WO2017044574A1 - Methods and compositions for nucleic acid library normalization - Google Patents

Methods and compositions for nucleic acid library normalization Download PDF

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Publication number
WO2017044574A1
WO2017044574A1 PCT/US2016/050694 US2016050694W WO2017044574A1 WO 2017044574 A1 WO2017044574 A1 WO 2017044574A1 US 2016050694 W US2016050694 W US 2016050694W WO 2017044574 A1 WO2017044574 A1 WO 2017044574A1
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nucleic acid
targets
library
acid targets
normalized
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French (fr)
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Craig Betts
Glenn Fu
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Cellular Research Inc
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Cellular Research Inc
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Priority to ES16770134T priority Critical patent/ES2745694T3/es
Priority to EP16770134.1A priority patent/EP3347465B1/en
Priority to JP2018512152A priority patent/JP6940484B2/ja
Priority to KR1020187008560A priority patent/KR102395450B1/ko
Priority to CN201680052330.2A priority patent/CN108026524A/zh
Publication of WO2017044574A1 publication Critical patent/WO2017044574A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/191—Modifications characterised by incorporating an adaptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/159—Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions

Definitions

  • the term "complementary" can refer to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of anoiher nucleic acid, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be "partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules.
  • label can refer to nucleic acid codes associated with a target within a sample.
  • a label can be, for example, a nucleic acid label.
  • a label can be an entirely or partially amplifiable label .
  • a label can be entirely or partially sequencable label.
  • a label can be a portion of a native nucleic acid that is identifiable as distinct.
  • a label can be a known sequence,
  • a label can comprise a junction of nucleic acid sequences, for example a junction of a native and non-native sequence.
  • label can be used interchangeably with the terms, "index", "tag,” or "label-tag.”
  • Labels can convey information. For example, in various embodiments, labels can be used to determine an identity of a sample, a source of a sample, an identity of a cell, and/or a target.
  • a nucleic acid can comprise polynucleotide backbones that are formed by- short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycioalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • sampling device can refer to a device which may take a section of a sample and/or place the section on a substrate.
  • a sample device can refer to, for example, a fluorescence activated cell sorting (FACS) machine, a cell sorter machine, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and/or a microtome.
  • FACS fluorescence activated cell sorting
  • stochastic barcode can refer to a polynucleotide sequence comprising labels of the disclosure.
  • a stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcoding.
  • Stochastic barcodes can be used to quantify targets within a sample.
  • Stochastic barcodes can be used to control for errors which may occur after a label is associated with a target.
  • a stochastic barcode can be used to assess amplification or sequencing errors.
  • a stochastic barcode associated with a target can be called a stochastic barcode-target or stochastic barcode-tag-target.
  • such enzymes include, but are not limited to, retroviral reverse transcriptase, retrotransposon reverse transcriptase, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intr on-derived reverse transcriptase, and mutants, variants or derivatives thereof.
  • Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transciptases, and group II intron reverse transcriptases.
  • Some embodiments disclosed herein provide methods of removing high abundance species from a plurality of nucleic acid molecules.
  • the methods disclosed herein can reduce the content of high abundance species from a plurality of nucleic acid molecules without significantly removing the low abundance species or the intermediate abundance species from the plurality of nucleic acid molecules.
  • "significantly removing” refers to removing at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more of a low abundance species or intermediate abundance species from the plurality of nucleic acid molecules.
  • the methods disclosed herein can remove high abundance species and the intermediate abundance species from a plurality of nucleic acid molecules without significantly removing the low abundance species from the plurality of nucleic acid molecules.
  • members of a species can hybridize to one another under moderate stringent hybridization conditions. In some embodiments, members of a species can hybridize to one another under low stringent hybridization conditions. In some embodiments, members of a species are transcripts from the same genetic locus and the transcripts can be of the same or different length. The species is, in some embodiments, cDNA or mRNA.
  • a "high abundance species” refers to a species that is present in high amount in the plurality of nucleic acids, for example the species can represent at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or more of the plurality of nucleic acid molecules.
  • the plurality of nucleic acid molecules can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1 ,000, or more, high abundance species, in some embodiments, the total of all the high abundance species represent at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more of the plurality of nucleic acid molecules.
  • high abundance species can comprise polynucleotides encoding one or more ribosomal proteins.
  • high abundance species can comprise polynucleotides encoding one or more mitochondrial proteins.
