EP3180445A1 - Multiplex-transkriptomanalyse - Google Patents

Multiplex-transkriptomanalyse

Info

Publication number
EP3180445A1
EP3180445A1 EP15753859.6A EP15753859A EP3180445A1 EP 3180445 A1 EP3180445 A1 EP 3180445A1 EP 15753859 A EP15753859 A EP 15753859A EP 3180445 A1 EP3180445 A1 EP 3180445A1
Authority
EP
European Patent Office
Prior art keywords
target
sequence
amplicons
rna
derived
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15753859.6A
Other languages
English (en)
French (fr)
Inventor
Kelli BRAMLETT
John Leamon
Mark Andersen
Michael Thornton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Life Technologies Corp
Original Assignee
Life Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Life Technologies Corp filed Critical Life Technologies Corp
Publication of EP3180445A1 publication Critical patent/EP3180445A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits comprising a multiplex nucleic acid amplification reaction that employs a plurality (e.g., hundreds, thousands, tens-of-thousands or hundreds-of-thousands) of different target- specific primer pairs that enable substantially simultaneous amplification of a plurality of different target sequences-of-interest in a single reaction mixture.
  • the multiplex nucleic acid amplification reaction generates a plurality of amplicons having sequences derived from a sample containing RNA or DNA, including whole transcriptome or genomic samples.
  • the sequences and abundances of at least some of the plurality of amplicons are characterized, optionally simultaneously or through a single assay, by suitable detection methods, including sequencing or other procedures known in the art.
  • Figure 1 shows a tally of the number of different amplicon sequences generated by multiplex amplification of transcripts contained in a Universal Human Reference or Human Brain Reference sample.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits, comprising a plurality of target polynucleotides and a plurality of target-specific primers.
  • the target-specific primers are complementary or identical to at least a portion of one or more target polynucleotides of the plurality of target polynucleotides.
  • At least one of the plurality of target-specific primers is a tailed primer having a portion that hybridizes to a target polynucleotide and a portion that does not hybridize to the target polynucleotide.
  • at least one of the plurality of target-specific primers is not a tailed primer.
  • At least one of the primers in the plurality of target-specific primers contains at least one cleavable group.
  • each of the plurality of target-specific primers contains at least one cleavable group.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits further comprising a cleaving agent capable of cleaving the at least one cleavable group of the plurality of target specific primers.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits further comprising at least one polymerase.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits further comprising a plurality of nucleotides.
  • the disclosure relates generally to compositions (and related methods of making and/or using, systems, apparatuses and kits) comprising (i) a plurality of target-specific primers each containing at least one cleavable group, (ii) a polymerase, (iii) a cleaving agent capable of cleaving the at least one cleavable group of the plurality of target-specific primers, and (iv) a plurality of target polynucleotides, wherein the target-specific primers are complementary or identical to at least a portion of one or more of the target polynucleotides of the plurality.
  • the compositions, systems, apparatuses and kits further include a plurality of nucleotides.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits, comprising a single reaction mixture which contains (i) a plurality of target- specific primers, (ii) a polymerase, and (iii) a plurality of target polynucleotides, wherein the target- specific primers are complementary or identical to at least a portion of one or more of the target polynucleotides of the plurality.
  • each target-specific primer in the single reaction mixture contains at least one cleavable group.
  • the single reaction mixture further comprises a cleaving agent capable of cleaving the at least one cleavable group of the plurality of target-specific primers.
  • the single reaction mixture further comprises a plurality of nucleotides.
  • the single reaction mixture contains at least 1000, 2500, 5000, 7500, 10,000, 12,000, 15,000, 17,500, 20,000, 25,000, 50,000, 100,000, 200,000 or 500,000 different target-specific primers.
  • the plurality of target polynucleotides comprises RNA, DNA or cDNA.
  • the plurality of target polynucleotides includes genomic DNA.
  • the plurality of target polynucleotides includes total RNA, polyA RNA or non- polyA RNA.
  • the plurality of target polynucleotides comprises single-stranded or double- stranded nucleic acids.
  • each of the target-specific primers can hybridize to at least a portion of one or more target polynucleotides of the plurality of target polynucleotides.
  • the plurality of target-specific primers includes at least 1000, 2500, 5000, 7500, 10,000, 12,000, 15,000, 17,500, 20,000, 25,000, 50,000, 100,000, 200,000 or 500,000 different target-specific primers.
  • the plurality of nucleotides includes a detectable label.
  • compositions comprising a single reaction mixture which contains: (i) a plurality of target-specific primer pairs each containing at least one cleavable group, (ii) a plurality of target cDNA polynucleotides, wherein the target-specific primer pairs are complementary or identical to at least a portion of one or more of the target cDNA polynucleotides of the plurality, (iii) a polymerase, and (iv) a plurality of nucleotides.
  • the single reaction mixture further comprises: (v) a cleaving agent capable of cleaving the at least one cleavable group of the plurality of target-specific primer pairs.
  • the plurality of target-specific primer pairs can hybridize to about 100-100,000 different target cDNA sequences.
  • each pair of target-specific primer pairs is configured to hybridize to one target polynucleotide.
  • compositions comprising any two or more, in any combination of: a plurality of target-specific primers each containing at least one cleavable group; a polymerase; a cleaving agent capable of cleaving the at least one cleavable group of the plurality of target-specific primers; a plurality of target polynucleotides wherein the target-specific primers are complementary or identical to at least a portion of one or more of the target polynucleotides of the plurality; and/or a plurality of nucleotides.
  • the plurality of target-specific primers includes at least 1000, 2500, 5000, 7500, 10,000, 12,000, 15,000, 17,500, 20,000, 25,000, 50,000, 100,000, 200,000 or 500,000 different target-specific primers.
  • at least a portion of each of the target-specific primers can hybridize to at least a portion of the one or more target
  • the plurality of target polynucleotides comprises single-stranded or double-stranded nucleic acids.
  • the plurality of target polynucleotides includes RNA, DNA, cDNA, a mixture of RNA and DNA, or genomic DNA.
  • the plurality of target polynucleotides includes naturally-occurring, recombinant or synthetically prepared forms.
  • the plurality of target polynucleotides includes amplification products (e.g., amplicons) or fragmentation products (e.g., fragments).
  • the plurality of target polynucleotides is derived from RNA, DNA, cDNA, a mixture of RNA and DNA, or genomic DNA.
  • the plurality of target polynucleotides includes total RNA, polyA RNA or non-polyA RNA.
  • the plurality of nucleotides includes a detectable label, or the plurality of nucleotides are unlabeled, or a mixture of labeled and unlabeled nucleotides.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits further comprising a ligase.
  • the ligase comprises a DNA or RNA ligase.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits further comprising one or more adaptors.
  • the one or more adaptors are not complementary or identical to the 5' end of the plurality of target-specific primers.
  • the one or more adapters do not include a nucleic acid sequence that is complementary or identical to the terminal 10 nucleotides at the 5' end of the plurality of target-specific primers.
  • the one or more adapters comprise a universal priming sequence, a tag, or a unique identifier sequence (e.g., barcode sequence).
  • the universal priming sequence comprises an amplification priming sequence or a sequencing priming sequence.
  • At least one of the one or more adaptors is phosphorylated at the 5' end.
  • a plurality of the one or more adaptors is single-stranded or double-stranded.
  • the plurality of target-specific primers includes at least 1000, 2500, 5000, 7500, 10,000, 12,000, 15,000, 17,500, 20,000, 25,000, 50,000, 100,000, 200,000 or 500,000 different target-specific primer pairs.
  • the plurality of target polynucleotides is derived from RNA.
  • the RNA is derived from a single cell or from a population of cells.
  • the RNA is derived from a cancer cell, oocyte, embryo, stem cell, or cell exposed to a companion diagnostic compound.
  • the plurality of target polynucleotides includes a plurality of cDNAs that are transcribed from a transcriptome.
  • the transcriptome comprises a population of RNA that is produced (e.g., transcribed) in one or more cells.
  • the transcriptome comprises a population of RNA that is produced by
  • the transcriptome comprises a population of RNA having a mixture of different sequences.
  • the transcriptome comprises different RNA sequences present in different amounts (e.g., abundance).
  • the population of RNA contained in a transcriptome represents one or more genes expressed in a single cell or in a plurality of cells.
  • the plurality of target polynucleotides contain sequences that are derived from one or more RNA sequences isolated from a single cell or from a plurality of cells.
  • the plurality of target polynucleotides contain sequences that are derived from one or more expressed genes a single cell or from a plurality of cells.
  • one or more target polynucleotides of the plurality contain sequences that represent one or more genes that are expressed in a single cell or in a plurality of cells.
  • the plurality of target polynucleotides includes a plurality of cDNAs that collectively represent RNA expression in a single cell or in a plurality of cells.
  • the plurality of target polynucleotides includes a plurality of cDNAs that represent mRNA expression in the transcriptome.
  • the plurality of target polynucleotides includes different sequences that are derived from a transcriptome, where the transcriptome represents one or more genes expressed in a single cell or in a plurality of cells.
  • the plurality of target-specific primers are complementary or identical to at least some portion of an RNA transcribed in vitro or in vivo from one or more of the genes selected from the group consisting of ABL1 ; AKT1 ; ALK; APC; ATM; BRAF; CDH1 ; CDKN2A; CSF1R; CTNNB 1 ; EGFR; ERBB2; ERBB4; FBXW7; FGFR1 ; FGFR2; FGFR3; FLT3; GNAS; HNF1A; HRAS; IDH1 ; JAK2; JAK3; KDR; KIT; KRAS; MET; MLH1 ; MPL; NOTCH1 ; NPM1 ; NRAS
  • the plurality of target-specific primers includes a plurality of target-specific primer pairs, at least one primer pair including a forward primer and a reverse primer and being configured to amplify at least some portion of a single target polynucleotide of the plurality of target polynucleotides.
  • the plurality of target-specific primer pairs includes at least two different primer pairs configured to amplify a polynucleotide sequence from different respective target polynucleotides.
  • each primer pair in the plurality of target-specific primer pairs is configured to amplify a polynucleotide sequence from a different target polynucleotide than any other primer pair.
  • the plurality of target-specific primers includes two or more pairs of target-specific primers configured to amplify any given target polynucleotide.
  • any given target polynucleotide can hybridize with two or more different pairs of target-specific primers, and be subjected to a primer extension reaction to yield two or more different amplification products.
  • each pair in the two or more pairs of target-specific primers comprises a forward and a reverse primer.
  • the plurality of target polynucleotides includes a first target polynucleotide
  • the plurality of target-specific primers includes only a single pair of target- specific primers configured to amplify the first target polynucleotide.
  • the single pair of target-specific primers comprises two different target-specific primers that are either complementary or identical to at least some portion of the first target polynucleotide.
  • any target polynucleotide of the plurality of target polynucleotides can hybridize to only a single pair of target-specific primers.
  • any target polynucleotide of the plurality of target polynucleotides contains a sequence that is complementary or identical to only a single pair of target-specific primers.
  • each of the different pairs of target-specific primers, in the plurality of target-specific primers is complementary or identical to a different target polynucleotide.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits further comprising a plurality of amplicons.
  • the amplicons are formed by hybridizing one or more of the plurality of target-specific primers to one or more of the plurality of target polynucleotides, and extending at least one of the one or more hybridized target-specific primers in a template dependent manner.
  • the amplicons include a polynucleotide formed by amplification of at least a portion of a target polynucleotide using only a single pair of the target-specific primers of the plurality of target-specific primers.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits, comprising (i) a plurality of target polynucleotides, (ii) 1000 target-specific primers, each primer including a cleavable group, (iii) at least one polymerase, (iv) a cleaving reagent capable of cleaving the cleavable group of the target-specific primers, and (v) a plurality of nucleotides.
  • the plurality of target polynucleotides is derived from a cell population.
  • the plurality of target polynucleotides is formed by reverse transcription of RNA extracted from a cell population.
  • the plurality of target polynucleotides includes a plurality of cDNAs formed by reverse transcription of total mRNA extracted from a cell population.
  • the total mRNA includes at least one RNA transcript having a mutant sequence and the plurality of target polynucleotides includes at least one cDNA derived from the mutant sequence.
  • the RNA transcript having the mutant sequence is associated with a disease or cancer.
  • the RNA transcript having the mutant sequence includes an abnormal splice junction sequence.
  • the abnormal splice junction sequence includes an abnormal exon-exon splice junction sequence, an abnormal exon-intron splice junction sequence, an abnormal intron splice junction sequence, an abnormal intra-exon splice junction sequence, or an abnormal intra-intron splice junction sequence
  • the RNA transcript having the mutant sequence includes an abnormal splice transcript sequence.
  • the RNA transcript having the abnormal splice junction sequence is associated with a disease or cancer.
  • only a single pair of primers of the 1000 target-specific primers hybridizes to any given target polynucleotide of the composition.
  • the plurality of target polynucleotides is obtained by reverse transcribing RNA.
  • the plurality of target polynucleotides is obtained by reverse transcribing a plurality of RNA transcripts from a sample.
  • the plurality of target polynucleotides includes DNA.
  • the plurality of target polynucleotides includes genomic DNA.
  • the plurality of nucleotides includes a detectable label.
  • the composition (as well as related methods, systems, apparatuses and kits) includes a plurality of target-specific primers each containing at least one cleavable group, a cleaving reagent capable of cleaving the at least one cleavable group of a plurality of the target-specific primers, a polymerase, and a plurality target polynucleotides where the target- specific primers are complementary to at least a portion of one or more target polynucleotides of the plurality of target polynucleotides.
  • the composition (as well as related methods, systems, apparatuses and kits) includes a plurality of target-specific primers each containing at least one cleavable group, a cleaving reagent capable of cleaving the at least one cleavable group of a plurality of the target-specific primers, a polymerase, and a plurality target polynucleotides where the target-specific primers are identical to at least a portion of one or more target polynucleotides of the plurality of target polynucleotides.
  • the composition further includes a plurality of nucleotides.
  • the composition includes 1000, 2000, 5000, 10000, 20000, 25000, 25000, 50000, 100000, 200000 or 500000 different target-specific primers. In yet another embodiment, the composition includes at least 1000, 2500, 5000, 7500, 10000, 12000, 15000, 17500, 20000, 25000, 50000, 100000, 200000 or 500000 target- specific primer pairs.
  • the composition includes at least some of the plurality of target-specific primers that are complementary or identical to at least some portion of an RNA transcribed in vitro or in vivo from one or more of the genes selected from the group consisting of ABL1; AKT1; ALK; APC; ATM; BRAF; CDH1; CDKN2A; CSF1R; CTNNB1; EGFR; ERBB2; ERBB4; FBXW7; FGFR1 ; FGFR2; FGFR3; FLT3; GNAS; HNF1A; HRAS; IDH1; JAK2; JAK3; KDR; KIT; KRAS; MET; MLH1; MPL; NOTCH1; NPM1; NRAS; PDGFRA; PIK3CA; PTEN; PTPN11; RBI; RET; SMAD4; SMARCB1; SMO; SRC; STK11; TP53; and VHL.
  • ABL1 AKT1; ALK
  • the target-specific primers are complementary to at least some portion of an RNA transcribed in vitro or in vivo from one or more active genes of the RNA transcriptome.
  • the composition includes a plurality of target-specific primers where only a single pair of target-specific primers is complementary to any target polynucleotide of the plurality of target polynucleotides.
  • the composition includes a plurality of amplicons formed by hybridizing one or more of the plurality of target-specific primers to one or more of the plurality of target polynucleotides and extending at least one of the one or more hybridized target- specific primers in a template dependent manner.
  • the plurality of amplicons formed by hybridizing one or more of the plurality of target-specific primers to one or more of the plurality of target polynucleotides and extending at least one of the one or more hybridized target-specific primers in a template dependent manner is formed via amplification of at least a portion of a target polynucleotide using only a single pair of target-specific primers from the plurality of target-specific primers.
  • the composition (as well as related methods, systems, apparatuses and kits) includes 1000 target-specific primers each including a cleavable group, a cleaving reagent capable of cleaving the cleavable group, a polymerase, a plurality target
  • the plurality of nucleotides includes a detectable label or nucleotide analog.
  • the plurality of target polynucleotides can be obtained by reverse transcribing a plurality of RNA transcripts from a sample.
  • the plurality of target polynucleotides can include genomic DNA or cDNA.
  • the cDNA represents mRNA expression in a RNA transcriptome.
  • the plurality of target polynucleotides includes RNA. In some embodiments, the plurality of target polynucleotides can include RNA derived from a single cell or from a population of cells. In some embodiments, the amount of DNA or cDNA required can be 200 pg to 1 microgram. In some embodiments, the amount of DNA or cDNA required for amplification of one or more of the target polynucleotides can be 200 pg to 100 ng, 500 pg to 50 ng, 1 ng to 25 ng, or 1 ng to 10 ng. In one embodiment, the amount of DNA or cDNA required for amplification of one or more of the plurality of target polynucleotides by one or more methods disclosed herein is 1 ng to 25 ng.
