetic particle.

20. The synthetic particle of any one of claims 1-8, wherein at least a portion of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes is encapsulated in the synthetic particle.

21. The synthetic particle of any one of claims 1-8, wherein the synthetic particle is destructible.

22. The synthetic particle of any one of claims 1-8, wherein the synthetic particle comprises a bead.

23. The synthetic particle of claim 22, wherein the beads comprise magnetic beads.

24. The synthetic particle of any one of claims 1-8 and 10, wherein the synthetic particle comprises a destructible hydrogel particle.

25. The synthetic particle according to any one of claims 1 to 8, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a linker functional group, wherein the synthetic particles comprise solid support functional groups, and wherein the support functional group and the linker functional group are associated with each other.

26. The synthetic particle of claim 25, wherein the linker functional group and the support functional group are individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof.

27. A kit comprising: A plurality of synthetic particles as claimed in any one of claims 1 to 8; and Instructions for using the plurality of synthetic particles.

28. The kit of claim 27, wherein the plurality of synthetic particles comprises at least 94 synthetic particles.

29. The kit of claim 27, comprising a plurality of partitions, each partition comprising a synthetic particle of the plurality of synthetic particles.

30. The kit of claim 29, wherein the plurality of partitions comprises microwells of a microwell array.

31. The kit of claim 29, wherein the plurality of partitions comprises a plurality of droplets.

32. The kit of claim 27, comprising a buffer.

33. The kit of claim 27, comprising a cassette.

34. The kit of claim 27, comprising one or more reagents for a reverse transcription reaction.

35. The kit of claim 27, comprising one or more reagents for an amplification reaction.

36. The kit of claim 27, wherein each synthetic particle comprises a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not comprise the target binding region and the target-specific target binding region.

37. The kit of claim 36, wherein each synthetic particle comprises a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise the target binding region and the target-specific target binding region.

38. The kit of claim 27, wherein at least one of the first plurality of oligonucleotide barcodes is shorter than at least one of the second plurality of oligonucleotide barcodes.

39. The kit of claim 27, wherein at least one of the second plurality of oligonucleotide barcodes is shorter than at least one of the first plurality of oligonucleotide barcodes.

40. The kit of claim 27, wherein at least 100 of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprise different molecular marker sequences.

41. The kit of claim 27, wherein the two target-specific target binding regions of two oligonucleotide barcodes in the second plurality of oligonucleotide barcodes comprise different target-specific sequences.

42. The kit of claim 27, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a spatial marker sequence, and wherein the spatial marker sequences differ from each other by at least one nucleotide.

43. The kit of claim 27, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a universal sequence, wherein the universal sequences are identical.

44. The kit of claim 27, wherein at least one of the plurality of synthetic particle oligonucleotides is immobilized on the synthetic particle.

45. The kit of claim 27, wherein at least a portion of the first plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

46. ​​The kit of claim 27, wherein at least one of the first plurality of oligonucleotide barcodes is encapsulated in the synthetic particle.

47. The kit of claim 27, wherein at least a portion of the first plurality of oligonucleotide barcodes is encapsulated in the synthetic particles.

48. The kit of claim 27, wherein the synthetic particles are destructible.

49. The kit of claim 27, wherein the synthetic particles comprise beads.

50. The kit of claim 49, wherein the beads comprise magnetic beads.

51. The kit of claim 27, wherein the synthetic particles comprise destructible hydrogel particles.

52. The kit according to claim 27, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a linker functional group, wherein the synthetic particles comprise solid support functional groups, and wherein the support functional group and the linker functional group are associated with each other.

53. The kit of claim 52, wherein the linker functional group and the support functional group are individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof.