  • high abundance species can comprise polynucleotides encoding one or more housekeeping proteins.
  • an "intermediate abundance species” refers to a species that is present in an amount in the plurality of nucleic acid that is lower than at least one species in the plurality of nucleic acid and is higher than at least one other species in the plurality of nucleic acid.
  • an intermediate abundance species can represent about 10%, 5%, 4%, 3%, 2%, 1%, 0. 1 %, 0.01%, or a range between any two of the above values, of the plurality of nucleic acid molecules.
  • the plurality of nucleic acid molecules can comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, or more, intermediate abundance species.
  • the total of all the intermediate abundance species represent about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, or a range between any two of the above values, of the plurality of nucleic acid molecules.
  • the total of all the low abundance species represent less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, or less of the plurality of nucleic acid molecules.
  • low abundance species can comprise polynucleotides encoding one or more transcription factors.
  • high abundance species can comprise polynucleotides encoding one or more T cell receptors.
  • high abundance species can comprise polynucleotides encoding one or more antibodies.
  • the methods and compositions disclosed herein can reduce the content of one or more high abundance species from the plurality of nucleic acid molecules.
  • the methods and compositions disclosed herein can reduce the content of at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1 ,000, or more, high abundance species.
  • the methods and compositions disclosed herein can reduce the content by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of each of the one or more high abundance species from the plurality of nucleic acid molecules.
  • the methods and compositions disclosed herein can reduce the content of one or more high abundance species from the plurality of nucleic acid molecules without significantly removing the low abundance species or the intermediate abundance species from the plurality of nucleic acid molecules. In some embodiments, the methods and compositions disclosed herein can reduce the content by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of each of the one or more high abundance species from the plurality of nucleic acid molecules without significantly removing the low abundance species or the intermediate abundance species from the plurality of nucleic acid molecules.
  • the methods and compositions disclosed herein can reduce the content by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the total of high abundance species from the plurality of nucleic acid molecules without significantly removing the low abundance species or the intermediate abundance species from the plurality of nucleic acid molecules.
  • the methods and compositions disclosed herein can reduce the content of one or more high abundance species from the plurality of nucleic acid molecules while keeping at least at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of each of the one or more low abundance species.
  • the methods and compositions disclosed herein can reduce the content of one or more high abundance species from the plurality of nucleic acid molecules while keeping at least at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of at least one of the one or more of low abundance species. In some embodiments, the methods and compositions disclosed herein can reduce the content of one or more high abundance species from the plurality of nucleic acid molecules while keeping at least at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the total of low abundance species. In some embodiments, the methods and compositions disclosed herein can reduce the content of one or more high abundance species from the plurality of nucleic acid
  • the methods and compositions disclosed herein can reduce the content of one or more high abundance species from the plurality of nucleic acid molecules while keeping at least at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the total of intermediate abundance species.
  • the methods and compositions disclosed herein can reduce the content of one or more high abundance species from the plurality of nucleic acid molecules while keeping at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of each of the intermediate abundance species from the plurality of nucleic acid molecules.
  • the plurality of nucleic acid molecules disclosed herein can comprise a variety of nucleic acid molecules.
  • the plurality of nucleic acid molecules can comprise, DNA molecules, RNA molecules, genomic DNA molecules, cDNA molecules, m NA molecules, rR ' NA molecules, siRNA molecules, or a combination thereof, and can be double-stranded or single-stranded.
  • the plurality of nucleic acid molecules comprise at least 100, at least 1,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000, at least 1,000,000, or more species.
  • the plurality of nucleic acid molecules can be from a sample, such as a single ceil, or a plurality of cells. In some embodiments, the plurality of nucleic acid molecules can be pooled from a plurality of samples, such as a plurality of single cells.
  • the plurality of nucleic acid molecules comprises an unnormalized nucleic acid library, a partially normalized nucleic acid library, or a nucleic acid libran,' that has been normalized by other methods, such as a cDNA library, a genomic DNA library, or the like.
  • the plurality of nucleic acid molecules can comprise a pooled unnormalized nucleic acid library, such as a pooled unnormalized nucleic acid library constructed from a plurality of unnormalized nucleic acid libraries each representing a single cell.
  • the unnormalized nucleic acid library is a cDNA library.
  • the unnormalized nucleic acid library is a genomic library.
  • the plurality of nucleic acid molecules can be subjected to amplification before removing the high abundance species.
  • the plurality of nucleic acid molecules can comprise an amplified nucleic acid library.