  • the number of target polynucleotides amplified by one or more of the methods using the compositions (as well as related kits, apparatuses and systems) disclosed herein can be hundreds, thousands, or hundreds of thousands of target polynucleotides in a single reaction mixture.
  • the number of different target polynucleotides amplified in a single multiplex amplification reaction can be at least 1000, 2000, 5000, 10000, 20000, 25000, 50000, 100000, 12500, 15000, 200000, 300000, 400000, or 500000, or greater.
  • the disclosure relates generally to methods (as well as related compositions, systems, apparatuses and kits) for synthesizing a plurality of polynucleotides in a sample, comprising synthesizing a plurality of amplicons, wherein the synthesizing includes forming a reaction mixture by contacting a plurality of target polynucleotides with a plurality of target- specific primer pairs.
  • the synthesizing includes forming a reaction mixture by contacting a plurality of target polynucleotides with a plurality of target- specific primer pairs.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • the methods can further include extending at least one primer of a target-specific primer pair to form one or more synthesized polynucleotides.
  • the methods can include extending both primers of a target specific primer pair, either simultaneously or sequentially, optionally using isothermal or non- isothermal conditions.
  • the extending can include extending in a template-dependent or template- directed manner.
  • the extending includes forming one or a plurality of amplicons. At least one amplicon is optionally formed via amplification of a single target polynucleotide using at least one pair of target-specific primers.
  • the methods include forming one or a plurality of amplicons, each amplicon being formed by amplifying a single target polynucleotide using only a single target-specific primer pair.
  • the methods further include detecting at least some of the amplicons, for example using optical or non-optical detection.
  • the methods further include obtaining sequence information from one or a plurality of the amplicons.
  • a sequence read assembly is not performed.
  • the plurality of target polynucleotides contains a mixture of different target sequences.
  • the plurality of target polynucleotides contains at least a first and a second target polynucleotide.
  • the sequence of the different target polynucleotides in the plurality can be directly extracted or otherwise derived from a sample containing RNA or DNA, or a mixture of both.
  • the sample comprises a whole transcriptome, or a portion of a whole transcriptome.
  • the sample comprises a genome or a portion of a genome.
  • the sample contains RNA, DNA, cDNA, or recombinant DNA or RNA derived from one or more cells.
  • the sample contains total RNA derived from one or more cells.
  • the different target polynucleotides include a plurality of RNA molecules forming a transcriptome, or a plurality of cDNA molecules formed via reverse transcription of a transcriptome, or a plurality of DNA molecules formed via amplification and/or fragmentation of a transcriptome or a genome.
  • the methods further include hybridizing different target-specific primer pairs from the plurality of target-specific primer pairs to different target polynucleotides.
  • the disclosed methods include hybridizing each pair of two or more different target-specific primer pairs to different target polynucleotides.
  • the methods further include hybridizing at least a portion of each primer of a target-specific primer pair to a portion of a corresponding target polynucleotide, or its complement.
  • the plurality of target-specific primers includes a plurality of target-specific primer pairs, at least one primer pair including a forward primer and a reverse primer and being configured to amplify at least some portion of a single target polynucleotide of the plurality of target polynucleotides.
  • the plurality of target-specific primer pairs includes at least two different primer pairs, each primer pair configured to amplify a polynucleotide sequence from a target polynucleotide, where no two primer pairs are configured to amplify a polynucleotide sequence from the same target polynucleotide.
  • each primer pair from the plurality of different primer pairs is configured to amplify polynucleotide sequences from different target polynucleotides.
  • each primer pair in the plurality of target-specific primer pairs is configured to amplify a polynucleotide sequence from a different target polynucleotide than any other primer pair.
  • the plurality of target polynucleotides includes a first target polynucleotide
  • the plurality of target-specific primers includes only a single pair of target- specific primers configured to amplify the first target polynucleotide.
  • the single pair of target-specific primers comprises a forward target-specific primer and a reverse target-specific primer that are either substantially complementary or substantially identical to at least some portion of the first target polynucleotide.
  • the first target-specific primer and the reverse target- specific primer hybridize to the first target polynucleotide or its complement under high-stringency hybridization conditions.
  • the methods further include hybridizing at least a portion of both target-specific primers of a first target-specific primer pair, each independently and separately, to a portion of a first target polynucleotide or its complement.
  • the disclosed methods include hybridizing at least a portion of both target-specific primers of a first target-specific primer pair to a portion of a first target polynucleotide, or its complement.
  • the hybridizing includes high stringency hybridization conditions.
  • the methods further include hybridizing at least a portion of both target-specific primers of a second target-specific primer pair, each independently and separately, to a portion of a second target polynucleotide, or its complement.
  • the disclosed methods include hybridizing at least a portion of both target-specific primers of a second target-specific primer pair to a portion of a second target polynucleotide, or its complement.
  • the methods further include hybridizing different pairs of target-specific primers from the plurality of target-specific primer pairs, each independently and separately, to different target polynucleotides, or their complements, to form a plurality of different target polynucleotides, or their complements, to form a plurality of different target polynucleotides, or their complements, to form a plurality of different target polynucleotides, or their complements, to form a plurality of different
  • the disclosed methods optionally include forming a plurality of different primer/polynucleotide complexes. In some embodiments, the forming includes hybridizing different pairs of target-specific primers from the plurality of target- specific primer pairs to different target polynucleotides or their complements. In some
  • the forming includes extending one or more target-specific primers within different primer/polynucleotide complexes, optionally in a template-dependent manner, for example by using a target polynucleotide of the complex as a template.
  • the methods further include hybridizing a plurality of target-specific primer pairs having extendible 3' ends in a primer extension reaction.
  • the disclosed methods including extending at least some of the target-specific primer pairs, optionally in a template-dependent manner, for example by using a target polynucleotide of the complex as a template.
  • the methods further include hybridizing a plurality of target polynucleotides with the plurality of target-specific primer pairs in a single reaction mixture.
  • the methods further include contacting a plurality of target polynucleotides with the plurality of target-specific primer pairs in a single reaction mixture.
  • the methods further include contacting, in a single reaction mixture, a first target polynucleotide with a first target-specific primer pair, and contacting a second target polynucleotide with a second target-specific primer pair.
  • At least one of the target-specific primer pairs has minimal cross- hybridization with any other pair of primers in the single reaction mixture.
  • the single reaction mixture contains 1000, 2500, 5000, 7500, 10,000, 12,000, 15,000, 17,500, 20,000, 25,000, 50,000, 100,000, 200,000, 500,000, or more than 500,000 different target-specific primer pairs.
  • the single reaction mixture contains at least 1000, 2500, 5000, 7500, 10,000, 12,000, 15,000, 17,500, 20,000, 25,000, 50,000, 100,000, 200,000 or 500,000, or more than 500000 different target-specific primer pairs.
  • the contacting step is conducted under nucleic acid hybridization conditions such that different target-specific primer pairs hybridize to their cognate target sequences.
  • the contacting includes contacting the target-specific primer pairs with target polynucleotides and hybridizing at least one member of each pair with a target polynucleotide or its complement.
  • the contacting is performed under standard nucleic acid hybridization conditions. In some embodiments, the contacting is performed using stringent hybridization conditions.
  • only a single pair of target-specific primers hybridize to any given target polynucleotide.
  • the disclosed methods optionally include hybridizing only a single pair of target-specific primer pairs to a given target polynucleotide.
  • the method further includes extending the plurality of
  • primer/polynucleotide complexes in a primer extension reaction.
  • the method further includes extending the target-specific primer pairs in primer extension reaction.
  • the method further includes extending the target-specific primer pairs in a template -dependent manner.
  • the method further includes extending the target-specific primer pairs to form a plurality of amplicons.
  • the method further includes conducting a primer extension reaction to form a plurality of amplicons containing sequences derived from the plurality of target polynucleotides. In some embodiments, the methods further include forming a plurality of amplicons containing sequences derived from the plurality of target polynucleotides by extending the target- specific primer pairs in a primer extension reaction.
  • the methods further include extending the first target-specific primer pair in a template -dependent manner to form a first amplicon, and extending the second target-specific primer pair in a template-dependent manner to form a second amplicon.
  • the each amplicon contains sequences derived from a target polynucleotide. [00105] In some embodiments, at least two of the plurality of amplicons have sequences that are less than 50% complementary to each other
  • the first amplicon contains sequences derived from the first target polynucleotide.
  • the second amplicon contains sequences derived from the second target polynucleotide.
  • the methods further include detecting the plurality of amplicons.
  • the detecting includes sequencing at least a portion of the amplicons.
  • a sequence read assembly is not performed.
  • the disclosed methods include quantifying or otherwise estimating the number of amplicons containing a sequence derived from a given target gene of interest (e.g., a first target gene).
  • the quantifying includes counting or otherwise estimating the number of amplicons containing a target polynucleotide sequence of interest to obtain a number.
  • the quantifying can include counting the number of amplicons containing a first polynucleotide sequence to obtain a first number.
  • the quantifying includes identifying a first number of amplicons as containing a first polynucleotide sequence. The first number can be the number of amplicons identified as containing the first polynucleotide sequence.
  • the disclosed methods further include using the first number to estimate the level of representation of the first target gene, or the first nucleic acid sequence, within the plurality of target polynucleotides.
  • the quantifying includes counting the number of amplicons containing a sequence that maps to the first target gene.
  • the first polynucleotide sequence is included in the first target gene.
  • the disclosed methods include estimating the number of polynucleotides containing of the first nucleic acid sequence within the plurality of target polynucleotides using the first number.
  • the quantifying can include counting the number of amplicons containing a second polynucleotide sequence to obtain a second number. In some embodiments, the quantifying includes identifying a second number of amplicons as containing a second polynucleotide sequence. The second number can be the number of amplicons identified as containing the second polynucleotide sequence.
  • the disclosed methods further include using the second number to estimate the level of representation of the second target gene, or the second nucleic acid sequence, within the plurality of target polynucleotides.
  • the quantifying includes counting the number of amplicons containing a sequence that maps to the second target gene.
  • the second polynucleotide sequence is included in the second target gene.
  • the disclosed methods include estimating the number of polynucleotides containing of the second nucleic acid sequence within the plurality of target polynucleotides using the second number.
  • the disclosed methods include determining the amount of the first target polynucleotide and/or the amount of the second target polynucleotide present in the reaction mixture.
  • the determining can include using the first number and the second number.
  • the methods can include inferring or otherwise determining the amount of first polynucleotide sequence and/or the amount of the second polynucleotide sequence in a biological sample.
  • the sample includes RNA, DNA or cDNA derived from one or more cells.
  • the reaction mixture can include at least some portion of the sample.
  • the plurality of target polynucleotides in the reaction mixture is extracted directly from the sample, or is derived via manipulation of polynucleotides extracted from the sample.
  • the plurality of target-specific primer pairs includes 2-100, or about 100-500, or about 500-1,000, or about 1,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different target-specific primer pairs.
  • the single reaction mixture contains about 2-100, or about
  • the primer extension reaction can form a plurality of amplicons containing sequences derived from 2- 100, or about 100-500, or about 500- 1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different target polynucleotides.
  • the plurality of polynucleotides can be detected by quantifying the number of amplicons containing sequence derived from each of the 2- 100, or about 100-500, or about 500-1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different target polynucleotides.
  • the sample includes nucleic acids (e.g., RNA, DNA or cDNA) derived from one or more cells and the method further includes quantifying the amounts for each of 2- 100, or about 100-500, or about 500- 1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000, or about 50,000- 100,000, or about 100,000-200,000, or about 200,000-500,000, or more different nucleic acids present in the sample.
  • nucleic acids e.g., RNA, DNA or cDNA
  • the sample includes cDNA derived from RNA (e.g., total cellular RNA) and the method further includes quantifying the amounts for each of the 2- 100, or about 100-500, or about 500- 1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000, or about 50,000- 100,000, or about 100,000-200,000, or about 200,000-500,000, or more different transcripts present in the sample.
  • RNA e.g., total cellular RNA
  • methods for detecting a plurality of polynucleotides in a sample further comprise hybridizing the plurality of amplicons with a labeled or un-labeled nucleic acid probe, with a microarray or with a nucleic acid having a reference sequence.
  • methods for detecting a plurality of polynucleotides in a sample further comprise re-amplifying the plurality of amplicons.
  • the method further comprises calculating a ratio of the first number and the second number.
  • the first number represents the number of amplicons derived from the first target gene
  • the second number of amplicons represents the number of amplicons derived from the second target gene.
  • the first number represents the number of amplicons containing a first polynucleotide sequence of interest
  • the second number represents the number of amplicons containing a second polynucleotide sequence of interest.
  • the disclosed methods further include sequencing at least some or substantially all of the plurality of adaptor-ligated amplified target sequences.
  • the sequencing comprises a massively parallel sequencing procedure or a gel electrophoresis procedure.
  • methods for detecting a plurality of polynucleotides in a sample further comprise comparing the number of plurality of adaptor-ligated amplified target sequences containing the sequence derived from the first polynucleotide with the number of plurality of adaptor-ligated amplified target sequences containing the sequence derived from the second polynucleotide.
  • methods for detecting a plurality of polynucleotides in a sample further comprise determining the abundance in the reaction mixture of the plurality of adaptor-ligated amplified target sequences containing the sequence derived from the first polynucleotide relative to the number of plurality of adaptor-ligated amplified target sequences containing the sequence derived from the second polynucleotide within the same reaction mixture or within a different reaction mixture.
  • methods for detecting a plurality of target polynucleotides in a sample further comprise calculating a ratio of the number of plurality of adaptor-ligated amplified target sequences containing the sequence derived from the first target polynucleotide and the number of plurality of adaptor-ligated amplified target sequences containing the sequence derived from the second target polynucleotide.
  • the extending step (e.g., primer extending) includes forming a plurality of amplicons , wherein each amplicon contains a primer-derived sequence on at least one end, and each amplicon contains a sequence derived from a target polynucleotide
  • the each amplicon contains sequences derived from at least one target specific primer.
  • the first and the second amplicons contain sequences derived from at least one target specific primer.
  • At least one of the primers from plurality of target-specific primer pairs includes a cleavable group.
  • the plurality of amplicons includes a primer-derived sequence on at least one end, and the primer-derived sequence includes at least one cleavable group.
  • the at least one cleavable group comprises uracil, uridine, inosine, or 7,8- dihydro-8-oxoguanine (8-oxoG) nucleobases.
  • the at least one cleavable group is cleavable with an enzyme, chemical compound, heat or light.
  • the at least one cleavable group is cleavable with uracil DNA glycosylase
  • UDG UDG
  • Fpg formamidopyrimidine DNA glycosylase
  • FuPa reagent a FuPa reagent
  • the methods for detecting a plurality of polynucleotides in a sample further comprise cleaving the cleavable groups on the primer-derived sequences on the ends of the plurality of amplicons thereby producing a plurality of cleaved amplified target sequences.
  • the methods for detecting a plurality of polynucleotides in a sample further comprise producing a plurality of adaptor-ligated amplified target sequences by ligating one or more adaptors to one or both ends of the plurality of cleaved amplified target sequences.
  • At least one of the one or more adaptors includes a unique identifier sequence.
  • At least one of the one or more adaptors includes a sequencing primer binding site, an amplification primer binding site or a universal sequence.
  • methods for detecting a plurality of polynucleotides in a sample further comprise hybridizing the plurality of adaptor-ligated amplified target sequences with a labeled or un-labeled nucleic acid probe, with a microarray or with a nucleic acid having a reference sequence.
  • methods for detecting a plurality of polynucleotides in a sample further comprise re-amplifying the plurality of adaptor-ligated amplified target sequences.
  • the method further comprises calculating a ratio of the number of a first adaptor-ligated amplified target sequence containing a first polynucleotide sequence derived from the first target polynucleotide, and the number of a second adaptor-ligated amplified target sequence containing a second polynucleotide sequence derived from the second target
  • methods for detecting a plurality of polynucleotides in a sample further comprise sequencing the plurality of adaptor-ligated amplified target sequences.
  • the sequencing comprises a massively parallel sequencing procedure or a gel electrophoresis procedure.
  • methods for detecting a plurality of polynucleotides in a sample further comprise comparing the number of plurality of adaptor-ligated amplified target sequences containing a first polynucleotide sequence derived from the first target polynucleotide with the number of plurality of adaptor-ligated amplified target sequences containing a second polynucleotide sequence derived from the second target polynucleotide.
  • methods for detecting a plurality of polynucleotides in a sample further comprise determining the relative abundance of adaptor-ligated amplified target sequences containing the first polynucleotide sequence derived from the first target polynucleotide relative to the number of adaptor-ligated amplified target sequences containing the second polynucleotide sequence derived from the second target polynucleotide.