54. A method for determining the amount of a target nucleic acid in a sample, the method comprising: barcoding a first plurality of copies of a target nucleic acid in a sample using a first plurality of oligonucleotide barcodes associated with the synthetic particles of any one of claims 1-8 to produce a first plurality of barcoded copies of the target nucleic acid; barcoding a second plurality of copies of the target nucleic acid in the sample using a second plurality of oligonucleotide barcodes associated with the synthetic particles to produce a second plurality of barcoded copies of the target nucleic acid; as well as assessing the quantity of each of the first plurality of target nucleic acids and the second plurality of target nucleic acids using the molecular marker sequences of the first plurality of oligonucleotide barcodes and the second plurality of oligonucleotide barcodes, The method is not intended for disease diagnosis.

55. The method of claim 54, wherein the first plurality of target nucleic acids comprises mRNA molecules of cells, and wherein the second plurality of target nucleic acids comprises DNA molecules of the cells.

56. The method of claim 54, wherein the first plurality of target nucleic acids comprises mRNA molecules of a cell, and wherein the second plurality of target nucleic acids comprises RNA molecules of a virus, bacteria and / or fungus that infects the cell.

57. The method of claim 54, wherein barcoding the copies of the first plurality of target nucleic acids in the sample using the first plurality of oligonucleotide barcodes associated with the synthetic particles comprises: The first plurality of target nucleic acids are hybridized to the first plurality of oligonucleotide barcodes on the synthetic particles, wherein barcoding copies of the second plurality of target nucleic acids in the sample using the second plurality of oligonucleotide barcodes associated with the synthetic particles comprises: hybridizing the second plurality of target nucleic acids to the second plurality of oligonucleotide barcodes on the synthetic particles.

58. The method of claim 54, wherein barcoding the first plurality of copies of the target nucleic acid comprises: The first plurality of target nucleic acids are reverse transcribed to generate barcoded cDNAs of the first plurality of target nucleic acids, and wherein barcoding copies of the second plurality of target nucleic acids comprises reverse transcribing the second plurality of target nucleic acids to generate barcoded cDNAs of the second plurality of target nucleic acids.

59. The method of claim 58, comprising: The barcoded cDNAs of the first plurality of target nucleic acids are amplified and / or the barcoded cDNAs of the second plurality of target nucleic acids are amplified using second strand synthesis to generate first double-stranded barcoded target nucleic acids and second double-stranded barcoded target nucleic acids, respectively.

60. The method of claim 59, comprising: The first double-stranded barcoded target nucleic acid and the second double-stranded barcoded target nucleic acid are amplified to produce a first labeled target amplicon and a second labeled target amplicon, respectively.

61. A method for capturing a target nucleic acid in a sample, the method comprising: hybridizing a first plurality of copies of a target nucleic acid in a sample using a first plurality of oligonucleotide barcodes associated with the synthetic particles of any one of claims 1-8 to produce a first plurality of barcoded copies of the target nucleic acid; hybridizing a second plurality of copies of the target nucleic acid using a second plurality of oligonucleotide barcodes associated with the synthetic particles to produce a second plurality of barcoded copies of the target nucleic acid, The method is not intended for disease diagnosis.

62. The method of claim 61, wherein the second plurality of copies of barcoded target nucleic acids comprises copies of at least 50% of the target nucleic acids in the second plurality of target nucleic acids.

63. The method of claim 61, wherein the second plurality of copies of barcoded target nucleic acids comprises copies of at least 75% of the target nucleic acids in the second plurality of target nucleic acids.

64. The method of claim 61, wherein the second plurality of copies of barcoded target nucleic acids comprises copies of at least 95% of the target nucleic acids in the second plurality of target nucleic acids.

65. The method of claim 61, wherein the copies of the second plurality of barcoded target nucleic acids comprise at least 50% of the copies of each target nucleic acid in the second plurality of target nucleic acids.

66. The method of claim 61, wherein the second plurality of copies of barcoded target nucleic acids comprises at least 75% of copies of each target nucleic acid in the plurality of target nucleic acids.

67. The method of claim 61, wherein the copies of the second plurality of barcoded target nucleic acids comprise at least 95% of the copies of each target nucleic acid in the second plurality of target nucleic acids.