  • the plurality of nucleic acid molecules can comprise at least 2, at least 4, at least 8, at least 16, at least 100, at least 1,000 or more copies of each nucleic acid molecules.
  • the binding moiety can be biotin, streptavidin, heparin, an aptamer, a click-chemistry moiety, digoxigenin, primary amine(s), carboxyl(s), hydroxyl(s), aldehyde(s), ketone(s), or any combination thereof.
  • the binding moieties as disclosed herein are capable of bind to capture moieties such as capture molecules.
  • the binding moiety and capture molecule can be members of a binding pair, for example, biotin/streptavidin.
  • the capture molecule can be immobilized on a solid support, such as a bead, a microparticle, or a nanoparticle.
  • the first oligonucleotides can be extended to generate a plurality of complementary strands of the plurality of nucleic acid targets comprising the binding moiety.
  • a second strand can be synthesized using a primer that binds to a binding site on the complementary strands to produce double stranded nucleic acid molecules.
  • Denaturation can be performed by a variety of methods including heating the double stranded nucleic acid molecules, treating the double stranded nucleic acid molecules with organic solvents (e.g., DMS or formamide), changing the salt concentration of the double stranded nucleic acid molecules, and/or changing the pH of the double stranded nucleic acid molecules.
  • organic solvents e.g., DMS or formamide
  • the single-stranded nucleic acid molecules can be partially reannealed. Partial reannealing can be performed by any method, for example, rapid cooling on ice, changing the salt concentration (e.g., reversing the salt concentration from the amount used in denaturation), and/or changing the pH (e.g., reversing the pH from the level used in denaturation), and the like.
  • Figure 1 depicts an exemplar)' embodiment of the methods of the disclosure.
  • a sample can comprise a plurality of nucleic acids. Some of the nucleic acids can be highly abundant 106 and some of the nucleic acids can be less abundant 105.
  • the nucleic acids can be transformed into a double-stranded cDNA library 125 that is asymmetrically labeled with a binding moiety 120.
  • the nucleic acids 105/106 can be mRNA that is reverse transcribed.
  • Second strand synthesis can be performed using a primer (e.g., gene-specific primer, or a random muitimer primer) comprising the binding moiety 120, thereby generating an asymmetrically labeled double-stranded nucleic acid library 125.
  • a primer e.g., gene-specific primer, or a random muitimer primer
  • the nucleic acids 105/106 can be DNA.
  • the DNA can be extended using a primer comprising the binding moiety 120, thereby generating an asymmetrically labeled double-stranded cDNA library 125,
  • the double-stranded cDNA libran,' can comprise highly abundant double-stranded cDNA species 116 and lowly abundant double-stranded cDNA species 115.
  • the double-stranded cDNA library 125 can be ligated to adaptors 130/135, thereby generating an un-normalized library 125.
  • the double-stranded cDNA library 125 can be heat denatured, thereby- separating the strands of the double-stranded cDNA.
  • the heat denatured library can be re- annealed (e.g., partially reanneal ed) 140.
  • the more abundant nucleic acids 106 can anneal faster than less abundant nucleic acids 105.
  • Denaturing and partial reannealing can result in a mixture of species 141 comprising re-annealed double-stranded cDNAs comprising said binding moiety 142, single-stranded molecules comprising said binding moiety 143 and single-stranded molecules lacking said binding moiety 144.
  • the nucleic acid can be an RNA and the primer can be a reverse transcription primer.
  • the reverse transcription primer can reverse transcribe the RNA, thereby generating an RNA-cDNA hybrid (e.g., first strand synthesis).
  • a second strand can be generated using standard second strand synthesis techniques.
  • the first cDNA strand can comprise the binding moiety.
  • the second strand can be the complement of the first strand.
  • the second strand may not comprise the binding moiety.
  • This cDNA can be referred to as an asymmetrically labeled cDNA (e.g., one strand of the cDNA is labeled with the binding moiety).
  • a primer comprising a binding moiety can be contacted to the nucleic acids to generate a first strand (e.g., complementary strand to the DNA template).
  • the first strand can comprise the binding moiety.
  • the primer can generate a second strand that is complementar to the first strand.
  • the second strand may not comprise the binding moiety.
  • the result can be an asymmetrically labelled double-stranded cDNA molecule where one of the strands comprises a binding moiety, and the other strand is the complement.
  • a group of asymmetrically labeled double-stranded cDNA molecules can be referred to as an un-normalized library.