  • methods for detecting a plurality of polynucleotides in a sample further comprise calculating a ratio of the number of adaptor-ligated amplified target sequences containing the first polynucleotide sequence derived from the first polynucleotide and the number of adaptor-ligated amplified target sequences containing the second polynucleotide sequence derived from the second target polynucleotide.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for characterizing a population of polynucleotides containing polynucleotide sequences of interest.
  • the polynucleotide population can, for example, include or be derived from a genome, whole transcriptome, or derived from a portion of a genome or whole transcriptome.
  • the disclosed methods include generating a plurality of amplicons having sequences derived from RNA.
  • the plurality of amplicons can be characterized.
  • the plurality of amplicons can be generated by amplifying a plurality of target polynucleotides in a single reaction mixture.
  • the plurality of polynucleotides can be extracted or otherwise derived from a biological sample including cells, tissue, stool, blood, lymph, plasma, serum or other bodily fluid.
  • the plurality of polynucleotides includes a transcriptome.
  • the plurality of polynucleotides is derived from a mixed sample (e.g., includes DNA from different individuals, tissue types or from a mixture of tumor and normal cells).
  • the plurality of polynucleotides includes a mixture of maternal and fetal DNA and/or RNA.
  • the plurality of polynucleotides include circulating DNA, e.g., circulating cell-free DNA (ccf-DNA), or circulating RNA (e.g., circulating cell free RNA) present in blood or plasma.
  • the ccf-DNA (or RNA) includes a mixture of maternal and fetal DNA (or RNA).
  • the ccf-DNA (or RNA) includes a mixture of DNA (or RNA) derived from tumor and non-tumor cells from a single individual.
  • the single reaction mixture contains a plurality of target-specific primer pairs.
  • each of the primer pairs in the plurality of different target-specific primer pairs hybridizes to a different target polynucleotide.
  • the plurality of amplicons can be characterized using any procedure including: hybridizing or sequencing the plurality of amplicons; detecting the presence of one or more sequences of interest; or determining the abundance of one or more sequences of interest in the reaction mixture.
  • the methods can include estimating the abundance of the one or more sequences of interest in the biological sample from which the plurality of polynucleotides were extracted or otherwise derived.
  • the methods can include comparing the abundance of a first polynucleotide sequence of interest to the abundance of a second polynucleotide sequence of interest, where the first and second polynucleotides are in the same reaction mixture or in different reaction mixtures. In some embodiments, the methods can include comparing the abundance of a first polynucleotide sequence of interest to the abundance of a second polynucleotide sequence of interest, where the first and second polynucleotides are in the same biological sample or in different biological samples. In some embodiments, the methods can include comparing the relative abundance of polynucleotides derived from different chromosomes.
  • the methods can include analyzing sequence data to determine the presence of a copy number variation, e.g., within a target sequence of interest. In some embodiments, the methods can include analyzing sequence data to determine the presence of one or more chromosomal aneuploidies.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides, comprising: (a) contacting, within a single reaction mixture, a plurality of target polynucleotides with a plurality of target- specific primer pairs under nucleic acid hybridization conditions such that different target-specific primer pair hybridizes to different target polynucleotides and only at most a single pair of target- specific primers is hybridized to any given target polynucleotide; (b) extending the target-specific primer pairs in a template -dependent fashion and forming a plurality of extension products, the extension products containing a sequence derived from a target polynucleotide; and (c) detecting the plurality of extension products.
  • the plurality of extension products includes a sequence derived from a target polynucleotide and a sequence derived from at least one target- specific primer of a primer pair. In some embodiments, the plurality of extension products comprises a plurality of amplicons. In some embodiments, at least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid. In some embodiments, at least one, some or all of the plurality of the target polynucleotides are separately hybridized to only a single pair of target-specific primers.
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target polynucleotides.
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target-specific primer pairs.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • the reaction mixture includes a single pair of target-specific primers configured to hybridize with each different target polynucleotide of the plurality of target polynucleotides.
  • the plurality of target polynucleotides is derived from a sample.
  • the detecting includes sequencing at least a portion of the extension products (e.g., amplicons). In some embodiments, since a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides, comprising: (a) contacting, within a single reaction mixture, a plurality of target polynucleotides with a plurality of target- specific primer pairs under nucleic acid hybridization conditions such that each different target- specific primer pair hybridizes to different target polynucleotides and only at most a single pair of target-specific primers is hybridized to any given target polynucleotide; (b) extending the target- specific primer pairs in a template-dependent fashion and forming a plurality of amplicons, each amplicon containing a sequence derived from a target polynucleotide; and (c) detecting the amplicons.
  • At least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • at least one, some or all of the plurality of the target polynucleotides are separately hybridized to only a single pair of target-specific primers. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more than 95% of the target
  • polynucleotides are each independently and separately hybridized to a single target-specific primer pair within the reaction mixture.
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target polynucleotides.
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target-specific primer pairs.
  • the reaction mixture includes a single pair of target-specific primers configured to hybridize with each different target polynucleotide of the plurality of target polynucleotides.
  • the plurality of target polynucleotides is derived from a sample.
  • the detecting includes sequencing at least a portion of the amplicons.
  • a sequence read assembly is not performed since a single primer pair is used to generate a single sequence for each target polynucleotide.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) contacting, within a single reaction mixture, a plurality of target polynucleotides derived from a sample with a plurality of target-specific primer pairs under nucleic acid hybridization conditions such that at least some of the target-specific primer pairs hybridize to at least some of the target polynucleotides and at least one of the target polynucleotides is hybridized to no more than one primer pair; (b) extending the target-specific primer pairs in a template-dependent fashion and forming a plurality of amplicons, each amplicon containing a sequence derived from a target polynucleotide; and (c) detecting the plurality of amplicons.
  • At least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • at least one, some or all of the plurality of the target polynucleotides are separately hybridized to only a single pair of target-specific primers.
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target polynucleotides. In some embodiments, the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target-specific primer pairs.
  • the reaction mixture includes a single pair of target-specific primers configured to hybridize with each different target polynucleotide of the plurality of target polynucleotides.
  • the detecting includes sequencing at least a portion of the amplicons.
  • a sequence read assembly is not performed since a single primer pair is used to generate a single sequence for each target polynucleotide.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) contacting, within a single reaction mixture, a plurality of target polynucleotides derived from a sample with a plurality of target-specific primer pairs under nucleic acid hybridization conditions such that different target-specific primer pairs hybridize to different target polynucleotides and only a single pair of target-specific primers hybridize to any given target polynucleotide; (b) extending the target-specific primer pairs in a template-dependent fashion and forming a plurality of amplicons, each amplicon containing a sequence derived from a target polynucleotide; and (c) detecting the plurality of amplicons.
  • At least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • at least one, some or all of the plurality of the target polynucleotides are separately hybridized to only a single pair of target-specific primers. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more than 95% of the target
  • polynucleotides are each independently and separately hybridized to a single target-specific primer pair within the reaction mixture.
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target-specific primer pairs.
  • the reaction mixture includes a single pair of target-specific primers configured to hybridize with each different target polynucleotide of the plurality of target polynucleotides.
  • the detecting includes sequencing at least a portion of the amplicons. In some embodiments, since a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides, comprising: (a) contacting, within a single reaction mixture, a plurality of target polynucleotides with a plurality of target- specific primer pairs under nucleic acid hybridization conditions such that different target-specific primer pairs hybridize to different target polynucleotides and at least some of the target
  • polynucleotides are hybridized to no more than one pair of target-specific primers; (b) extending the target-specific primer pairs in a template-dependent fashion and forming a plurality of amplicons, each amplicon containing a sequence derived from a target polynucleotide; and (c) detecting the amplicons.
  • at least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • At least one, some or all of the plurality of the target polynucleotides are separately hybridized to only a single pair of target-specific primers. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more than 95% of the target
  • polynucleotides are each independently and separately hybridized to a single target-specific primer pair within the reaction mixture.
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target
  • the reaction mixture includes at least 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target-specific primer pairs.
  • the reaction mixture includes a single pair of target-specific primers configured to hybridize with each different target polynucleotide of the plurality of target polynucleotides.
  • the detecting includes sequencing at least a portion of the amplicons. In some embodiments, when a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) contacting, within a single reaction mixture, (i) a plurality of target-specific primer pairs, with (ii) a plurality of target polynucleotides derived from a sample, where the contacting is performed under nucleic acid hybridization conditions such that different target-specific primer pairs hybridize to different target polynucleotides, where the plurality of target polynucleotides contains at least a first and a second target polynucleotide, and a first target-specific primer pair hybridizes to the first target polynucleotide and a second target-specific primer pair hybridizes to the second target
  • At least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • at least one of the target polynucleotides hybridizes to only a single pair of target- specific primers.
  • the first target polynucleotide hybridizes only to the first target-specific primer pair and not to any other target-specific primers in the reaction mixture.
  • the second target polynucleotide hybridizes only to the second target-specific primer pair and not to any other target-specific primers in the reaction mixture.
  • the detecting includes sequencing at least a portion of the amplicons. In some embodiments, when a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) contacting, within a single reaction mixture, (i) a plurality of different target-specific primer pairs, with (ii) a plurality of target polynucleotides, where the contacting is performed under nucleic acid hybridization conditions such that the plurality of different target-specific primer pairs hybridizes to different target polynucleotides, wherein the single reaction mixture includes 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, or 500,000 different target specific primer pairs; (b) extending the target-specific primer pairs in a template-dependent fashion and forming a plurality of amplicons, where each amplicon includes a sequence derived from a target
  • the polynucleotide and at least one of the target-specific primer pairs comprising at least one of the target-specific primer pairs; and (c) detecting the plurality of amplicons.
  • at least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • at least one of the target polynucleotides hybridizes to only a single pair of target-specific primers.
  • at least some of the target polynucleotides hybridize to a single corresponding target-specific primer pair and not to any other target-specific primers in the reaction mixture.
  • the detecting includes sequencing at least a portion of the amplicons. In some embodiments, when a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the plurality of target polynucleotides in the reaction mixture includes a population of genomic DNA or RNA extracted directly from a biological sample.
  • the biological sample can include cells, tissue, stool, lymph, blood, plasma, serum, cerebrospinal fluid, cell or tissue exudate or other bodily fluid.
  • the plurality of target polynucleotides in the reaction mixture includes a population of polynucleotides derived from such total genomic DNA or total RNA.
  • the plurality of target polynucleotides can include specific sequences derived via reverse transcription and/or selective or non-selective amplification of such total genomic DNA or RNA.
  • the plurality of target polynucleotides in the reaction mixture can be the products of additional manipulations such as restriction digestion, fragmentation, end polishing and/or adapter ligation, or any combination of the foregoing.
  • the plurality of target polynucleotides in the reaction mixture includes a population of DNA fragments substantially representing an entire genome or any portion thereof.
  • the plurality of target polynucleotides in the reaction mixture includes a population of cDNA fragments derived from RNA transcripts and substantially representing an entire transcriptome or any portion thereof.
  • the plurality of target-specific primers includes at least one target-specific primer pair for each different DNA or cDNA fragment present (or expected to be present) in the reaction mixture.
  • the plurality of target-specific primers includes only a single target- specific primer pair for each different DNA or cDNA fragment present (or expected to be present) in the reaction mixture.
  • [00175] includes 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000,
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) contacting, within a single reaction mixture, (i) a plurality of target-specific primer pairs containing 20,000 different primer pairs, with (ii) a plurality of target polynucleotides having sequences derived from RNA from one or more cells, where the contacting is performed under nucleic acid hybridization conditions such that the 20,000 different target-specific primer pairs hybridizes to different target polynucleotides, and only a single pair of target-specific primers hybridizes to any given target polynucleotide; (b) extending the target-specific primer pairs in a template-dependent fashion and forming a plurality of amplicons, where each amplicon includes a sequence derived from a target polynucleotide and at least one of the target-specific primer pairs; and (c) detecting the plurality
  • At least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non- tailed primer.
  • at least some of the target polynucleotides hybridize to a single corresponding target-specific primer pair and not to any other target-specific primers in the reaction mixture.
  • the detecting includes sequencing at least a portion of the amplicons. In some embodiments, since a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) contacting, within a single reaction mixture, (i) a plurality of different target-specific primer pairs having a cleavable group, with (ii) a plurality of target polynucleotides derived from RNA from one or more cells, where the contacting is performed under nucleic acid hybridization conditions such that the plurality of different target-specific primer pairs hybridizes to different target
  • polynucleotides and only a single pair of target-specific primers hybridizes to any given target polynucleotide; (b) extending the target-specific primer pairs in a template-dependent fashion and forming a plurality of amplicons, where each amplicon includes a sequence derived from a target polynucleotide and a primer-derived sequence having the cleavable group; (c) cleaving the cleavable group in the primer-derived sequence to produce a cleaved amplified target sequence; (d) ligating at least one adaptor to an end of at least one cleaved amplified target sequence to produce a adaptor- ligated amplified target sequence; and (e) detecting the adaptor-ligated amplified target sequence.
  • At least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • the detecting includes sequencing at least some of the adaptor-ligated amplified target sequences. In some embodiments, since a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) generating a plurality of target polynucleotides having sequences derived from a plurality of RNA in a sample, by reverse-transcribing the plurality of RNA with a plurality of primers to produce the plurality of target polynucleotides; (b) contacting, within a single reaction mixture, a plurality of target polynucleotides derived from a sample with a plurality of target-specific primer pairs under nucleic acid hybridization conditions such that different target-specific primer pairs hybridize to different target polynucleotides and only a single pair of target-specific primers hybridize to any given target polynucleotide; (c) extending the target-specific primer pairs in a template-dependent fashion and forming a plurality of amplicons, each amplicon containing
  • the plurality of RNA includes RNA sequences present in a biological sample.
  • the plurality of RNA sequences includes different RNA sequences.
  • the plurality of RNA represents total RNA, or a portion of total RNA, from a biological sample.
  • the plurality of RNA sequences includes a transcriptome derived from a biological sample.
  • at least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • the detecting includes sequencing at least a portion of the amplicons. In some embodiments, since a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) generating a plurality of target polynucleotides having sequences derived from a plurality of RNA in a sample, by reverse-transcribing the plurality of RNA with a plurality of primers to produce the plurality of target polynucleotides; (b) contacting, within a single reaction mixture, (i) a plurality of target-specific primer pairs, with (ii) a plurality of target polynucleotides derived from a sample, where the contacting is performed under nucleic acid hybridization conditions such that different target-specific primer pairs hybridize to different target polynucleotides, where the plurality of target polynucleotides contains at least a first and a second target polynucleotide, and a first target-specific primer pair hybridizes to the
  • At least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non-tailed primer.
  • the detecting includes sequencing at least some of the amplicons. In some embodiments, since a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) generating a plurality of target polynucleotides having sequences derived from a plurality of RNA in a sample, by reverse-transcribing the plurality of RNA with a plurality of primers to produce the plurality of target polynucleotides; (b) contacting, within a single reaction mixture, (i) a plurality of different target-specific primer pairs, with (ii) a plurality of target polynucleotides derived from RNA from one or more cells, where the contacting is performed under nucleic acid hybridization conditions such that the plurality of different target-specific primer pairs hybridizes to different target polynucleotides, and only a single pair of target-specific primers hybridizes to any given target polynucleotide, wherein the single reaction mixture includes 100-
  • the polynucleotide and at least one of the target-specific primer pairs comprising at least one of the target-specific primer pairs; and (d) detecting at least a sub- population of the amplicons.
  • at least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non- tailed primer.
  • the detecting includes sequencing at least some of the amplicons.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) generating a plurality of target polynucleotides having sequences derived from a plurality of RNA in a sample, by reverse-transcribing the plurality of RNA with a plurality of primers to produce the plurality of target polynucleotides; (b) contacting, within a single reaction mixture, (i) a plurality of target-specific primer pairs containing at least 20,000 different primer pairs, with (ii) the plurality of target polynucleotides, where the contacting is performed under nucleic acid hybridization conditions such that the at least 20,000 different target-specific primer pairs hybridizes to different target polynucleotides
  • At least one of the plurality of target- specific primers is a tailed primer. In some embodiments, at least one of the plurality of target- specific primers is a non-tailed primer. In some embodiments, a single pair of target-specific primers hybridizes to any one of at least 20,000 different target polynucleotide sequences. In some embodiments, the reverse transcribing is conducted with a plurality of random sequence primers. In some embodiments, at least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid. In some embodiments, the detecting includes sequencing at least some of the plurality of amplicons. In some embodiments, since a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for detecting a plurality of polynucleotides in a sample, comprising: (a) generating a plurality of target polynucleotides having sequences derived from a plurality of RNA in a sample, by reverse-transcribing the plurality of RNA with a plurality of primers to produce the plurality of target polynucleotides; (b) contacting, within a single reaction mixture, (i) a plurality of different target-specific primer pairs having a cleavable group, with (ii) the plurality of target polynucleotides derived from the RNA, where the contacting is performed under nucleic acid hybridization conditions such that the plurality of different target-specific primer pairs hybridizes to different target polynucleotides, and only a single pair of target-specific primers hybridizes to any given target polynucleotide; (c) extending
  • polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • at least one of the plurality of target-specific primers is a tailed primer.