68. The method of claim 61, wherein the first plurality of target nucleic acids and the second plurality of target nucleic acids comprise mRNA, the method comprising: reverse transcribing the first plurality of target nucleic acids to generate first barcoded cDNAs of the first plurality of target nucleic acids; and The second plurality of target nucleic acids is reverse transcribed to generate second barcoded cDNAs of the second plurality of target nucleic acids.

69. The method of claim 68, comprising: The first barcoded cDNA and the second barcoded cDNA are amplified using second strand synthesis to generate a first barcoded double-stranded target and a second barcoded double-stranded target, respectively.

70. The method of claim 69, comprising: The first barcoded target nucleic acid and the second barcoded target nucleic acid are amplified to produce a first labeled target amplicon and a second labeled target amplicon, respectively.

71. The method of claim 54 or 61, wherein the first plurality of target nucleic acids and the second plurality of target nucleic acids are from a single cell, the method comprising associating the synthetic particles with the single cell.

72. The method of claim 71, wherein the single cell comprises a rare cell, a tumor cell, a cell infected with a viral polynucleotide, or any combination thereof.

73. The method of claim 71, wherein the single cell comprises a cell from a human.

74. The method of claim 71, wherein the single cell comprises a cell from a tissue.

75. The method of claim 71, wherein the single cell comprises a cell from a tumor.

76. The method of claim 71, comprising: After associating the synthetic particles with the single cells, the single cells are lysed.

77. The method of claim 76, wherein lysing the single cells comprises heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

78. The method of claim 71, wherein the synthetic particles and the single cells are in the same well.

79. The method of claim 71, wherein the synthetic particles and the single cells are in the same droplet.

80. The method of claim 54 or 61, wherein the first plurality of target nucleic acids and / or the second plurality of target nucleic acids comprises ribonucleic acid (RNA), RNA degradation products, and any combination thereof.

81. The method of claim 54 or 61, wherein the first plurality of target nucleic acids and / or the second plurality of target nucleic acids comprise messenger RNA (mRNA), micro RNA, small interfering RNA (siRNA), viral RNA, and any combination thereof.

82. The method of claim 54 or 61, wherein the first plurality of target nucleic acids and / or the second plurality of target nucleic acids comprise RNAs each comprising a poly(A) tail or RNAs that do not contain any poly(A) tail.

83. The method of claim 54 or 61, wherein the synthetic particles comprise a first plurality of spacer oligonucleotides, wherein each of the first plurality of spacer oligonucleotides does not comprise the target binding region and the target-specific target binding region.

84. The method of claim 83, wherein the synthetic particles comprise a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise the target binding region and the target-specific target binding region.

85. The method of claim 54 or 61, wherein at least one of the first plurality of oligonucleotide barcodes is shorter than at least one of the second plurality of oligonucleotide barcodes.

86. The method of claim 54 or 61, wherein at least one of the second plurality of oligonucleotide barcodes is shorter than at least one of the first plurality of oligonucleotide barcodes.

87. The method of claim 54 or 61, wherein at least 100 of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprise different molecular marker sequences.

88. The method of claim 54 or 61, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a spatial marker sequence, and wherein the spatial marker sequences differ from each other by at least one nucleotide.

89. The method of claim 54 or 61, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a universal sequence, wherein the universal sequences are the same.

90. The method of claim 54 or 61, wherein at least one of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

91. The method of claim 54 or 61, wherein at least a portion of the first plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

92. The method of claim 54 or 61, wherein at least one of the first plurality of oligonucleotide barcodes is encapsulated in the synthetic particle.

93. The method of claim 54 or 61, wherein at least a portion of the first plurality of oligonucleotide barcodes is encapsulated in the synthetic particle.

94. The method of claim 54 or 61, wherein the synthetic particles are destructible.

95. The method of claim 54 or 61, wherein the synthetic particles comprise beads.

96. The method of claim 95, wherein the beads comprise magnetic beads.

97. The method of claim 54 or 61, wherein the synthetic particles comprise destructible hydrogel particles.

98. The method according to claim 54 or 61, wherein each of the first plurality of oligonucleotide barcodes and / or the second plurality of oligonucleotide barcodes comprises a linker functional group, wherein the synthetic particles comprise solid support functional groups, and wherein the support functional group and the linker functional group are associated with each other.