  • the un-normaiized library (e.g., comprising nucleic acid target cDNAs) can be denatured. Denaturation can be performed by a variety of methods including heating the sample, treating the sample with organic solvents (e.g., DMS or formamide), changing the salt concentration of the sample, and/or changing the pH of the sample.
  • organic solvents e.g., DMS or formamide
  • Partial re-annealing can comprise re-annealing of at most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the strands of the denatured sample. At least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of strands from highly abundant nucleic acids can be re-annealed during the step of partial re-annealing. At most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of strands from highly abundant nucleic acids can be re-annealed during the step of partial re-annealing.
  • Strands from higher abundant species can re-anneal at most 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500% or more quickly than strands from lower abundant species. Strands from higher abundant species can re-anneal at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold or more than strands from lower abundant species. Strands from higher abundant species can re-anneal at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold or more than strands from lower abundant species.
  • the solid support can bind to the molecules of the re-annealed samples that comprise the binding moiety.
  • the solid support can bind to double-stranded cDNAs (e.g., comprising the binding moiety).
  • the solid support can bind to single-stranded un- annealed strands (e.g., comprising the binding moiety).
  • the solid support can be used to purify the sample.
  • the solid support can be separated from the sample (i.e., supernatant) (e.g., by centrifugation, magnetism).
  • the leftover sample i.e., supernatant
  • the normalized library can comprise single-stranded nucleic acid molecules that may not comprise a binding moiety.
  • the single-stranded nucleic acid molecules can be molecules that did not anneal during the partial re-annealing step of the method.
  • a first target 405 and a second target 410 can each comprise universal sequences 415/420 and a binding moiety 421.
  • targets with difference sequences 405/410 can anneal together through the universal sequences that each of them comprise 415/420.
  • Blockers can be used to prevent this from happening.
  • the sample can be contacted 425 with blockers 430.
  • the blockers 430 can hybridize to one or more universal sequences of the targets.
  • One or more different types of blockers 430 can be used.
  • the blockers can aid partial re-annealing by forcing strands to associate (e.g., hybridize) through their gene sequences. In this way, blockers can be used to aid library normalization methods of the disclosure,
  • the library normalization methods of the disclosure can be performed on a solid support.
  • a library can be generated wherein the amplicons of the library are asymmetrically labeled with one of the molecules involved in click chemistry (e.g., azide, alkyne, for the azide-alkyne cycloaddition).
  • the solid support can comprise the other molecule in the click chemistry.
  • the amplicon can comprise an alkyne and the solid support can comprise an azide.
  • the amplicons can be attached to the solid support (e.g., by click chemistry).
  • the solid support can be heated thereby inducing denaturation of the attached amplicons.
  • the amplicons that are more abundant can re-anneal to the molecules attached to the solid support.
  • the amplicons that are less abundant can be left in solution (e.g., by centrifugation, magnetism, chromatography).
  • the solid supports can be removed from the solution, thereby leaving by a normalized library.
  • One or more nucleic acid amplification reactions may be performed to create multiple copies of the normalized target nucleic acid molecules. Amplification 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 adaptors to the nucleic acid molecules.
  • the amplification reactions may comprise amplifying at least a portion of a sample label, if present.
  • the amplification reactions may comprise amplifying at least a portion of the cellular and/or molecular label.
  • the amplification reactions may comprise amplifying at least a portion of a sample tag, a cellular label, a spatial label, a molecular label, a target nucleic acid, or a combination thereof.
  • Sequencing may comprise at least about 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more sequencing reads per run. Sequencing may comprise at most about 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more sequencing reads per run. In some instances, sequencing comprises sequencing at least about 1,500; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or more sequencing reads per run. In some instances, sequencing comprises sequencing at most about 1,500; 2,000; 3,000; 4,000; 5,000; 6,000, 7,000; 8,000; 9,000; or 10,000 or more sequencing reads per run.
  • Sequencing can comprise sequencing at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 2000, 3000, 4000, or 5000 or more millions of sequencing reads in total .
  • Sequencing can comprise sequencing at most 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 2000, 3000, 4000, or 5000 or more millions of sequencing reads in total.
  • Sequencing may comprise less than or equal to about 1,600,000,000 sequencing reads per run. Sequencing may comprise less than or equal to about 200,000,000 reads per run.
  • less abundant (e.g., rarer) transcripts can be identified more easily than in an un-normalized library.