  • at least one of the plurality of target-specific primers is a non- tailed primer.
  • the detecting includes sequencing at least some of the adaptor- ligated amplified target sequences. In some embodiments, since a single primer pair is used to generate a single sequence for each target polynucleotide, a sequence read assembly is not performed.
  • the sample includes RNA, DNA or cDNA derived from one or more cells.
  • At least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • one primer of at least one target-specific primer pair hybridizes to a sequence that is complementary to the sequence of any give target polynucleotide.
  • detecting a plurality of polynucleotides in a sample further comprises: determining an amount of amplicons containing a sequence derived from a first target polynucleotide.
  • the determining includes counting a number of amplicons derived from the first target polynucleotide.
  • at least a portion of the amplicons is analyzed to count the number of amplicons derived from the first target polynucleotide.
  • the first target polynucleotide is present within, or derived from, a first chromosome.
  • detecting a plurality of polynucleotides in a sample further comprises: determining an amount of amplicons containing a sequence derived from a second target polynucleotide.
  • the determining includes counting a number of amplicons derived from the second target polynucleotide.
  • at least a portion of the amplicons is analyzed to count the number of amplicons derived from the second target polynucleotide.
  • the second target polynucleotide is present within, or derived from, a second chromosome.
  • detecting a plurality of polynucleotides in a sample further comprises: quantifying the number of amplicons containing a sequence derived from a first target polynucleotide.
  • the quantifying includes counting the number of amplicons containing a polynucleotide sequence of interest (e.g., a first polynucleotide sequence) that is derived from the first target polynucleotide.
  • detecting a plurality of polynucleotides in a sample further comprises: quantifying the number of amplicons containing sequence derived from a second target polynucleotide.
  • the quantifying includes counting the number of amplicons containing a polynucleotide sequence of interest (e.g., a second polynucleotide sequence) that is derived from the second target polynucleotide.
  • the second target polynucleotide sequence of interest e.g., a second polynucleotide sequence
  • polynucleotide is present within, or derived from, a second chromosome.
  • first and second chromosomes are different.
  • detecting a plurality of polynucleotides in a sample further comprises: quantifying the amount of the first target polynucleotide and the amount of the second target polynucleotide present in the sample.
  • the sample includes RNA or cDNA extracted or otherwise derived from the biological sample.
  • the single reaction mixture includes 10, 50, 100, 250, 500,
  • the single reaction mixture includes 10, 50, 100, 250, 500, 1000, 5000, 10,000, 15,000, 25,000, 50,000, 100,000, 500,000, or 1 ,000,000 different DNA or cDNA fragments and about the same number of different target specific primer pairs.
  • the plurality of target-specific primer pairs includes 2-100, or about 100-500, or about 500- 1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different target-specific primer pairs.
  • the forming step further includes forming a plurality of amplicons containing sequences derived from 2- 100, or about 100-500, or about 500- 1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different target polynucleotides.
  • detecting a plurality of polynucleotides in a sample further comprises: quantifying the number of amplicons containing sequence derived from each of the 2- 100, or about 100-500, or about 500-1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different target polynucleotides.
  • the sample includes nucleic acids (e.g., RNA, DNA or cDNA) derived from one or more cells and the method (and related compositions, systems, apparatuses and kits) includes quantifying the amounts for each of 2- 100, or about 100-500, or about 500- 1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different nucleic acids present in the sample.
  • nucleic acids e.g., RNA, DNA or cDNA
  • the sample includes cDNA derived from RNA (e.g., total cellular RNA) and the method (and related compositions, systems, apparatuses and kits) includes quantifying the amounts for each of 2- 100, or about 100-500, or about 500- 1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000- 25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different transcripts present in the sample.
  • RNA e.g., total cellular RNA
  • the method further comprises re-amplifying the plurality of amplicons.
  • the method further comprises calculating a ratio of the number of amplicons derived from the first target polynucleotide, and the number of amplicons derived from the second target polynucleotide.
  • the reverse transcribing can be conducted with a plurality of random sequence primers, target-specific primers, or polyT primers.
  • the reverse transcribing can be conducted by directly ligating the RNA to a plurality of double-stranded RNA/DNA or DNA/DNA adaptors, heating to remove one strand of the double-stranded adaptors, and conducting a reverse transcription reaction with primers that hybridize at least one adaptor sequence.
  • the reverse transcribing can be conducted according to an RNA-Seq procedure described in U.S. patent No. 8, 192,941 , which is incorporated by reference in its entirety.
  • At least one of the primers from the pairs of the target-specific primers includes a cleavable group.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprising conducting a multiplex nucleic acid amplification reaction on target polynucleotide sequences that represent RNA or DNA.
  • the target polynucleotide sequences that represent RNA include cDNA sequences derived from a whole transcriptome, or from a portion of a whole transcriptome.
  • the multiplex nucleic acid amplification reaction can be performed after any procedure that converts RNA to a plurality of cDNA.
  • the target polynucleotides e.g., plurality of DNA
  • the target polynucleotides can be produced in any reverse transcription reaction.
  • the target polynucleotides can be subjected to a multiplex nucleic acid amplification reaction to produce a plurality of amplicons having sequences derived from RNA.
  • amplification reaction uses a plurality of target-specific primer pairs.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprising conducting a multiplex nucleic acid amplification reaction with a plurality of target polynucleotides and a plurality of target-specific primer pairs in a single reaction mixture to produce a plurality of different amplicons.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for amplifying a plurality of target polynucleotides to produce a plurality of amplicons.
  • the plurality of amplicons can be generated by amplifying the plurality of target polynucleotides with a plurality of target-specific primer pairs in a single amplification mixture.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for amplifying one or more target polynucleotides within a sample containing a plurality of different target polynucleotides.
  • a plurality of different target polynucleotides for example at least 500, 1000, 2000, 2500, 5000, 7500, 10000, 15000, 20000, 25000, 50000, 100000, 200000, 400000 or 500000, are amplified within a single amplification reaction.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprising a reaction mixture.
  • the reaction mixture contains a single type of nucleic acid or a mixture of different types of nucleic acids.
  • the reaction mixture contains a plurality of nucleic acids having the same sequence or different sequences.
  • the reaction mixture contains single- stranded or double-stranded nucleic acids.
  • the sample contains RNA, cDNA or DNA.
  • the reaction mixture contains a plurality of nucleic acids that are naturally-occurring, recombinant or synthetically-prepared.
  • the reaction mixture contains nucleic acids that are isolated from a single fresh or archived cell, fresh cells, fresh tissues, or archived cells or tissues that are formalin-treated and/or embedded in paraffin or plastic, or cells or tissues that are formalin fixed paraffin-embedded (FFPE).
  • the reaction mixture contains nucleic acids that are isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, and viruses; cells; tissues; normal or diseased cells or tissues or organs, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen;
  • the reaction mixture contains nucleic acids that are unfragmented, or fragmented by mechanical force, chemical, enzyme or heat. In some embodiments, the reaction mixture contains nucleic acids that are depleted of, or enriched for, one or more nucleic acid species.
  • the reaction mixture includes polynucleotides derived from whole-genome amplification (WGA) of genomic DNA extracted from a single cell, multiple cells, whole tissue, blood or other bodily fluid.
  • WGA whole-genome amplification
  • the single cell is taken from a fertilized zygote, blastocyst or embryo, or is a fetal cell extracted from maternal tissue or blood, or is a tumor cell (e.g., a circulating tumor cell).
  • the disclosure relates generally to methods (as well as related compositions, systems, apparatus and kits) for performing multiplex amplification of target polynucleotides.
  • the method includes amplifying a plurality of target polynucleotides within a single reaction mixture including two or more target polynucleotides.
  • multiple different target polynucleotides of interest can be amplified in a single reaction mixture using one or more target-specific primers in the presence of a polymerase under amplification conditions to produce a plurality of different target amplicons.
  • the amplifying optionally includes contacting a nucleic acid molecule including at least one target polynucleotide with one or more target-specific primers and at least one polymerase under amplification conditions, thereby producing one or more target amplicons.
  • at least one of the target- specific primers includes a cleavable group.
  • the cleavable group is cleavable with uracil DNA glycosylase (UDG, also referred to as UNG), formamidopyrimidine DNA glycosylase (Fpg), or a FuPa reagent.
  • UDG uracil DNA glycosylase
  • Fpg formamidopyrimidine DNA glycosylase
  • FuPa reagent a FuPa reagent
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for amplifying a plurality of target polynucleotides comprising contacting a plurality of target polynucleotides with a plurality of target-specific primer pairs.
  • the plurality of target polynucleotides and the plurality of target-specific primer pairs are contacted together in a single reaction mixture.
  • at least one of the target- specific primers in the plurality of target-specific primer pairs includes a cleavable group.
  • the cleavable group is cleavable with uracil DNA glycosylase (UDG, also referred to as UNG), formamidopyrimidine DNA glycosylase (Fpg), or a FuPa reagent.
  • UDG uracil DNA glycosylase
  • Fpg formamidopyrimidine DNA glycosylase
  • FuPa reagent a FuPa reagent
  • the single reaction mixture comprises any one or any combination of a plurality of target polynucleotides, a plurality of target-specific primer pairs, at least one polymerase, and a plurality of nucleotides.
  • the plurality of nucleotides includes one or more non-labeled nucleotides or at least one nucleotide labeled with a detectable moiety.
  • the plurality of target polynucleotides are contacted with a plurality of target-specific primer pairs in a single reaction mixture, where the plurality of target-specific primer pairs contains 2- 100, or about 100-500, or about 500- 1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different target-specific primer pairs.
  • At least one of the target-specific primers in the plurality of target-specific primer pairs includes a cleavable group.
  • each target-specific primer in one or more pairs includes a cleavable group.
  • the cleavable group is cleavable with uracil DNA glycosylase (UDG, also referred to as UNG), formamidopyrimidine DNA glycosylase (Fpg), or a FuPa reagent.
  • the single reaction mixture further includes RNase H to degrade any RNA that may be present.
  • the single reaction mixture further comprises any one or any combination of: magnesium, manganese, formamide, DMSO, betaine, trehalose, spermidine, sulfones, sodium pyrophosphate, low molecular amides, and/or single-stranded binding proteins.
  • the single reaction mixture includes a plurality of target polynucleotides which comprise a plurality of single-stranded or double-stranded nucleic acids (e.g., cDNA).
  • the plurality of target polynucleotides and the plurality of target-specific primer pairs are contacted together, in a single reaction mixture, under nucleic acid hybridization conditions so that different target-specific primer pairs hybridize to different target polynucleotides.
  • At least one target-specific primer can hybridize under stringent conditions to at least some portion of a corresponding target polynucleotide sequence.
  • At least one target specific primer in a target specific primer pair can include at least one sequence that is substantially complementary or substantially identical to at least a portion of a corresponding target polynucleotide sequence or its complement. In some embodiments, at least a portion of each of the different target-specific primer pairs can be substantially complementary to a target sequence in a polynucleotide. [00218] In some embodiments, the plurality of target specific primer pairs includes at least a first and a second target specific primer pair that are different from each other. In some
  • the first target specific primer pair can be substantially non-complementary to another target sequence in the sample. In some embodiments, the first target specific primer pair can be substantially non-complementary to a second target polynucleotide sequence.
  • the different target-specific primer pairs hybridize to the different target polynucleotides to form a plurality of different primer/polynucleotide complexes.
  • each primer pair in the plurality of target-specific primer pairs is configured or designed to hybridize to a different target polynucleotide sequence of interest, optionally under high- stringency hybridization conditions.
  • a single pair of target-specific primers can hybridize to one target polynucleotide sequence.
  • only a single pair of target-specific primers hybridize to any given target polynucleotide.
  • more than one pair of target- specific primers hybridize to any given target polynucleotide.
  • each primer pair in the plurality of target-specific primer pairs is designed to hybridize to a different target polynucleotide sequence of interest. For example, if there are N different target polynucleotides sequences of interest, then the plurality of target-specific primer pairs will contain N different primer pairs.
  • the single reaction mixture can contain 2- 100, or about 100-500, or about 500-1 ,000, or about 1 ,000- 5,000, or about 5,000- 10,000, or about 10,000- 15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000- 100,000, or more different target-specific primer pairs. In some
  • the plurality of target-specific primer pairs includes about 20,000 different target-specific primer pairs.
  • At least one of the target-specific primer pairs has minimal cross-hybridization with any other pair of primers in the single reaction mixture.
  • the multiplex nucleic acid amplification reaction includes contacting a plurality of polynucleotides with a plurality of target-specific primer pairs, under suitable nucleic acid hybridization conditions.
  • the suitable hybridization conditions can include the plurality of polynucleotides and the plurality of target-specific primer pairs in an aqueous solution containing salts (e.g., sodium), magnesium, buffers, and/or formamide.
  • the hybridization can be conducted at a temperature that is about 5-30° C below the melting temperature.
  • hybridization conditions include high-stringency hybridization conditions.
  • high-stringency conditions can include any conditions whereby duplexes only form between strands (e.g., target polynucleotide and primers) having perfect one-to-one complementarity.
  • the disclosure relates generally to methods (as well as related compositions, systems, apparatus and kits) for performing nucleic acid synthesis of target polynucleotides.
  • the method includes synthesizing a plurality of target polynucleotides within a single reaction mixture including two or more target polynucleotides.
  • multiple different target polynucleotides of interest can be synthesized in a single reaction mixture using one or more target-specific primers in the presence of a catalyst (e.g., an enzyme that can catalyze the polymerization of nucleotides and nucleotides, such as dNTP's to promote extension of the one or more target-specific primers) under synthesis conditions to produce a plurality of different target amplicons.
  • a catalyst e.g., an enzyme that can catalyze the polymerization of nucleotides and nucleotides, such as dNTP's to promote extension of the one or more target-specific primers
  • the synthesizing optionally includes contacting a nucleic acid molecule including at least one target polynucleotide with one or more target-specific primers and at least one polymerase under synthesis conditions, thereby producing one or more target amplicons.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for conducting a primer extension reaction to amplify a plurality of different target polynucleotides in a the multiplex nucleic acid amplification reaction, where the plurality of different target polynucleotides are amplified substantially simultaneously in a single reaction mixture containing a plurality of different target specific primer pairs.
  • the multiplex nucleic acid amplification reaction of the present teachings can substantially simultaneously amplify at least a first target sequence and at least a second target sequence that are less than 50% complementary to each other.
  • the first target sequence and the second target sequence are substantially non-complementary to each other.
  • at least one of the target-specific primer pairs has minimal cross- hybridization with any other pair of primers in the single reaction mixture.
  • one or more of the methods of amplifying disclosed herein includes performing a target-specific amplification.
  • Performing the target-specific amplification can include amplifying one or more target polynucleotides using one or more exclusively target-specific primers, i.e., primers that do not include a shared or universal sequence motif with other target- specific primers or other target polynucleotides in the reaction mixture.
  • a target polynucleotide can be amplified using no more than a single pair of target-specific primers.
  • one or more of the target-specific primers are substantially complementary, or complementary, to at least some portion of the corresponding target polynucleotide, or to some portion of the nucleic acid molecule including the corresponding target polynucleotide.
  • one, some or all of the target-specific primers (or primer pairs) are substantially complementary, or complementary, to at least some portion of their corresponding target polynucleotide, or to some portion of the nucleic acid molecule including the corresponding target polynucleotide, across their (i.e., the target specific primers') entire length.
  • the plurality of target polynucleotides is amplified by conducting a primer extension reaction on the primer/polynucleotide complexes.
  • the primer extension reaction comprises incorporating one nucleotide onto a primer that is part of a primer/polynucleotide complex.
  • the nucleotide is incorporated onto the primer in a template-based manner, which can include complementary base pairing, including standard A-T or C-G base pairing, or optionally other forms of base-pairing interactions.
  • the primer extension reaction includes successively
  • nucleotides onto a primer that is part of the primer/polynucleotide complex.
  • the primer extension reaction includes the target-specific primer pairs, the target polynucleotides, at least one polymerase, and a plurality of nucleotides.
  • the polymerase comprises a DNA-dependent DNA polymerase.
  • the polymerase exhibits RNA-dependent DNA polymerase activity.
  • the plurality of nucleotides comprises unlabeled nucleotides, or at least one labeled nucleotide.
  • the primer extension reaction can be conducted in a single reaction mixture.
  • the primer extension reaction produces at least one amplicon or a plurality of amplicons.