99. The method of claim 98, wherein the linker functional group and the support functional group are individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof.

100. A synthetic particle comprising: a plurality of oligonucleotide barcodes coupled to the synthetic particle, wherein each of the plurality of oligonucleotide barcodes comprises the same cellular marker sequence, molecular marker sequence, and target binding region that hybridizes to a target nucleic acid in a sample, and wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences, wherein each cellular marker sequence comprises at least 6 nucleotides, wherein each molecular marker sequence comprises at least 6 nucleotides; and a first plurality of spacer oligonucleotides coupled to the synthetic particle, wherein each of the first plurality of spacer oligonucleotides does not comprise the target binding region, wherein the plurality of oligonucleotide barcodes and the first plurality of spacer oligonucleotides are different molecules, and wherein the first plurality of spacer oligonucleotides coupled to the synthetic particle reduces the density of the plurality of oligonucleotide barcodes on the synthetic particle compared to a synthetic particle coupled only to the plurality of oligonucleotide barcodes, wherein the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides is in the range of from 1:10 to 10:

1.

101. The synthetic particle of claim 100, wherein each of the first plurality of spacer oligonucleotides comprises a cellular marker sequence or a molecular marker sequence.

102. The synthetic particle of claim 100, wherein at least one of the first plurality of spacer oligonucleotides is shorter than at least one of the plurality of oligonucleotide barcodes.

103. The synthetic particle of claim 100, wherein each of the first plurality of spacer oligonucleotides comprises a region corresponding to the target binding region, and wherein the region corresponding to the target binding region and the target binding region have different sequences.

104. The synthetic particle of claim 103, wherein the region corresponding to the target binding region and the target binding region have at most 50% sequence identity.

105. The synthetic particle of claim 100, wherein at least two of the first plurality of spacer oligonucleotides have different lengths.

106. The synthetic particle of claim 100, wherein the sequence of at least two of the first plurality of spacer oligonucleotides are identical.

107. The synthetic particle of claim 100, wherein at least two of the first plurality of spacer oligonucleotides comprise the same sequence.

108. The synthetic particle of any one of claims 100-107, wherein the synthetic particle comprises a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise the target binding region.

109. The synthetic particle of claim 108, wherein at least one of the second plurality of spacer oligonucleotides is shorter than at least one of the first plurality of spacer oligonucleotides.

110. The synthetic particle of claim 108, wherein at least one of the second plurality of spacer oligonucleotides is shorter than at least one of the plurality of oligonucleotide barcodes.

111. The synthetic particle of claim 108, wherein at least two of the second plurality of spacer oligonucleotides have different lengths.

112. The synthetic particle of claim 108, wherein the sequence of at least two of the second plurality of spacer oligonucleotides are identical.

113. The synthetic particle of claim 108, wherein at least two of the second plurality of spacer oligonucleotides comprise the same sequence.

114. The synthetic particle of any one of claims 100-107, wherein at least 100 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences.

115. The synthetic particle of any one of claims 100-107, wherein the target binding region comprises a gene-specific sequence or an oligo (dT) sequence.

116. The synthetic particle of any one of claims 100-107, wherein the two target binding regions of two oligonucleotide barcodes in the plurality of oligonucleotide barcodes comprise different gene-specific sequences.

117. The synthetic particle of any one of claims 100-107, wherein each of the plurality of oligonucleotide barcodes comprises a spatial marker sequence, and wherein the spatial marker sequences of the plurality of oligonucleotide barcodes differ from each other by at least one nucleotide.

118. The synthetic particle of any one of claims 100-107, wherein each of the plurality of oligonucleotide barcodes comprises a universal tag sequence, wherein the universal tag sequences of the plurality of oligonucleotide barcodes are the same.