  • Sequencing reads of less abundant transcripts in a normalized library can comprise a larger portion of total reads of than in an un-normalized library.
  • Sequencing reads of a less abundant transcript in a normalized library can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500% or more reads compared to reads of the same transcript in an un- normalized library.
  • Sequencing reads of a less abundant transcript in a normalized library can be at least 1, 2, 3, 4, 5, or 6 or more fold than sequencing reads for the same transcript in an un-normalized library.
  • a target-binding region can be 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 or more nucleotides in length.
  • a target-binding region can be from 5-30 nucleotides in length.
  • the stochastic barcode can be referred to as a gene-specific stochastic barcode.
  • the length of the nucleic acid subsequences used for creating error correction codes may vary, for example, they may be at least 3 nucleotides, at least 7 nucleotides, at least 15 nucleotides, or at least 31 nucleotides in length. In some embodiments, nucleic acid sub-sequences of other lengths may be used for creating error correction codes.
  • the solid support may comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof.
  • a solid support may take the form of resins, gels, microspheres, or other geometric configurations.
  • a solid support can comprise silica chips, microparticles, nanoparticles, 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, microweli plates, slides, plastic materials including multiwell plates or membranes (e.g., formed of polyethylene, polypropylene, polyamide, polyvinylidenedifluoride), and/or wafers, combs, pins or needles (e.g., arrays of pins suitable for combinatorial synthesis or analysis) or beads in an array of pits or nanoliter wells of flat surfaces such as wafers (e.g., silicon wafers), wafers with pits with or without filter
  • the cells are bacteria. These may include either gram-positive or gram-negative bacteria. Examples of bacteria that may be analyzed using the disclosed methods, devices, and systems include, but are not limited to, Actinomedurae, Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Coiynebacterium, Enterocoecus faecal is, Listeria monocytogenes, Nocardia, Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epiderm, Streptococcus mutans, Streptococcus pneumoniae and the like.
  • the cells are fungi.
  • fungi that may be analyzed using the disclosed methods, devices, and systems include, but are not limited to, Aspergilii, Candidae, Candida albicans, Coccidioides immitis, Cryptococci, and combinations thereof.
  • the cells are protozoans or other parasites.
  • parasites to be analyzed using the methods, devices, and systems of the present disclosure include, but are not limited to, Balantidium coli, Cryptosporidium parvum, Cyclospora cayatanensis, Encephaiitozoa, Entamoeba histolytica, Enterocytozoon bieneusi, Giardia lamblia, Leishmaniae, Plasmodii, Toxoplasma gondii, Trypanosomae, trapezoidal amoeba, worms (e.g., helminthes), particularly parasitic worms including, but not limited to, Nematoda (roundworms, e.g., whipworms, hookworms, pinworms, ascarids, filarids and the like), Cestoda (e.g., tapeworms).
  • Nematoda roundworms, e.g., whipworms, hookworms, pinworm
  • a first ceil sample is obtained from a person not having a disease or condition
  • a second cell sample is obtained from a person having the disease or condition.
  • the persons are different.
  • the persons are the same but cell samples are taken at different time points.
  • the persons are patients, and the cell samples are patient samples.
  • the disease or condition can be a cancer, a bacterial infection, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a parasitic disease, a genetic disorder, or any combination thereof.
  • the sample comprises an immune cell.
  • An immune ceil can include, for example, T ceil, B cell, lymphoid stem cell, myeloid progenitor cell, lymphocyte, granulocyte, B-cell progenitor, T cell progenitor, Natural Killer cell, Tc cell, Th cell, plasma cell, memory ceil, neutrophil, eosinophil, basophil, mast ceil, monocyte, dendritic cell and/or macrophage, or any combination thereof.
  • T cells can be isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
  • a specific subpopulation of T cells such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
  • T cells can be isolated by incubation with anti-CD3/anti-CD28 (i.e., 3 x28)-conjugated beads, such as D YN ABE AD S ® M- 50 CD3/CD28 T, or XCYTE DYNABEADSTM for a time period sufficient for positive selection of the desired T cells.
  • Immune cells e.g., T cells and B ceils
  • can be antigen specific e.g., specific for a tumor.
  • cDNA synthesis can incorporate the label information from the labels in the stochastic barcode into the new cDNA target molecule being synthesized, thereby generating a target- barcode molecule.
  • the target-barcode molecules can be amplified using PGR,
  • the sequence of the targets and the labels of the stochastic barcode on the target-barcode molecule can be determined by sequencing methods.