  • the primer extension reaction produces at least two different amplicons that include sequences that are less than 50% complementary to each other.
  • the primer extension reaction produces at least on amplicon containing a sequence having at least a portion of a target polynucleotide sequence.
  • each amplicon also includes the sequence of at least one primer of a target-specific primer pair.
  • the primer extension reaction produces at least one amplicon containing a primer- derived sequence on at least one end of the amplicon.
  • the primer-derived sequence on the at least one end of the plurality of amplicons includes at least one cleavable group.
  • the cleavable group comprises a modified nucleoside, nucleotide or nucleobase.
  • the cleavable group comprises: uracil, uridine, inosine, or 7,8-dihydro-8-oxoguanine (8- oxoG) nucleobases.
  • the cleavable group is cleavable with uracil DNA glycosylase (UDG, also referred to as UNG), formamidopyrimidine DNA glycosylase (Fpg), or a FuPa reagent.
  • UDG uracil DNA glycosylase
  • Fpg formamidopyrimidine DNA glycosylase
  • FuPa reagent a FuPa reagent
  • the methods, compositions, systems, apparatuses and kits for amplifying one or more target polynucleotides in a single amplification reaction include at least two amplified target polynucleotides that are not complementary along their length to a different amplified target polynucleotide in the single reaction mixture.
  • the methods, compositions, systems, apparatuses and kits for amplifying one or more target amplicons include at least two target amplicons that are not complementary along their length to a different target amplicon in the single reaction mixture.
  • the methods, compositions, systems, apparatuses and kits for amplifying a plurality of target polynucleotides in a single amplification reaction include a plurality of target polynucleotides that are not complementary along their length to a different amplified target polynucleotide in the single reaction mixture.
  • the amplification conditions can produce at least two different target amplicons that are less than 50 % complementary to each other along their length.
  • at least one target amplicon is substantially non-complementary, or non- complementary, along its length to another target amplicon in the reaction mixture.
  • a target amplicon can be substantially non-complementary, or non-complementary, along its length to any one or more target polynucleotides in the sample that do not correspond to the target amplicon nucleic acid sequence.
  • the at least two different target amplicons are not complementary along their length to any other target amplicon in the reaction mixture.
  • the at least two different target amplicons are not complementary to another nucleic acid molecule in the amplification reaction mixture. In another embodiment, the at least two different target amplicons are not complementary along their length to any target-specific primer in the amplification reaction mixture.
  • the multiplex nucleic acid amplification reactions produce at least one amplicon containing a sequence having at least a portion of a target polynucleotide sequence, where the target polynucleotide contains wild-type or mutant sequences, fusion sequences, spliced sequences, unspliced sequences, splice isoforms, allelic variant sequences, single nucleotide variant sequences, or cell or tissue-specific expressed sequences.
  • the multiplex nucleic acid amplification reactions produce a first amplicon having at least a portion of a first target polynucleotide sequence, and a second amplicon having at least a portion of a second target polynucleotide sequence.
  • the sequences of the first and the second amplicons are substantially non- complementary to each other.
  • the multiplex nucleic acid amplification reactions produce at least two different amplicons that include sequences that are less than 50% complementary to each other.
  • the multiplex nucleic acid amplification reactions produce at least one amplicon containing a sequence having at least a portion of a target polynucleotide sequence, and the at least one amplicon also includes the sequence of at least one primer (a primer-derived sequence) of a target-specific primer pair. In some embodiments, the multiplex nucleic acid amplification reactions produce at least one amplicon containing a primer- derived sequence on at least one end of the amplicon.
  • the primer-derived sequence on the at least one end of the plurality of amplicons includes at least one cleavable group.
  • the cleavable group comprises a modified nucleoside, nucleotide or nucleobase.
  • the cleavable group comprises: uracil, uridine, inosine, or 7,8-dihydro-8-oxoguanine (8-oxoG) nucleobases.
  • the cleavable group is cleavable with uracil DNA glycosylase (UDG, also referred to as UNG), formamidopyrimidine DNA glycosylase (Fpg), or a FuPa reagent.
  • a nucleic acid molecule in a sample, an amplified target polynucleotide, an adapter or a target-specific primer includes a 5' end and a 3' end.
  • the 5' end can include a free 5' phosphate group or its equivalent; the 3' end can include a free 3' hydroxyl group or its equivalent.
  • the ends of an amplified target polynucleotide can be non-complementary to the ends of another amplified target polynucleotide in the reaction mixture.
  • the 3' end can include about 30 nucleotides, or about 15 nucleotides, or about 10 nucleotides, or about 8 nucleotides from the 3' hydroxyl group.
  • the 5' end can include about 30 nucleotides, or about 15 nucleotides, about 10 nucleotides, or about 8 nucleotides from the 5' phosphate group.
  • any one amplified target polynucleotide having a 3' end and a 5 'end can be substantially non-complementary, or non-complementary, to any portion of any other amplified target polynucleotide in the reaction mixture.
  • the amplicons can be phosphorylated.
  • phosphorylation of the amplicons can be conducted using a FuP reagent.
  • the FuP reagent can include a DNA polymerase, a DNA ligase, at least one uracil cleaving or modifying enzyme, and/or a storage buffer.
  • the FuP reagent can further include at least one of the following: a preservative and/or a detergent.
  • phosphorylation of the amplicons can be conducted using a
  • the FuPa reagent can include a DNA polymerase, at least one uracil cleaving or modifying enzyme, an antibody and/or a storage buffer. In some embodiments, the FuPa reagent can further include at least one of the following: a preservative and/or a detergent. In some embodiments, the antibody is provided to inhibit the DNA polymerase and 3 '-5' exonuc lease activities at ambient temperature.
  • the disclosure relates generally to methods for performing amplification of a target polynucleotide or target amplicon (as well as related compositions, systems, apparatuses and kits using the disclosed methods) and can include a digestion step.
  • the methods also include a ligating step, and the digestion step is performed prior to the ligating step.
  • an amplified target polynucleotide can be partially digested prior to performing the ligation step.
  • an amplified target polynucleotide can be digested by enzymatic, thermal, chemical, or other suitable means.
  • an amplified target polynucleotide can be digested prior to the ligating to produce a blunt-end or sticky-ended amplified target polynucleotide.
  • a blunt-ended amplified target polynucleotide can include a 5' phosphate group at the 5' end of the digested amplified target polynucleotide.
  • a blunt-ended amplified target amplicon can include a 5' phosphate group at the 5' end of the digested amplified target amplicon.
  • a target-specific primer, adapter, target amplicon, amplified target polynucleotide or nucleic acid molecule can include one or more cleavable moieties, also referred to herein as cleavable groups.
  • the methods can further include cleaving at least one cleavable group of the target-specific primer, adapter, target amplicon, amplified target polynucleotide or nucleic acid molecule.
  • the cleaving can be performed before or after any of the other steps of the disclosed methods.
  • the cleavage step occurs after the amplifying and prior to a ligating step.
  • the cleaving includes cleaving at least one amplified target polynucleotide or target amplicon prior to the ligating.
  • the cleaving can include cleaving at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more of the target-specific primers present in the single reaction mixture.
  • the cleavable moiety can be present as a modified nucleotide, nucleoside or nucleobase.
  • the cleavable moiety can include a nucleobase not naturally occurring in the target sequence of interest.
  • uracil or uridine can be incorporated into a DNA as a cleavable group.
  • a uracil DNA glycosylase can be used to cleave the cleavable group from a nucleic acid including uracil.
  • inosine can be incorporated into a DNA-based nucleic acid as a cleavable group.
  • EndoV can be used to cleave near the inosine residue and a further enzyme, such as Klenow, can be used to create blunt-ended fragments capable of blunt-ended ligation.
  • the enzyme hAAG can be used to cleave inosine residues from a nucleic acid creating abasic sites that can be further processed by one or more enzymes, such as Klenow, to create blunt-ended fragments capable of blunt-ended ligation.
  • the cleavable moiety can include an enzymatic restriction recognition sequence, such as the Hind III, Spel, Hpal or DpnII, located within the nucleic acid sequence of the target polynucleotide, amplicon, or target-specific primer.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprising conducting a multiplex nucleic acid amplification reaction and further comprising a cleaving step.
  • the plurality of amplicons comprises a primer-derived sequence on at least one end of the amplicon, and the primer-derived sequence contains a cleavable group.
  • the cleavable group comprises: uracil, uridine, inosine, or 7,8-dihydro- 8-oxoguanine (8-oxoG) nucleobases.
  • the primer-derived sequence (which contains a cleavable group) on at least one end of the plurality of amplicons can be cleaved with a cleaving agent.
  • the cleaving agent comprises uracil DNA glycosylase (UDG, also referred to as UNG), formamidopyrimidine DNA glycosylase (Fpg), or a FuPa reagent.
  • UDG uracil DNA glycosylase
  • Fpg formamidopyrimidine DNA glycosylase
  • FuPa reagent uracil DNA glycosylase
  • EndoV can be used to cleave near the inosine residue and a further enzyme such as Klenow can be used to create blunt- ended fragments capable of blunt-ended ligation.
  • the enzyme hAAG can be used to cleave inosine residues from a nucleic acid creating abasic sites that can be further processed by one or more enzymes such as Klenow to create blunt-ended fragments capable of blunt-ended ligation (see for example U.S patent Nos. 8,673,560, 8,728,728 and 8,728,736 which are incorporated herein in their entireties).
  • the cleaving the cleavable group produces a population of cleaved amplified nucleic acids. In some embodiments, the cleaving the cleavable group produces a plurality of cleaved amplified nucleic acids having at least one blunt end or at least one overhang end.
  • one or more target-specific primers, target polynucleotides, target amplicons or adapters can include a cleavable moiety. Furthermore, a cleavable moiety can be located at a nucleotide position at, or near, the terminus of a target-specific primer, target polynucleotide, target amplicon or adapter.
  • a cleavable moiety can be located within 15, within 10, within 8, within 5, within 4, within 3, nucleotides of the 3' end or the 5' end of the nucleic acid having the cleavable moiety. In some embodiments, a cleavable moiety can be located at or near a central nucleotide in a target-specific primer. In some embodiments, one or more cleavable moieties can be present in a target-specific primer, target amplicon or adapter. In some embodiments, cleavage of one or more cleavable moiety in a target-specific primer, target amplicon or adapter can generate a plurality of nucleic acid fragments with differing melting temperatures.
  • the placement of one or more cleavable moieties in a target- specific primer, target amplicon or adapter can be regulated or manipulated by determining a melting temperature for each nucleic acid fragment, after cleavage of the cleavable moiety.
  • the cleavable moiety can include a cleavable group such as uracil or uridine.
  • the cleavable group can include an inosine moiety.
  • at least 25% of the target-specific primers or target amplicons can include at least one cleavable group.
  • at least 50% of the target-specific primers or target amplicons can include at least one cleavable group.
  • At least 75% of the target-specific primers can include at least one cleavable group. In some embodiments, at least 90% of the target-specific primers can include at least one cleavable group. In some embodiments, at least 95% of the target-specific primers can include at least one cleavable group. In some embodiments, at least 98% of the target-specific primers can include at least one cleavable group. In some embodiments, each target- specific primer includes at least one cleavable group. In some embodiments, one target specific primer from a primer pair includes at least one cleavable group. In another embodiment, each target- specific primer from each primer pair can include at least one cleavable group.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprising conducting a multiplex nucleic acid amplification reaction and further comprising ligation of an adapter.
  • at least one end of the cleaved amplified nucleic acid can be ligated to at least one adaptor to produce at least one adapter-ligated amplified nucleic acid.
  • the cleaved amplified nucleic acid can have at least one end having a substantially blunt end, which can be created by cleaving the cleavable group, optionally followed by digestion of overhangs, end-polishing or some other process whereby a blunt end is created.
  • At least one end of one or more adaptors includes a blunt end. In some embodiments, at least one end of the cleaved amplified nucleic acid can be ligated to at least one adaptor in a blunt-end ligation reaction.
  • the cleaved amplified nucleic acid can have at least one end having an overhang end, which can be created by cleaving the cleavable group, or via restriction digestion, terminal tailing, exonuclease digestion or endonuclease digestion or via other suitable means.
  • at least one end of one or more adaptors includes an overhang end. In some embodiments, at least one end of the cleaved amplified nucleic acid can be ligated to at least one adaptor in an overhang-end ligation reaction.
  • the first end of the cleaved amplified nucleic acid can be ligated to a first adaptor, and the second end of the cleaved amplified nucleic acid can be ligated to a second adaptor, where the first and the second adaptor contain the same sequence or different sequences.
  • the adaptor includes an amplification primer binding site, a sequencing primer binding site, a universal sequence and/or a unique identifier sequence (e.g., barcode sequence).
  • the amplification primer binding site on the adapter-ligated amplified nucleic acid can hybridize to an amplification primer.
  • the sequencing primer binding site on the adapter-ligated amplified nucleic acid can hybridize to a sequencing primer.
  • the unique identifier sequence on the adapter-ligated amplified nucleic acid can hybridize to an amplification primer or a sequencing primer.
  • the disclosed methods can include ligating at least one adapter, where the at least one adapter includes a nucleic acid sequence that is substantially non-complementary (or non-complementary) under stringent hybridizing conditions to the target polynucleotide, to the amplified target sequence, to the target amplicon and/or to any other nucleic acid molecule in the reaction mixture.
  • the at least one adapter includes a single-stranded linear oligonucleotide.
  • the at least one adapter includes a double-stranded adapter.
  • the at least one adapter includes a plurality of different single-stranded and/or double-stranded adapters in the same reaction mixture.
  • the disclosed methods can include ligating at least one adapter to at least one of the amplified target polynucleotides to produce one or more adapter-ligated amplified target polynucleotides.
  • the disclosed methods can include ligating at least one adapter to at least one of the target amplicons to produce one or more adapter-ligated amplicons.
  • the ligating can include ligating an adapter to the 5' end of the at least one amplified target polynucleotide or target amplicon.
  • the ligating can include ligating an adapter to the 3 ' end of the at least one amplified target polynucleotide or target amplicon. In some embodiments, the ligating can include ligating an adapter to the 5' end of the at least one amplified target polynucleotide or target amplicon and ligating an adapter to the 3' end of the at least amplified target polynucleotide or target amplicon. In some embodiments, the ligating can include ligating the same adapter to the 5' end and the 3' end of the amplified target polynucleotide or target amplicon.
  • the ligating can include ligating different adapters to the 5' end and the 3' end of the amplified target polynucleotide or target amplicon. In some embodiments, ligation of an adapter to the 3' end and ligation of an adapter to the 5' end of the amplified target sequence or target amplicon can occur simultaneously. In some embodiments, ligation of an adapter at the 3' end and ligation of an adapter at the 5' end can occur sequentially.
  • the methods disclosed herein can include contacting an amplified target polynucleotide or target amplicon having a 3' end and a 5'end with a ligation reaction mixture.
  • a ligation reaction mixture can include one or more adapters and a ligase to produce at least one adapter-ligated amplified target polynucleotide.
  • the ligation reaction can include a DNA ligase and at least one pair of adapters, each of the pair of adapters including a different, and non-complementary, nucleic acid sequence to the other adapter in the pair of adapters.
  • none of the adapters in the ligation mixture, prior to the ligating includes a target-specific sequence that is complementary along its length to one or more of the amplified target polynucleotides or target amplicons. In some embodiments, none of the adapters in the ligation mixture, prior to ligating, includes a sequence that is substantially complementary, or
  • the one or more adapters are not complementary or identical to the 5' end of the plurality of target-specific primers. In another embodiment, the one or more adapters do not include a nucleic acid sequence that is complementary or identical to the terminal 10 nucleotides at the 5' end of the plurality of target-specific primers.
  • the 3' end of an amplified target sequence or target amplicon includes about the terminal 30 nucleotides, and in some instances refers to about the terminal 15 nucleotides, or about the terminal 10 nucleotides from the 3' end of an amplified target polynucleotide or target amplicon.
  • the 5' end of an amplified target polynucleotide or target amplicon includes about the terminal 30 nucleotides, and in some instances refers to about the terminal 15 nucleotides, or about the terminal 10 nucleotides from the 5' end of an amplified target polynucleotide or target amplicon.
  • the ligation reaction can include one or more adapters that further include a barcode, tag, or universal priming sequence.
  • the ligation reaction can include one or more adapters that are phosphorylated at the 5' end.
  • none of the adapters in the ligation mixture, prior to ligating can hybridize under high stringency, to some portion of an amplified target polynucleotide or target amplicon.
  • ligating can include direct ligation of one or more adapters to one or more amplified target polynucleotide or target amplicons.
  • the ligation reaction can include a single-stranded or double-stranded adapter.
  • ligating can include performing a blunt-ended ligation.
  • the process of blunt-ended ligation can include ligating a blunt-end double-stranded amplified target polynucleotide to a blunt-ended double- stranded adapter.