119. The synthetic particle of any one of claims 100-107, wherein at least one of the plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

120. The synthetic particle of any one of claims 100-107, wherein at least a portion of the plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

121. The synthetic particle of any one of claims 100-107, wherein at least one of the plurality of oligonucleotide barcodes is encapsulated in the synthetic particle.

122. The synthetic particle of any one of claims 100-107, wherein at least a portion of the plurality of oligonucleotide barcodes is encapsulated within the synthetic particle.

123. The synthetic particle of any one of claims 100-107, wherein the synthetic particle is destructible.

124. The synthetic particle of any one of claims 100-107, wherein the synthetic particle comprises a bead.

125. The synthetic particles of claim 124, wherein the beads comprise agarose beads, magnetic beads, conjugated beads, silica beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof.

126. The synthetic particles of claim 124, wherein the beads comprise agarose gel beads, streptavidin protein beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica-like beads, or any combination thereof.

127. The synthetic particle of any one of claims 100-107, wherein the synthetic particle comprises a material selected from the group consisting of: Glass, hydrogel, paramagnetic substances, ceramics, plastics, titanium, latex, cellulose, nylon, silicone, and any combination thereof.

128. The synthetic particle of any one of claims 100-107, wherein the synthetic particle comprises a material selected from the group consisting of: Polydimethylsiloxane (PDMS), polystyrene, polypropylene, agarose, gelatin, and any combination thereof.

129. The synthetic particle of any one of claims 100-107, wherein the synthetic particle comprises a material selected from the group consisting of methyl styrene, acrylic polymer, agarose gel, and any combination thereof.

130. The synthetic particle of any one of claims 100-107, wherein the synthetic particle comprises a destructible hydrogel particle.

131. The synthetic particle according to any one of claims 100 to 107, wherein each of the plurality of oligonucleotide barcodes comprises a linker functional group, wherein the synthetic particles comprise solid support functional groups, and wherein the support functional group and the linker functional group are associated with each other.

132. The synthetic particle of claim 131, wherein the linker functional group and the support functional group are individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof.

133. A kit comprising: A plurality of synthetic particles according to any one of claims 100-107; and Instructions for using the plurality of oligonucleotide barcodes.

134. The kit of claim 133, wherein the plurality of synthetic particles comprises at least 94 synthetic particles.

135. The kit of claim 133, comprising a microwell array, wherein each microwell of the microwell array comprises a synthetic particle of the plurality of synthetic particles.

136. The kit of claim 133, comprising a buffer.

137. The kit of claim 133, comprising a box.

138. The kit of claim 133, comprising one or more reagents for a reverse transcription reaction.

139. The kit of claim 133, comprising one or more reagents for an amplification reaction.

140. The kit of claim 133, comprising a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise the target binding region.

141. The kit of claim 140, wherein at least one of the second plurality of spacer oligonucleotides is shorter than at least one of the first plurality of spacer oligonucleotides.

142. The kit of claim 140, wherein at least one of the second plurality of spacer oligonucleotides is shorter than at least one of the plurality of oligonucleotide barcodes.

143. The kit of claim 140, wherein at least two of the second plurality of spacer oligonucleotides have different lengths.

144. The kit of claim 140, wherein the sequence of at least two of the second plurality of spacer oligonucleotides are identical.

145. The kit of claim 140, wherein at least two of the second plurality of spacer oligonucleotides comprise the same sequence.

146. The kit of claim 133, wherein at least 100 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences.

147. The kit of claim 133, wherein the target binding region comprises a gene-specific sequence or an oligo (dT) sequence.

148. The kit of claim 133, wherein the two target binding regions of two oligonucleotide barcodes in the plurality of oligonucleotide barcodes comprise different gene-specific sequences.

149. The kit of claim 133, wherein each of the plurality of oligonucleotide barcodes comprises a spatial marker sequence, and wherein the spatial marker sequences of the plurality of oligonucleotide barcodes differ from each other by at least one nucleotide.