  • the cells can be lysed to liberate the target molecules.
  • Cell lysis may be accomplished by any of a variety of means, for example, by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis, or optical lysis.
  • Cells may be lysed by addition of a cell lysis buffer comprising a detergent (e.g. SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP- 40), an organic solvent (e.g. methanol or acetone), or digestive enzymes (e.g. proteinase K, pepsin, or trypsin), or any combination thereof.
  • a detergent e.g. SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP- 40
  • an organic solvent e.g. methanol or acetone
  • digestive enzymes e.g. proteinase K, peps
  • the nucleic acid molecules may randomly associate with the stochastic barcodes of the co-localized solid support. Association may comprise hybridization of a stochastic barcode's target recognition region to a complementar' portion of the target nucleic acid molecule (e.g., oligo dT of the stochastic barcode can interact with a poly- A tail of a target).
  • the assay conditions used for hybridization e.g. buffer pH, ionic strength, temperature, etc.
  • Attachment may further comprise ligation of a stochastic barcode's target recognition region and a portion of the target nucleic acid molecule.
  • the stochastically barcoded cDNA molecule can be subjected to downstream methods such as amplification (e.g., by universal and/or gene-specific primers) and the library normalization methods of the disclosure.
  • downstream methods such as amplification (e.g., by universal and/or gene-specific primers) and the library normalization methods of the disclosure.
  • kits for performing library normalization methods of the disclosure can comprise a second strand synthesis primer comprising a binding moiety.
  • a kit can comprise a solid support comprising capture moieties that can bind to the binding moiety on the second strand synthesis primer.
  • a kit can comprise a magnet to capture the solid support.
  • a kit can comprise reagents for cleaning up an amplification reaction (e.g., AmpureXP beads and/or a purification spin column).
  • a kit can comprise adaptors and/or primers comprising sequencing flow cell sequences. The kit may further comprise reagents (e.g.
  • Kits of the disclosure can generally include instructions for carrying out one or more of the methods described herein. Instmctions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the disclosure. Such media can include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), RF tags, and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.
  • reagents e.g. enzymes, universal primers, sequencing primers, target- specific primers, or buffers
  • a target comprises a poiy-A tail.
  • the target is an mRNA.
  • the target is hybridized to a stochastic barcode.
  • the stochastic barcode comprises a number of labels.
  • the stochastic barcode comprises a target-specific region (e.g., oligo dT for binding to poiy-A tails of mRNAs), a molecular label, a cellular label, and a first universal label.
  • the stochastic barcode is reverse transcribed using a reverse transcriptase, thereby generating a labelled-cDNA molecule.
  • Excess stochastic barcodes are treated with a degradation enzyme.
  • the degradation enzyme is an exonuclease.
  • the labelled-cDNA molecule undergoes second strand synthesis thereby- generating a double- stranded labeled cDNA molecule.
  • Second strand synthesis is performed by contacting the labelled cDNA molecule-mRNA hybrid with a nicking enzyme (e.g., RNaseH) that can nick the mRNA hybridized to the labelled cDNA molecule, thereby- generating nicked mRNA.
  • a nicking enzyme e.g., RNaseH
  • the nicked mRNA is used as a primer and extended using a polymerase (e.g., DNA Pol I), thereby incorporating the sequence of the first strand.
  • the polymerase comprises 5 '-3' exonuclease activity.
  • the polymerase degrades the downstream mRNA nicks that serve as the primers for the second strand synthesis.
  • a ligase is used to ligate the extended sequences together, thereby generating a second strand (e.g., double- stranded labeled cD ' NA molecule).
  • the first set of amplicons can be amplified in a second amplification reaction using a second gene-specific nested PCR primer and the universal primer comprising the azide or alkyne moiety.
  • This reaction generates an asymmetrically labeled amplicon comprising an azide or alkyne moiety at one end.
  • Re-annealed amplicons will be attached to the solid support through the click chemistry.
  • the solid support is removed (e.g., by centrifugation or magnetism).
  • the remaining strands (e.g., that have not re-annealed) will not comprise an azide or alkyne moiety.
  • the remaining strands represent sequences that are lower in abundance and are the complement of the strands with the azide or alkyne. These strands represent a normalized library.
  • the library is regenerated with PCR primers.
  • the PCR primers can comprise sequencing flow ceil primer sequences.
  • the normalized library is sequenced.

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