  • ligating can include performing a sticky-ended ligation.
  • the process of sticky-ended ligation can include ligating a sticky-end double-stranded amplified target polynucleotide to a blunt-ended double-stranded adapter.
  • the ligating can include a single-stranded adapter.
  • the process of direct single-stranded ligation can include ligating a single-stranded amplified target polynucleotide or target amplicon to a single-stranded adapter.
  • the ligated single-stranded adapter can be used as a template in the presence of an appropriate primer (e.g., a universal primer) to extend the appropriate primer in a template dependent manner, using the single-stranded ligation product as the template.
  • the adapter can include a double-stranded adapter that contains a partially single- stranded region, such as a single-stranded overhang.
  • the partially-single stranded region can include an "A" or "T” overhang, or a "G" or "C” overhang.
  • the ligating does not include one or more additional oligonucleotide adapters (i.e., bridging or patch oligonucleotides) prior to ligating an adapter to an amplified target polynucleotide or target amplicon.
  • additional oligonucleotide adapters i.e., bridging or patch oligonucleotides
  • the disclosed methods can further include ligating one or more adapters including a universal priming sequence to the amplified product formed as a result of the target- specific primer amplification.
  • the universal priming sequence can be used in any applicable downstream process, such as universal amplification, nucleic acid enrichment, clonal amplification, bridge PCR, or nucleic acid sequencing.
  • one or more adapters can be ligated to an amplified target polynucleotide.
  • an adapter that is ligated to an amplified target polynucleotide is susceptible to exonuclease digestion.
  • an adapter susceptible to exonuclease digestion can be ligated to the 3' end of an amplified target polynucleotide.
  • an adapter ligated to an amplified target polynucleotide does not include a protecting group.
  • the one or more adapters do not include a protecting group that can prevent nucleic acid degradation or digestion under degrading or digesting conditions. For example, subsequent enzymatic digestion of the adapter- ligated amplified target polynucleotide in the presence of nucleic acids that do not include a protecting group, offers a means for selective digestion of the unprotected nucleic acids.
  • the one or more adapters can further include a DNA barcode or tag for any suitable method used in downstream processing.
  • the disclosure relates generally to methods, (as well as compositions, systems, apparatuses and kits) for performing multiplex nucleic acid amplification.
  • the methods include amplifying one or more target polynucleotides using one or more target-specific primers in the presence of polymerase under amplification conditions to produce an amplified target polynucleotide and, ligating an adapter to the amplified target polynucleotide.
  • the method can include reamplifying an adapter-ligated amplified target polynucleotide to form a reamplified adapter-ligated amplified target polynucleotide.
  • a reamplified adapter-ligated amplified target polynucleotide can be produced using no more than two rounds of target-specific selection.
  • a first target-specific primer can be used under amplification conditions to produce a first amplified target polynucleotide (e.g., hybridizing the first target-specific primer to a target polynucleotide under amplification conditions and extending the hybridized first target-specific primer in a template dependent manner).
  • a second target-specific primer can be used that is specific for a region (e.g., the 3' or 5' end) of the first amplified target polynucleotide, and the second target specific primer can be used under amplification conditions to produce a second amplified target polynucleotide using no more than two rounds of target-specific amplification.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for avoiding or reducing the formation of amplification artifacts (for example primer-dimers and non-specific priming) during selective amplification of one or more target polynucleotides in a population of nucleic acid molecules.
  • amplification artifacts for example primer-dimers and non-specific priming
  • the disclosure relates generally to the synthesis of multiple target polynucleotides from a population of nucleic acid molecules.
  • the method comprises hybridizing one or more target-specific primer pairs to the target polynucleotide, extending a first primer of the primer pair, denaturing the extended first primer product from the population of nucleic acid molecules, hybridizing to the extended first primer product the second primer of the primer pair, extending the second primer to form a double stranded product, and digesting the target-specific primer pair away from the double stranded product to generate a plurality of amplified target polynucleotides.
  • the amplified target polynucleotide can be denatured to form single stranded
  • the digesting step includes digesting one or more of the target-specific primers from the amplified target polynucleotides to create blunt-ended or sticky-end polynucleotides.
  • the double-stranded or single-stranded amplified target polynucleotides can be ligated to one or more adapters.
  • the one or more adapters can include one or more DNA barcodes or tagging sequences.
  • the amplified target polynucleotides once ligated to an adapter can undergo a nick translation reaction and/or further amplification to generate a library of adapter-ligated amplified target polynucleotides.
  • the amplified target polynucleotides can undergo a further amplification step, for example using a nucleic acid sequence within the adapter that can act as a universal priming sequence to allow further amplification of the single stranded adapter-ligated polynucleotide with an appropriate primer, thereby generating a library of adapter-ligated amplified target polynucleotides.
  • the target-specific primer pairs when hybridized to a target polynucleotide and amplified as outlined herein can generate a library of adapter-ligated amplified target polynucleotides that are from 100 to 1 ,000 base pairs in length, 150 to 800 base pairs in length, or 200 to 700 base pairs in length.
  • the multiplex nucleic acid amplification reaction comprises: contacting a first plurality of target polynucleotides with a first plurality of target-specific primer pairs in a first reaction mixture, and contacting a second plurality of target polynucleotides with a second plurality of target-specific primer pairs in a second reaction mixture.
  • the first and the second reaction mixtures are contained in separate reaction vessels.
  • the first and the second reaction mixtures can undergo separate primer extension reactions to produce a first and a second plurality of amplicons.
  • the first and a second plurality of amplicons can be pooled.
  • the pooled plurality of amplicons can be characterized.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for characterizing the plurality of amplicons, using any procedure, including: hybridizing (e.g., subtractive hybridization or microarray analysis), sequencing, detecting or determining the abundance of one or more sequences of interest.
  • the subtractive hybridization reaction can be conducted by hybridizing the plurality of amplicons with a nucleic acid having a reference sequence.
  • the microarray analysis can be conducted by hybridizing the plurality of amplicons with one or more capture probes on a microarray. Any procedure that can be used to characterize an amplicon can also be used to characterize a plurality of adapter-li gated amplified nucleic acids.
  • the number of amplicons that contain sequences derived from a target polynucleotide can be counted and used to determine the complexity and abundance of RNA sequences of interest that are present in the sample, or can be used to calculate ratios of abundances of two different RNA sequences of interest.
  • the sequence information can be used for additional downstream analyses, including: detecting the presence of one or more RNA sequences-of-interest in the sample; detecting wild-type sequences; detecting mutant sequences; detecting gene fusion sequences; detecting splice isoforms; detecting differences in abundance levels of one or more RNA sequences compared to wild type levels; identifying mutant RNA sequences; identifying allelic variant RNA sequences; identifying single nucleotide variant RNA sequences; determining the sequence of a splice junction; determining the terminal 5' or 3 ' boundary of an RNA; or determining the abundance, or relative abundance, of an RNA; gene expression profiling; differential gene expression; or preparation of arrays by immobilizing the plurality of amplicons to a support.
  • the detecting comprises hybridizing a nucleic acid probe with the plurality of amplicons. In some embodiments, the detecting further comprises detecting the presence of a complex formed by hybridization of the nucleic acid probe with at least one amplicon.
  • the nucleic acid probe includes a detectable moiety.
  • the detecting comprises re-amplifying the plurality of amplicons.
  • the re-amplifying comprises hybridizing an amplification primer to the amplification primer binding site on the amplicon or the adapter-ligated amplified nucleic acid, and conducting a nucleic acid amplification reaction.
  • the number of amplicons or adaptor-ligated amplified nucleic acids that contain sequences derived from a target polynucleotide can be quantified by hybridization in a microarray analysis, or by sequencing.
  • the sequences of the different amplicons e.g., a sequencing read
  • the number of a sequencing read from a particular amplicon that aligns to a particular reference sequence can be used to generate a read count of that amplicon sequence.
  • a read count of different amplicon sequences can be generated.
  • the read counts of two or more amplicon sequences can be converted to relative abundance, or can be used to calculate a ratio of two different amplicon sequences within the same reaction mixtures, or within different reaction mixtures.
  • the sequencing data is counted and tallied.
  • a plurality of amplicons or adaptor-ligated amplified nucleic acids is generated according to the present teachings. At least some, or all, of the plurality of amplicons or adaptor- ligated amplified nucleic acids are sequenced to generate a plurality of sequence reads.
  • each sequence read represents a target polynucleotide sequence (or a portion thereof) which is contained in the amplicons or adaptor-ligated amplified nucleic acids.
  • some or all of the plurality of amplicons or adaptor-ligated amplified nucleic acids are sequenced.
  • the sequence reads are compared and/or aligned with sequences of interest in a reference list.
  • polynucleotides are counted using a software program, for example using an RNA plugin from Torrent Suite (Torrent SuiteTM Software, version 4.0.2, user interface guide, document revision November 2013 Rev. A).
  • a software program for example using an RNA plugin from Torrent Suite (Torrent SuiteTM Software, version 4.0.2, user interface guide, document revision November 2013 Rev. A).
  • sequence reads can be aligned to one or more reference sequences and compared to a reference list to determine a read count for one or more sequences of interest.
  • a sequence variant includes a sequencing read that differs from one or more reference sequences.
  • the sequencing reads that differ from the reference sequence e.g., a sequence variant
  • the reads that align to the reference sequences that do not correspond to the one or more sequences of interest can be retained or discarded.
  • 2013/0073214 and 2013/0268207 can be used to identify sequencing read variants.
  • the reference list contains nucleic acid sequences of interest that are associated with a healthy cell, or any disease or cancer.
  • reconstruction of a longer target sequence can be achieved by assembling two or more sequence reads.
  • Sequence assembly includes alignment of two or more sequence reads against a reference sequence of interest, or alignment of overlapping sequences in two or more sequence reads.
  • the need for sequence assembly is substantially reduced or obviated, because a single pair of target-specific primers is configured to generate a single sequence for each target polynucleotide.
  • a sequence read assembly is not performed.
  • the number of a sequence read that aligns with a particular sequence of interest is counted. For example, the number of a first sequence read that aligns with a first sequence of interest is counted, and the number of a second sequence read that aligns with a second sequence of interest is counted.
  • the sequence reads are counted. The count of the number of first sequence reads is tallied, and the count of the number of second sequence reads is also tallied.
  • the total number of tallied first and second reads are compared to each other, and the comparison can be expressed as a ratio or percentage.
  • the relative abundance of a first transcript and a second transcript can be obtained by comparing the total number of tallied first and second reads.
  • the sequence reads have perfect or imperfect alignment with their respective sequence of interest in the reference lists.
  • the sequence reads have one or more mutations that result in imperfect alignment with the reference sequence of interest.
  • at least one sequence read includes mutations comprising one or more deletions, insertions, or substitutions of one or more nucleotides, inversions, rearrangements, fusions, truncations, and/or variant or abnormal splice junction sequences.
  • sequence coverage includes the number of reads that map to a location of a reference genome.
  • sequence coverage is used to calculate percentage of an allele (e.g., an allelic variant or a mutant allele). For example, calculating a percentage of an allele includes the count of an allele divided by the coverage at that locus in the reference genome.
  • the percentage of a first and a second allele can be compared and expressed as a ratio or percentage. In some embodiments, the percentage of a normal allele and a cancer allele can be compared.
  • the percentage of the cancer allele decreases while the percentage of the normal allele increases.
  • the percentage of the cancer allele increases while the percentage of the normal allele decreases.
  • the frequency of at least one sequence variant can be determined.
  • low frequency sequence variants includes sequence variants that occur in fewer than about 60%, or about 50%, or about 40%, or lower percent occurrence of the sequencing reads.
  • the first and second sequencing reads may correspond to different amplicon sequences generated by common primer or pair of primers.
  • the first and second reads may be distinguished by a single nucleotide polymorphism, an indel, the presence or absence of a gene fusion, or the like.
  • relative abundance of the first read can be calculated by comparing the read count for the first read with the total count of reads associated with the common primer.
  • the relative abundance of a gene fusion amplicon can be determined by comparing to the read count for the gene fusion amplicon to the total number of reads for amplicons associated with the first primer from the first gene and the second primer from the second gene. In some embodiments, the read count for an amplicon with a large deletion or gene fusion can be compared to the average of read counts for reads associated with the first primer and reads associated with the second primer.
  • the relative abundance of the same transcript within samples taken before and after treatment of interest can be compared and determined. In some embodiments, relative abundances of different transcripts can be compared.
  • the detecting comprises sequencing the plurality of amplicons.
  • the identity of the sequences of the plurality of amplicons can be determined.
  • the sequencing procedure comprises hybridizing a sequencing primer to the sequencing primer binding site on the amplicon or the adapter-ligated amplified nucleic acid, and conducting a sequencing reaction.
  • the sequencing comprises a massively parallel sequencing procedure, or a gel electrophoresis procedure.
  • the detecting comprises determining the abundance of an
  • RNA sequence of interest by quantifying the number of the amplicons containing sequences derived from RNA or derived from the plurality of target polynucleotides.
  • the abundance of a first RNA sequence of interest can be determined by quantifying the number of the amplicons containing sequences derived from a first RNA or derived from a first target
  • the abundance of a second RNA sequence of interest can be determined by quantifying the number of the amplicons containing sequences derived from a second RNA or derived from a second target polynucleotide.
  • the quantifying includes counting the number of amplicons containing sequences derived from RNA or derived from the plurality of target polynucleotides.
  • the detecting further comprises comparing the abundance of the amplicons containing sequences derived from a first RNA or derived from a first target polynucleotide, with the abundance of the amplicons containing sequences derived from a second RNA or derived from a second target polynucleotide.
  • the detecting comprises determining differences in abundance levels of one or more RNA sequences compared to wild type or normal levels, by quantifying the number of the amplicons containing sequences derived from RNA or derived from the plurality of target polynucleotides.
  • normal levels of an RNA sequence of interest can be determined by quantifying the number of the amplicons containing sequences derived from the RNA from a first sample (e.g., a sample of normal cells).
  • different levels of the same RNA sequence of interest can be determined by quantifying the number of the amplicons containing sequences derived from the RNA from a second sample (e.g., a sample of abnormal or diseased cells).
  • the detecting further comprises comparing the levels
  • the abnormal cells include diseased cells, tumor cells, cells challenged with nutrient starvation, or cells challenged with a chemical compound or physical stress.
  • determining the difference in abundance levels of one or more RNA sequences in a sample is used to detect changes in the expression level of a gene in a first cell (or in a first plurality of cells) compared to the expression level of the same gene in a second cell (or in a second plurality of cells).
  • the expression level of the gene increases or decreases.
  • the detecting comprises determining a ratio of the number of amplicons containing a sequence derived from a first RNA, and the number of amplicons containing a sequence derived from a second RNA.
  • determining the ratio includes counting the number of amplicons containing a sequence derived from the first RNA and the number of amplicons containing a sequence derived from the second RNA.
  • the detecting comprises determining a ratio of the number of amplicons containing a sequence derived from a first target polynucleotide, and the number of amplicons containing a sequence derived from a second target polynucleotide.
  • determining the ratio includes counting the number of amplicons containing a sequence derived from the first target polynucleotide and the number of amplicons containing a sequence derived from the second target polynucleotide.
  • the number of amplicons or adaptor-ligated amplified nucleic acids that contain sequences derived from a target polynucleotide can be counted and tallied, and used to determine the amount of amount of one or more RNA transcripts of interest that are present in any cell or tissue.
  • the cell or tissue is subjected to an extraction procedure to produce an RNA sample. Some or all of the RNA in the sample is converted to a plurality of cDNA using any suitable procedure.
  • the plurality of cDNA can be generated by conducting a reverse transcription reaction, comprising: contacting some or all of the RNA with primers, at least one enzyme having RNA-dependent DNA polymerase activity, and a plurality of nucleotides, under conditions suitable for reverse transcription.
  • the primers can be random-sequence primers, polyT primers, or target-specific primers.
  • the RNA- dependent DNA polymerase enzyme can be a reverse transcriptase.
  • the plurality of cDNA can be generated by ligating some or all of the RNA to double-stranded adaptors to produce ligation products having single-stranded RNA joined, at one end or at both ends, to one strand of a double-stranded adaptor.
  • the double-stranded adaptors comprise RNA/DNA or DNA/DNA.
  • the ligation products can be heated to remove one of the strands of the adaptors, thereby generating a single-stranded RNA template.
  • some or all of the single- stranded RNA templates are converted to a plurality of cDNA using a reverse transcription procedure.
  • the plurality of cDNA contains different sequences derived from different RNA in the sample.
  • the plurality of cDNA contains at least a first cDNA derived from a first RNA transcript in the sample, and a second cDNA derived from a second RNA transcript in the sample.
  • the sequence complexity and amounts of different cDNAs reflects the sequence complexity and amounts of different RNA sequences found in the RNA sample from which the cDNA was derived.