150. The kit of claim 133, wherein each of the plurality of oligonucleotide barcodes comprises a universal tag sequence, wherein the universal tag sequences of the plurality of oligonucleotide barcodes are identical.

151. The kit of claim 133, wherein at least one of the plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

152. The kit of claim 133, wherein at least a portion of the plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

153. The kit of claim 133, wherein at least one of the plurality of oligonucleotide barcodes is encapsulated in the synthetic particle.

154. The kit of claim 133, wherein at least a portion of the plurality of oligonucleotide barcodes is encapsulated within the synthetic particle.

155. The kit of claim 133, wherein the synthetic particles are destructible.

156. The kit of claim 133, wherein the synthetic particles comprise beads.

157. The kit of claim 156, wherein the beads comprise agarose beads, magnetic beads, conjugated beads, silica beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof.

158. A kit according to claim 156, wherein the beads include agarose gel beads, streptavidin protein beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo (dT) conjugated beads, silica-like beads, or any combination thereof.

159. The kit of claim 133, wherein the synthetic particles comprise a material selected from the group consisting of: Glass, hydrogel, paramagnetic substances, ceramics, plastics, titanium, latex, cellulose, nylon, silicone, and any combination thereof.

160. The kit of claim 133, wherein the synthetic particles comprise a material selected from the group consisting of: Polydimethylsiloxane (PDMS), polystyrene, polypropylene, agarose, gelatin, and any combination thereof.

161. The kit of claim 133, wherein the synthetic particles comprise a material selected from the group consisting of methyl styrene, acrylic polymers, agarose gel, and any combination thereof.

162. The kit of claim 133, wherein the synthetic particles comprise destructible hydrogel particles.

163. The kit according to claim 133 wherein each of the plurality of oligonucleotide barcodes comprises a linker functional group, wherein the synthetic particles comprise solid support functional groups, and wherein the support functional group and the linker functional group are associated with each other.

164. The kit of claim 163, wherein the linker functional group and the support functional group are individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof.

165. A method for determining the amount of a target nucleic acid in a sample, the method comprising: barcoding a plurality of copies of a target nucleic acid in a sample using a plurality of oligonucleotide barcodes associated with the synthetic particle of any one of claims 100-107 to produce a plurality of barcoded copies of the target nucleic acid, and Using the molecular marker sequences of the plurality of oligonucleotide barcodes, the quantity of each of the plurality of target nucleic acids in the sample is assessed, The method is not intended for disease diagnosis.

166. The method of claim 165, wherein barcoding the copies of the plurality of target nucleic acids in the sample using the plurality of oligonucleotide barcodes associated with the synthetic particles comprises: The plurality of target nucleic acids are hybridized to the plurality of oligonucleotide barcodes on the synthetic particles.

167. The method of claim 165, wherein barcoding the plurality of copies of the target nucleic acid comprises: The plurality of target nucleic acids are reverse transcribed to generate barcoded cDNAs of the plurality of target nucleic acids.

168. The method of claim 167, comprising: The barcoded cDNA is amplified using second strand synthesis to generate a barcoded target nucleic acid, wherein the barcoded target nucleic acid is double-stranded.

169. The method of claim 168, comprising: The barcoded target nucleic acid is amplified to produce labeled target amplicons.

170. A method for capturing a target nucleic acid in a sample, the method comprising: hybridizing a plurality of copies of a target nucleic acid in a sample using a plurality of oligonucleotide barcodes associated with the synthetic particles of any one of claims 100-107 to produce a plurality of barcoded copies of the target nucleic acid, The method is not intended for disease diagnosis.

171. The method of claim 170, wherein the plurality of copies of the barcoded target nucleic acids comprises copies of at least 50% of the target nucleic acids in the plurality of target nucleic acids in the sample.

172. The method of claim 170, wherein the plurality of copies of the barcoded target nucleic acids comprises copies of at least 75% of the target nucleic acids in the plurality of target nucleic acids in the sample.

173. The method of claim 170, wherein the plurality of barcoded target nucleic acid copies comprises at least 95% of the copies of the target nucleic acids in the plurality of target nucleic acids in the sample.

174. The method of claim 170, wherein the plurality of copies of barcoded target nucleic acids comprises at least 50% of the copies of each target nucleic acid in the plurality of target nucleic acids in the sample.

175. The method of claim 170, wherein the plurality of copies of barcoded target nucleic acids comprises at least 75% of the copies of each target nucleic acid in the plurality of target nucleic acids in the sample.

176. The method of claim 170, wherein the plurality of copies of barcoded target nucleic acids comprises at least 95% of the copies of each target nucleic acid in the plurality of target nucleic acids in the sample.

177. The method of claim 170, comprising: The plurality of target nucleic acids are reverse transcribed to generate barcoded cDNAs of the plurality of target nucleic acids.

178. The method of claim 177, comprising: The barcoded cDNA is amplified using second strand synthesis to generate a barcoded target nucleic acid, wherein the barcoded target nucleic acid is double-stranded.

179. The method of claim 178, comprising: The barcoded target nucleic acid is amplified to produce labeled target amplicons.

180. The method of claim 165 or 170, wherein the sample comprises a single cell, the method comprising associating the synthetic particle with the single cell in the sample.

181. The method of claim 180, wherein the single cell comprises a rare cell, a tumor cell, a cell infected with a viral polynucleotide, and any combination thereof.

182. The method of claim 180, wherein the single cell comprises a cell from a human.

183. The method of claim 180, wherein the single cell comprises a cell from a tissue.

184. The method of claim 180, wherein the single cell comprises a cell from a tumor.

185. The method of claim 180, comprising: After associating the synthetic particles with the single cells, the single cells are lysed.

186. The method of claim 185, wherein lysing the single cells comprises heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

187. The method of claim 180, wherein the synthetic particles and the single cells are in the same well.

188. The method of claim 180, wherein the synthetic particles and the single cells are in the same droplet.

189. The method of claim 165 or 170, wherein the plurality of target nucleic acids comprises ribonucleic acid (RNA), RNA degradation products, and any combination thereof.

190. The method of claim 165 or 170, wherein the plurality of target nucleic acids comprises messenger RNA (mRNA), micro RNA, small interfering RNA (siRNA), and any combination thereof.

191. The method of claim 165 or 170, wherein the plurality of target nucleic acids comprises RNAs each containing a poly(A) tail.

192. The method of claim 165 or 170, wherein the synthetic particle comprises a second plurality of spacer oligonucleotides, wherein each of the second plurality of spacer oligonucleotides does not comprise the target binding region.

193. The method of claim 192, wherein at least one of the second plurality of spacer oligonucleotides is shorter than at least one of the first plurality of spacer oligonucleotides.

194. The method of claim 192, wherein at least one of the second plurality of spacer oligonucleotides is shorter than at least one of the plurality of oligonucleotide barcodes.

195. The method of claim 192, wherein at least two of the second plurality of spacer oligonucleotides have different lengths.

196. The method of claim 192, wherein the sequences of at least two of the second plurality of spacer oligonucleotides are identical.

197. The method of claim 192, wherein at least two of the second plurality of spacer oligonucleotides comprise the same sequence.

198. The method of claim 165 or 170, wherein at least 100 of the plurality of oligonucleotide barcodes comprise different molecular marker sequences.

199. The method of claim 165 or 170, wherein the target binding region comprises a gene-specific sequence or an oligo (dT) sequence.

200. The method of claim 165 or 170, wherein the two target binding regions of two oligonucleotide barcodes in the plurality of oligonucleotide barcodes comprise different gene-specific sequences.

201. The method of claim 165 or 170, wherein each of the plurality of oligonucleotide barcodes comprises a spatial marker sequence, and wherein the spatial marker sequences of the plurality of oligonucleotide barcodes differ from each other by at least one nucleotide.