  • the amount of the first cDNA relative to the amount of the second cDNA is similar to the relative amounts of the first and second RNA transcripts in the sample.
  • the plurality of cDNA is contacted with a plurality of target- specific primer pairs, under conditions suitable to hybridize at least one of the target-specific primer pairs to at least one cDNA to form at least one nucleic acid duplex.
  • each of the plurality of target-specific primer pairs hybridizes to a different target cDNA sequence.
  • a single pair of target-specific primers will hybridize to any give target cDNA sequence.
  • at least one cDNA molecule that is generated from the RNA sample contains a target sequence.
  • a single pair of target-specific primers will hybridize to a cDNA sequence and mediate amplification to produce amplicons that represent a transcript of interest.
  • the plurality of cDNA may, or may not, contain all target cDNA sequences.
  • a primer extension reaction is conducted on the nucleic acid duplexes, in a template-dependent fashion, to form a plurality of amplicons.
  • each amplicon contains a sequence derived from an RNA in the sample.
  • the plurality of amplicons contains an amount of different sequences derived from that reflects the sequence complexity and relative amounts of different polynucleotide sequences found in the sample from which the plurality of amplicons was derived.
  • the amount of the first amplicon relative to the amount of the second amplicon is similar to the relative amounts of the first and second polynucleotides in the sample.
  • the plurality of amplicons contains different sequences derived from different sources within a mixed sample.
  • the plurality of amplicons can contain at least a first amplicon derived from a normal cell in the sample, and the plurality of amplicons contains a second amplicon derived from a tumor cell in the sample.
  • the plurality of amplicons can contain at least a first amplicon derived from maternal polynucleotide in the sample, and the plurality of amplicons can contain a second amplicon derived from a fetal polynucleotide in the sample.
  • the plurality of amplicons can contain at least a first amplicon derived from a first chromosome, and the plurality of amplicons can contain a second amplicon derived from a second chromosome in the sample.
  • the number of amplicons containing sequence corresponding to, or derived from, the first and second amplicons (or first and second template polynucleotides) is counted and tallied for each of the first and second amplicons. For example, the number of amplicons containing a first target sequence of interest can be counted and tallied to obtain a first number.
  • the number of amplicons containing a second target sequence of interest are counted and tallied to obtain a second number.
  • the resulting counts, tallies and/or numbers e.g., first and second numbers
  • the resulting counts, tallies or numbers can be used to estimate the relative abundance of the first and second target sequences of interest within the sample.
  • the resulting counts, tallies or numbers e.g., first and second numbers
  • the resulting counts, tallies or numbers can be used to determine the presence of a chromosomal aneuploidy, or a copy number change, or the percentage of polynucleotides including a variant or substitution at a given position.
  • the resulting counts, tallies or numbers can be used to determine the proportion of minor DNA sequences present amongst a majority.
  • the resulting counts, tallies or numbers e.g., first and second numbers
  • the plurality of amplicons contains different sequences derived from different RNA in the sample.
  • the plurality of amplicons contains at least a first amplicon derived from a first RNA transcript in the sample, and the plurality of amplicons contains a second amplicon derived from a second RNA transcript in the sample.
  • the plurality of amplicons contains an amount of different sequences that reflects the sequence complexity and amounts of different RNA sequences found in the RNA sample from which the plurality of amplicons was derived.
  • the amount of the first amplicon relative to the amount of the second amplicon is similar to the relative amounts of the first and second RNAs in the sample.
  • the amplicons are ligated to nucleic acid adaptors to generate adaptor- ligated amplified nucleic acids.
  • the amplicons (or the adaptor-ligated amplified nucleic acids) are characterized, for example, by sequencing to generate sequencing data.
  • the amplicons can be characterized by massively parallel sequencing or sequencing using gel electrophoresis.
  • different target sequences are identified from the sequencing data.
  • the number of different target sequences is counted and tallied, to generate information pertaining the different transcript sequences, and abundances of the transcripts, contained in the initial RNA sample. For example, the number of a first amplicon derived from a first RNA transcript are counted and tallied.
  • the number of a second amplicon derived from a second RNA transcript are counted and tallied.
  • the number of first and second amplicons can be expressed as a percentage or a ratio relative to each other.
  • One skilled in the art will readily recognize that more than two transcripts of interest can be analyzed using any of the methods described herein.
  • the counted and tallied sequencing information is used to determine gene expression of one or more transcripts of interest contained in a single RNA sample or in two or more RNA samples.
  • gene expression includes transcription of at least one DNA sequence of interest in one or more cells.
  • the RNA transcripts present in a cell, at a given time represent steady-state RNA levels resulting from transcription of DNA sequences in the cells, and post-transcriptional modification and/or degradation of the RNA. At least some of the RNA transcripts in the cells may be post-transcriptionally modified (including splicing) and/or degraded (e.g., RNA turnover).
  • RNA transcripts present in the cells at different abundances.
  • the types of RNA sequences, and their abundances can change with onset of cell cycle progression, cell differentiation, cell development, abnormality or a disease, or can change in response to stimuli with a physical or chemical challenge.
  • the types of RNA transcripts and their abundances may differ in different types of cells (e.g., pancreas vs. ovary cells).
  • the RNA present in the cells may include coding and/or non-coding transcripts.
  • the sequencing information is used to determine the presence or absence of one or more RNA transcripts of interest in the one or more samples.
  • the counted and tallied sequencing information is used to measure the abundance of transcripts of interest contained in a single RNA sample, by comparing the amount of a first amplicon of interest with the amount of a second amplicon of interest, where the first and second amplicons of interest are derived from first and second RNA transcripts
  • RNA sample (respectively) present in the same RNA sample. It will be appreciated by the skilled artisan that the amount of more than two different amplicons present in the same sample can be compared. Analysis of the counted and tallied sequencing information may show that expression levels of the first and second transcripts of interest in the sample is the same or is different. The difference in the amounts of the first and second transcripts of interest can be mathematically expressed as a -fold change or percent change.
  • the counted and tallied sequencing information is used to measure the abundance of transcripts of interest contained in a reference RNA sample and contained in one or more test samples, by comparing the amount of a first amplicon of interest from the reference sample with the amount of a second amplicon of interest from the test sample, where the first amplicons of interest are derived from a first RNA transcript present in the reference sample and the second amplicons of interest are derived from a second RNA transcript present in the test sample.
  • the first and second RNA transcripts can be the same or different transcripts of interest. It will be appreciated by the skilled artisan that the amount of two or more different amplicons from the reference and the test samples can be compared.
  • Analysis of the counted and tallied sequencing information may show that expression levels of the transcripts of interest in the reference and test samples changes, or remains unchanged.
  • the changes in the transcripts of interest in the reference and test samples can be mathematically expressed as a -fold change or percent change.
  • the changes in abundance of the transcripts of interest may correlate with a change within the cells, or correlate with an abnormal or diseased cell, or correlate with a physical- or chemical-induced challenge.
  • the test samples can be derived from cells suspected of containing different types and/or different abundances of at least one transcript of interest.
  • the counted and tallied sequencing information is used to determine copy number changes of one or more transcripts of interest contained in a single RNA sample or in two or more RNA samples.
  • changes in copy number of a transcript in a cell can arise from transcription of a DNA sequence in a cell, where the DNA sequence has an increase or decrease in copy number (e.g., aneuploidy).
  • a cell may contain a trisomic chromosome arm resulting in three copies of a DNA sequence of interest, or the cell may contain a missing chromosome arm resulting in one copy of the DNA sequence of interest.
  • a diploid cell may contain one extra copy of a DNA sequence of interest on a chromosome arm resulting in three copies of a DNA sequence of interest, or the cell may contain a deletion of a DNA sequence of interest resulting in one copy of the DNA sequence of interest.
  • transcription of the DNA sequence of interest may result in an abnormal copy number of the RNA transcript of interest.
  • the DNA sequence of interest on both paired chromosomes is transcribed.
  • the amount of steady-state RNA transcript of interest produced from the paired chromosomes is approximately equal.
  • the counted and tallied sequencing information is used to measure the copy number of transcripts of interest contained in a reference RNA sample and contained in one or more test samples, by comparing the amount of a first amplicon of interest from the reference sample with the amount of a second amplicon of interest from the test sample, where the first amplicons of interest are derived from a first RNA transcript present in the reference sample and the second amplicons of interest are derived from a second RNA transcript present in the test sample.
  • the first and second RNA transcripts of interest can have the same or different sequences. It will be appreciated by the skilled artisan that the amount of two or more different amplicons from the reference and the test samples can be compared. Analysis of the counted and tallied sequencing information may show that the copy number of the transcripts of interest in the reference and test samples changes, or remains unchanged. The changes in the transcripts of interest can be mathematically expressed as a -fold change or percent change.
  • the counted and tallied sequencing information may yield N copy number of the transcript of interest from the reference sample, and 1.5 x N (1.5 times N) copy number of the transcript of interest from the test sample. This data indicates that the test sample contains one extra copy number of the transcript of interest, which correlates with a test sample containing three copies of the transcript of interest.
  • the counted and tallied sequencing information may yield N copy number of the transcript of interest from the reference sample, and a 0.5 x N (0.5 times N) copy number of the transcript of interest from the test sample. This data indicates that the test sample is missing one copy of the transcript of interest, which correlates with a test sample containing one copy of the transcript of interest.
  • the changes in copy number of the transcripts of interest may correlate with a change within the cells, or correlate with an abnormal or diseased cell, or correlate with a physical- or chemical-induced challenge.
  • the test samples can be derived from cells suspected of containing different types and/or different copy numbers of at least one transcript of interest.
  • the counted and tallied sequencing information is used to detect gene fusion RNA transcripts contained in a single RNA sample or in two or more RNA samples.
  • a gene fusion RNA transcript includes a chimeric RNA transcript having two or more sequences joined together that are not normally found joined together in a transcript.
  • the gene fusion RNA transcripts can arise from transcription of DNA gene fusion sequences in the cell.
  • the DNA and RNA gene fusion sequences need not include entire genes, or entire exons or introns of genes.
  • the DNA gene fusion sequences can contain a promoter from a first gene joined to the coding region of a second gene.
  • the promoter causes altered transcription (e.g., increase or decrease) of the co-joined coding region in the cell.
  • the RNA gene fusion transcripts contain a junction sequence containing at least part of the promoter sequence or a sequence of the first gene joined to the second gene sequence.
  • the DNA and RNA gene fusion sequences contain at least one exon or intron region of a first gene joined to a second gene sequence, which may lead to altered RNA splicing events.
  • the RNA gene fusion sequence contains abnormal spliced or unspliced sequences.
  • the DNA and RNA gene fusion sequences contain a first gene sequence joined to a second gene sequence.
  • the RNA gene fusion transcript undergoes altered folding into a secondary structure that causes an abnormality in the cell.
  • the DNA and RNA gene fusion sequences contain a first gene sequence joined to a second gene sequence.
  • the RNA gene fusion transcript undergoes altered degradation rates that causes an abnormality in the cell.
  • the presence of abnormal RNA gene fusion sequences or abnormal amounts of an RNA gene fusion transcripts in the cell may cause cellular abnormality such as abnormal cell growth or function, or tumor formation, or can lead to diseased tissue development, or can lead to cell death.
  • the sequencing information is used to determine the presence or absence of one or more RNA gene fusion transcripts in the one or more RNA samples.
  • the counted and tallied sequencing information is used to measure the abundance of RNA gene fusion transcripts contained in a single RNA sample, by comparing the amount of a reference amplicon of interest (e.g., having a normal sequence) with the amount of a second amplicon having an RNA gene fusion sequence.
  • the reference amplicons and gene fusion amplicons are derived from a reference and second RNA transcript (respectively) present in the same RNA sample. It will be appreciated by the skilled artisan that the amount of more than two different amplicons can be compared.
  • Analysis of the counted and tallied sequencing information may show that expression levels of the reference and gene fusion transcripts of interest in the sample is the same or is different.
  • the difference in the amounts of the reference and gene fusion transcripts of interest can be mathematically expressed as a -fold change or percent change.
  • the counted and tallied sequencing information is used to measure the abundance of a reference transcript contained in a reference RNA sample and the abundance of RNA gene fusion transcripts contained in one or more test samples, by comparing the amount of a reference amplicon of interest (e.g., a normal transcript) from the reference sample with the amount of a second amplicon having a fusion sequence from the test sample.
  • the reference amplicons are derived from a first RNA transcript present in the reference sample and the second amplicons are derived from one or more RNA fusion transcripts present in the test sample. It will be appreciated by the skilled artisan that the amount of two or more different amplicons from the reference and the test samples can be compared. Analysis of the counted and tallied sequencing information may show that expression levels of the transcripts of interest in the reference and test samples changes, or remains unchanged. The changes in the transcripts of interest can be mathematically expressed as a -fold change or percent change.
  • the presence and abundance of fusion gene transcripts may correlate with a change within the cells, or may correlate with an abnormal or diseased cell.
  • the test samples can be derived from cells suspected of containing gene fusion transcripts, including normal, abnormal or diseased cells.
  • the disclosure relates generally to method, and related compositions, systems, kits and apparatuses, for attaching one or more amplicons to a support.
  • any procedure that can be used to attach an amplicon to a support can also be used to attach one or more adapter-ligated amplified nucleic acid to a support.
  • the amplicon can be modified for attachment to a support.
  • the amplicon can be amino-modified for attachment to a support (e.g., particles or a planar support).
  • an amino-modified nucleic acid fragment can be attached to a support that is coated with a carboxylic acid.
  • an amino-modified nucleic acid can be reacted with EDC (or ED AC) for attachment to a carboxylic acid coated support (with or without NHS).
  • the amplicon can be attached to particles, such as Ion
  • a support can include an outer or top-most layer or boundary of an object.
  • a support includes a solid surface or semi-solid surface.
  • a support can be porous or non-porous.
  • a support can be a planar surface, as well as concave, convex, or any combination thereof.
  • a support can be a bead, particle, sphere, filter, flowcell, or gel.
  • a support includes the inner walls of a capillary, a channel, a well, groove, channel, reservoir.
  • a support can have texture (e.g., etched, cavitated, pores, three-dimensional scaffolds or bumps).
  • a support can be made from materials such as glass, borosilicate glass, silica, quartz, fused quartz, mica, polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics, silicon, semiconductor, high refractive index dielectrics, crystals, gels, polymers, or films (e.g., films of gold, silver, aluminum, or diamond).
  • the amplicons can be arranged on a support in a random pattern, organized pattern, rectilinear pattern, hexagonal pattern, or addressable array pattern.
  • the amplicons can be modified to attach to one member of a binding partner (e.g., biotin).
  • a biotinylated nucleic acid fragment can be attached to another member of a binding partner (e.g., avidin-like, such as streptavidin) which is attached to a support.
  • molecules that function as binding partners include: biotin
  • the disclosure relates generally to method, and related compositions, systems, kits and apparatuses, comprise sequencing any of the amplified target nucleic acids (e.g., amplicons or adapter-ligated amplified nucleic acids) generated according to the present teachings.
  • amplified target nucleic acids e.g., amplicons or adapter-ligated amplified nucleic acids
  • any type of sequencing platform can be employed, including: size- separation via gel electrophoresis, sequencing by oligonucleotide probe ligation and detection (e.g., SOLiDTM from Life Technologies, WO 2006/084131), probe-anchor ligation sequencing (e.g., Complete GenomicsTM or PolonatorTM), sequencing- by-synthesis (e.g., Genetic Analyzer and HiSeqTM, from Illumina), pyrophosphate sequencing (e.g., Genome Sequencer FLX from 454 Life Sciences), ion-sensitive sequencing (e.g., Personal Genome Machine (PGMTM) and Ion ProtonTM Sequencer, both from Ion Torrent Systems, Inc.), and single molecule sequencing platforms (e.g., HeliScopeTM from HelicosTM).
  • SOLiDTM from Life Technologies, WO 2006/084131
  • probe-anchor ligation sequencing e.g., Complete GenomicsTM or PolonatorTM
  • sequencing- by-synthesis e
  • a multiplex nucleic acid amplification method includes (a) amplifying one or more target polynucleotides using one or more target-specific primers in the presence of polymerase to produce an amplified target polynucleotide, and (b) ligating an adapter to the amplified target polynucleotide to form an adapter-ligated amplified target polynucleotide.
  • amplifying can be performed in solution such that an amplified target polynucleotide or a target-specific primer is not linked to a solid support or surface.
  • ligating can be performed in solution such that an amplified target polynucleotide or an adapter is not linked to a solid support or surface.
  • amplifying and ligating can be performed in solution such that an amplified target polynucleotide, a target-specific primer or an adapter is not linked to a solid support or surface.
  • the amplifying can be performed on a solid support or surface, such as a flow cell, an array, a nucleic acid sequencing bead, and the like.