202. The method of claim 165 or 170, wherein each of the plurality of oligonucleotide barcodes comprises a universal tag sequence, wherein the universal tag sequences of the plurality of oligonucleotide barcodes are the same.

203. The method of claim 165 or 170, wherein at least one of the plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

204. The method of claim 165 or 170, wherein at least a portion of the plurality of oligonucleotide barcodes is immobilized on the synthetic particle.

205. The method of claim 165 or 170, wherein at least one of the plurality of oligonucleotide barcodes is encapsulated in the synthetic particle.

206. The method of claim 165 or 170, wherein at least a portion of the plurality of oligonucleotide barcodes is encapsulated within the synthetic particle.

207. The method of claim 165 or 170, wherein the synthetic particles are destructible.

208. The method of claim 165 or 170, wherein the synthetic particles comprise beads.

209. The method of claim 208, wherein the beads comprise agarose beads, magnetic beads, conjugated beads, silica beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof.

210. The method of claim 208, wherein the beads comprise agarose gel beads, streptavidin protein beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica-like beads, or any combination thereof.

211. The method of claim 165 or 170, wherein the synthetic particles comprise a material selected from the group consisting of: Glass, hydrogel, paramagnetic substances, ceramics, plastics, titanium, latex, cellulose, nylon, silicone, and any combination thereof.

212. The synthetic particle of any one of claims 165 or 170, wherein the synthetic particle comprises a material selected from the group consisting of: Polydimethylsiloxane (PDMS), polystyrene, polypropylene, agarose, gelatin, and any combination thereof.

213. The synthetic particle of any one of claims 165 or 170, wherein the synthetic particle comprises a material selected from the group consisting of: Methylstyrene, acrylic polymer, agarose gel, and any combination thereof.

214. The method of claim 165 or 170, wherein the synthetic particles comprise destructible hydrogel particles.

215. The method according to claim 165 or 170, wherein each of the plurality of oligonucleotide barcodes comprises a linker functional group, wherein the synthetic particles comprise solid support functional groups, and wherein the support functional group and the linker functional group are associated with each other.

216. The method of claim 215, wherein the linker functional group and the support functional group are individually selected from the group consisting of: C6, biotin, streptavidin, one or more primary amines, one or more aldehydes, one or more ketones, and any combination thereof.

217. The synthetic particle of any one of claims 1-8, wherein a ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is in a range from 1:3 to 2:

1.

218. The synthetic particle of any one of claims 1-8, wherein the ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is at most 2:

1.

219. The kit of claim 27, wherein the ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is in the range of from 1:3 to 2:

1.

220. The kit of claim 27, wherein the ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is at most 2:

1.

221. The method of claim 54, wherein the ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is in the range of from 1:3 to 2:

1.

222. The method of claim 54, wherein the ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is at most 2:

1.

223. The method of claim 61, wherein the ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is in the range of from 1:3 to 2:

1.

224. The method of claim 61, wherein the ratio of the first plurality of oligonucleotide barcodes to the second plurality of oligonucleotide barcodes is at most 2:

1.

225. The synthetic particle of any one of claims 100-107, wherein the ratio of the plurality of oligonucleotide barcodes and the first plurality of spacer oligonucleotides is in the range from 1:3 to 2:

1.

226. The synthetic particle of any one of claims 100-107, wherein the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides is at least 1:

3.

227. The kit of claim 133, wherein the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides is in the range of from 1:3 to 2:

1.

228. The kit of claim 133, wherein the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides is at least 1:

3.

229. The method of claim 165, wherein the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides is in the range of from 1:3 to 2:

1.

230. The method of claim 165, wherein the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides is at least 1:

3.

231. The method of claim 170, wherein the ratio of the plurality of oligonucleotide barcodes to the first plurality of spacer oligonucleotides is in the range of from 1:3 to 2:

1.

232. The method of claim 170, wherein the plurality of oligonucleotide barcodes and the first plurality of The ratio of spacer oligonucleotides is at least 1:3.

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