  • the ligating can be performed on an amplified target polynucleotide that is attached to a solid support or surface, such as a flow cell, an array, a nucleic acid sequencing bead, and the like.
  • a solid support or surface such as a flow cell, an array, a nucleic acid sequencing bead, and the like.
  • one of more of the plurality of target polynucleotides amplified using one or more of the disclosed methods can be used in DNA sequencing, such as any applicable next-generation sequencing platform.
  • a variety of next-generation sequencing platforms are available that can use of one or more of the products from the amplification and synthesis methods disclosed herein.
  • next generation sequencing platforms made by Life Technologies (CA)(e.g., Ion Torrent' s PGM and Proton platforms), Illumina (CA)(e.g., MiSeq, HiSeq, and X- 10 platforms), Roche, Helicos, and Pacific Biosciences sequencing platforms are capable of utilizing the methods (as well as compositions, systems, apparatuses and kits) as disclosed herein for nucleic acid sequencing and/or nucleic acid analysis.
  • one or more of the plurality of target polynucleotides amplified or synthesized using one or more of the methods disclosed herein can be used to determine (or estimate) the level of mRNA expression of one or more active genes in a RNA transcriptome.
  • the level of mRNA expression of one or more active genes in a RNA transcriptome may be determined as an over-expression or under-expression of mRNA as compared to a known, matched, or reference sample.
  • the over-expression of one or more active genes in the RNA transcriptome can be indicative of a diseased or abnormal state.
  • the under-expression of one or more active genes in the RNA transcriptome can be indicative of a diseased or abnormal state.
  • any amplified target nucleic acids (e.g., amplicons or adapter- ligated amplified target nucleic acids) that has been generated according to the present teachings, can be attached to a solid support.
  • a bridge amplification reaction can be conducted to attach the adapter-ligated amplified target nucleic acids to a planar support (e.g., flowcell) or beads.
  • Individual amplified target nucleic acids are ligated to a first universal adaptor at one end and a second universal adaptor at the other end to generate a population of adapter-ligated amplified target nucleic acids.
  • the first and second adaptors have different sequences.
  • the first and/or second adaptor includes a universal sequencing primer sequence.
  • at least two of the amplified target nucleic acids have different sequences.
  • the population of adapter-ligated amplified target nucleic acids is rendered single-stranded. At least a portion of the population of single-stranded adapter-ligated amplified target nucleic acids is hybridized to capture primers that are attached to a support.
  • the support can include a plurality of first and second capture primer having different sequences.
  • the first universal adaptor hybridizes with the first capture primer, and a primer extension reaction extends the first capture primer to generate a first capture primer extension product having a complementary sequence of the second adaptor at one end.
  • the primer extension reaction employs the adapter- ligated amplified target nucleic acid as a template.
  • the template molecule is removed.
  • the first capture primer extension product bends (e.g., arches) so that the second adaptor complementary sequence can hybridize to a nearby second capture primer, and a primer extension reaction extends the second capture primer to generate a second capture primer extension product having a complementary sequence of the first adaptor at one end, and forming a double-stranded bridge molecule.
  • the double-stranded bridge is denatured to yield two single-stranded, immobilized target nucleic acids.
  • One of the single-stranded, immobilized target nucleic acids has a first primer (or complementary sequence thereof) which is attached to the support and the other end of the molecule has a second primer sequence (or complementary sequence thereof) that can hybridize to a nearby second capture primer to start another bridge amplification reaction.
  • the other single-stranded, immobilized target nucleic acids has a second primer (or complementary sequence thereof) which is attached to the support and the other end of the molecule has a first primer sequence (or
  • any amplified target nucleic acids (e.g., amplicons or adapter- ligated amplified target nucleic acids) that has been generated according to the present teachings, can be attached to a solid support.
  • a template walking reaction can be conducted to attach the adapter-ligated amplified target nucleic acids to a planar support (e.g., flowcell) or beads.
  • first and second adaptors have different sequences.
  • the first and/or second adaptor includes a universal sequencing primer sequence.
  • the first and second adaptors have different sequences.
  • the first adaptor includes a universal amplification primer sequence that differs from the universal amplification sequence in the second adaptor.
  • at least two of the amplified target nucleic acids have different sequences.
  • the population of adapter-ligated amplified target nucleic acids is rendered single-stranded. At least a portion of the population of single- stranded adapter-ligated amplified target nucleic acids is hybridized to capture primers that are attached to a support.
  • the support can include a plurality of immobilized capture primers, where the 3' end of the capture primers includes the same sequence. In some embodiments, the 3' end of the capture primers includes a sequence having a low T m (melting temperature) sequence.
  • the first universal adaptor hybridizes with a first immobilized capture primer
  • a primer extension reaction extends the first capture primer to generate a first capture primer extension product having a complementary sequence of the second adaptor at one end.
  • the primer extension reaction employs the adapter-ligated amplified target nucleic acid as a template.
  • the template molecule (which is hybridized along its length to the extension product) undergoes localized denaturation at the first adaptor region that contains the low T m region, and the first adaptor region rehybridizes to a nearby capture primer (second capture primer), while the remainder of the template molecule is hybridized to the extension product.
  • Primer extension of the second capture primer serves to denature the portion of the template molecule that is still hybridized with the first extension product, and generates a second capture primer extension product.
  • Repeat cycles of template walking include hybridizing the first adaptor region to a nearby capture primer, primer extension, localized denaturation, re -hybridization with a different nearby capture primer, and primer extension, produces a plurality of amplified target nucleic acids that are attached to the support.
  • the cycles of template walking can be conducted under isothermal conditions. Examples of compositions and methods for nucleic acid template walking are found in U.S. published application Nos.
  • any amplified target nucleic acids (e.g., amplicons or adapter- ligated amplified target nucleic acids) that has been generated according to the present teachings, can be attached to a solid support.
  • a recombinase-polymerase amplification (RPA) reaction can be conducted under aqueous conditions to attach the adapter-ligated amplified target nucleic acids to a planar support (e.g., flowcell) or to beads.
  • RPA recombinase-polymerase amplification
  • Individual amplified target nucleic acids are ligated to a first universal adaptor at one end and a second universal adaptor at the other end to generate a population of adapter-ligated amplified target nucleic acids.
  • the first and second adaptors have different sequences. In some embodiments, the first and/or second adaptor includes a universal sequencing primer sequence. In some embodiments, the first and second adaptors have different sequences. In some embodiments, the first adaptor includes a universal amplification primer sequence that differs from the universal amplification sequence in the second adaptor. In some embodiments, at least two of the amplified target nucleic acids have different sequences. The population of adapter-ligated amplified target nucleic acids is rendered single- stranded.
  • the single-stranded nucleic acids are reacted with: (i) a plurality of beads having a plurality of capture primers attached thereon; (ii) a plurality of soluble reverse primers; (iii) polymerase; and (iv) a plurality of nucleotides.
  • the single reaction mixture also includes a forward fusion primer serves as a splint molecule that can hybridize to a capture primer and the first adaptor sequence which is joined to the target nucleic acid.
  • the first adaptor sequence which is joined to the target nucleic acid can hybridize with a portion of the fusion primer, but the first adaptor lacks a sequence that can hybridize to the capture primer on the bead.
  • the single reaction mixture further includes a recombinase (e.g., T4 uvsX), and optionally accessory proteins, including recombinase loading factor (e.g., T4 uvsY) and/or single-stranded binding protein (T4 gp32).
  • T4 uvsX e.g., T4 uvsX
  • accessory proteins e.g., T4 uvsY
  • T4 gp32 single-stranded binding protein
  • the fusion primer hybridizes to the first adaptor sequence, and a primer extension reaction yields a fusion primer extension product which includes a sequence that can hybridize to the capture primer on the bead.
  • the soluble reverse primer hybridizes with the fusion primer extension product, and a primer extension reaction yields a reverse primer extension product.
  • the reverse primer extension product can hybridize to one of the plurality of capture primers on the bead, and a primer extension reaction yields a capture primer extension product which is attached to the bead and includes a sequence that is complementary to the reverse primer extension product.
  • the adapter-ligated amplified target nucleic acid hybridizes to one of the plurality of capture primers on the bead, and primer extension produces a capture primer extension product.
  • a reverse primer hybridizes to the capture primer extension product, and a primer extension reaction produces a reverse primer extension product.
  • the reverse primer extension product can dissociate (e.g., denature) from the capture primer extension product, and re -hybridize with a different capture primer on the same bead, for another primer extension reaction.
  • Repeat cycles of the RPA-bead amplification reaction yields beads that are attached with multiple copies of the adapter-ligated amplified target nucleic acids.
  • individual beads are attached with substantially monoclonal copies of one adapter-ligated amplified target nucleic acid.
  • different beads are attached with copies of a different adapter-ligated amplified target nucleic acid.
  • the RPA-bead method includes an water-and-oil emulsion, where droplets of the aqueous reaction mixture are surrounded by an immiscible fluid (e.g., oil) so that the aqueous droplets provide compartmentalized reaction mixtures containing one or more beads that are attached with capture primers, template nucleic acids, fusion primers (or lacking fusion primers), reverse primers, polymerase, nucleotides, recombinase and accessory proteins.
  • an immiscible fluid e.g., oil
  • the capture primers are attached to a support (e.g., planar-like support) and the recombinase-polymerase reaction is conducted in a manner similar to the RPA-bead method, where the aqueous single reaction mixture contacts the surface of the support having the attached capture primers, where the aqueous single reaction mixture contains template nucleic acids, fusion primers (or lacking fusion primers), reverse primers, polymerase, nucleotides, recombinase and accessory proteins.
  • a support e.g., planar-like support
  • the recombinase-polymerase reaction is conducted in a manner similar to the RPA-bead method, where the aqueous single reaction mixture contacts the surface of the support having the attached capture primers, where the aqueous single reaction mixture contains template nucleic acids, fusion primers (or lacking fusion primers), reverse primers, polymerase, nucleotides, recombinase and accessory proteins.
  • cycles of an RPA reaction, using beads or a support, with or without an emulsion can be conducted under isothermal amplification conditions.
  • RPA recombinase-polymerase amplification
  • any amplified target nucleic acids (e.g., amplicons or adapter- ligated amplified nucleic acids) that has been generated according to the present teachings, can be used as a template molecule for sequencing using any sequencing method, including sequencing-by- synthesis methods using natural nucleotides or nucleotide analogs.
  • the sequence-by-synthesis methods comprise successively incorporating nucleotides onto a terminal 3 ' OH end of a primer or self-priming template, using a polymerase, detecting the incorporated nucleotides, and determining the identity of the newly incorporated nucleotide.
  • the nucleotide analogs include terminator nucleotides that are optionally reversibly blocked at the 2' or 3 ' OH sugar position on the nucleotide.
  • the reversibly blocked nucleotides include a blocking moiety attached to the 2' or 3 ' OH position via a linker that is cleavable with a chemical compound, enzyme, light or heat.
  • the nucleotide analog can also include a detectable label (e.g., a fluorphore) attached to the 2' or 3 ' OH position, or attached to the base.
  • Each of the four types of nucleotides can be attached to a different label that is distinguishable from the other labels.
  • the polymerase incorporates the in-coming terminator nucleotide onto an existing 3 ⁇ end, but cannot incorporate the next nucleotide until the linker is cleaved to release the blocking moiety and restore the 3 ⁇ on the newly-incorporated nucleotide.
  • the identity of the newly- incorporated nucleotide is determined by detecting the type of fluorphore attached to the nucleotide analog.
  • the newly-incorporated nucleotide is treated with a cleaving agent to release the blocking moiety and restore the 3 ⁇ .
  • the polymerase can now incorporate another terminator nucleotide.
  • the sequence of the template molecule is determined by performing repeated cycles of nucleotide incorporation, detection and identification of the newly incorporated nucleotide, and removal of the blocking moiety.
  • the blocking moiety comprises an allyl, alkyl, substitute alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, acyl, cyano, alkoxy, aryloxy, or heteroaryloxy moiety.
  • the nucleotide analog includes a 3 ' O allyl blocking moiety (see U.S. patent Nos. 8,796,432 and 7,883,869).
  • the blocking moiety comprises -O, -S, -P, -F, -NH 2 , -OCH 3 , -
  • the nucleotide analog comprises a 3 ' azido blocking moiety which is cleavable with a phosphine compound (see U.S. patent No. 7,635,578).
  • the nucleotide analogs include blocking moieties attached via a disulfide linkage, acid labile linkers (e.g., dialkoxybenzyl linkers), Sieber linkers, indole linkers, or t-butyl Sieber linkers.
  • the linkers are cleavable linkers, and include: electrophilically- cleavable linkers, nucleophilically-cleavable linkers, photocleavable linkers, and linkers cleavable under reductive or oxidative conditions.
  • the linkers are cleavable via use of safety-catch linkers, and linkers cleavable by elimination mechanisms. See for example U.S. patent No.
  • the nucleotide analogs include blocking moieties attached via a photocleavable linker.
  • the cleavable linker comprises a nitrobenzyl moiety.
  • MOM -CH 2 OCH 3
  • the nucleotides analogs include a detectable label attached to the base.
  • a 7-deazapurine base can be linked at the 7-position.
  • the linker attaching the base to the detectable label can be an acid labile linker, a photocleavable linker, disulfide linkage, dialkoxybenzyl linkers, Sieber linkers, indole linkers, or t-butyl Sieber linkers.
  • the linker that attaches the base to the detectable label can be cleavable under oxidation conditions, or cleavable with a palladium compound, or cleavable with thiophilic metals, including nickel, silver or mercury.
  • the terminator nucleotides also include a blocking moiety linked to the 2' or 3 ' sugar position by a linker.
  • the blocking moiety includes an azido group.
  • the linker attached to the base and the linker attached to the 2' or 3 ' sugar position are cleavable under the same conditions. See for example, U.S. patent Nos. 7,057,026; 7,566,537; 8, 158,346; 7,541 ,444; 7,057,026; 7,592,435; 7,414, 1 16; 7,427,673 and 8,399, 188, and U.S. published application No. 2014/0249036.
  • any amplified target nucleic acids (e.g., amplicons or adapter- ligated amplified nucleic acids) that has been generated according to the present teachings, can be sequenced by any sequencing method, including sequencing-by-synthesis, ion-based sequencing involving the detection of sequencing byproducts using field effect transistors (e.g., FETs and ISFETs), chemical degradation sequencing, ligation-based sequencing, hybridization sequencing, pyrophosphate detection sequencing, capillary electrophoresis, gel electrophoresis, next-generation, massively parallel sequencing platforms, sequencing platforms that detect hydrogen ions or other sequencing by-products, and single molecule sequencing platforms.
  • a sequencing reaction can be conducted using at least one sequencing primer that can hybridize to any portion of the polynucleotide constructs, including a nucleic acid adaptor or a target polynucleotide.
  • any amplified target nucleic acids that has been generated according to the present teachings can be sequenced using methods that detect one or more byproducts of nucleotide incorporation.
  • the detection of polymerase extension by detecting physicochemical byproducts of the extension reaction can include pyrophosphate, hydrogen ion, charge transfer, heat, and the like, as disclosed, for example, in U.S. Pat. No. 7,948,015 to Rothberg et al.; and Rothberg et al, U.S. Patent Publication No. 2009/0026082, hereby incorporated by reference in their entireties.
  • Other examples of methods of detecting polymerase-based extension can be found, for example, in Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006);
  • reactions involving the generation and detection of ions are widely performed.
  • the use of direct ion detection methods to monitor the progress of such reactions can simplify many current biological assays.
  • template -dependent nucleic acid synthesis by a polymerase can be monitored by detecting hydrogen ions that are generated as natural byproducts of nucleotide incorporations catalyzed by the polymerase.
  • Ion-sensitive sequencing also referred to as "pH- based" or "ion-based” nucleic acid sequencing
  • ionic byproducts such as hydrogen ions
  • the nucleic acid to be sequenced can be captured in a microwell, and nucleotides can be flowed across the well, one at a time, under nucleotide incorporation conditions.
  • the polymerase incorporates the appropriate nucleotide into the growing strand, and the hydrogen ion that is released can change the pH in the solution, which can be detected by an ion sensor that is coupled with the well. This technique does not require labeling of the nucleotides or expensive optical components, and allows for far more rapid completion of sequencing runs.
  • Examples of such ion-based nucleic acid sequencing methods and platforms include the Ion Torrent PGMTM or ProtonTM sequencer (Ion TorrentTM Systems, Life Technologies Corporation).
  • amplified target nucleic acids produced using the methods, systems and kits of the present teachings can be used as a substrate for a biological or chemical reaction that is detected and/or monitored by a sensor including a field-effect transistor (FET).
  • FET field-effect transistor
  • the FET is a chemFET or an ISFET.
  • a "chemFET” or chemical field-effect transistor is a type of field effect transistor that acts as a chemical sensor. It is the structural analog of a MOSFET transistor, where the charge on the gate electrode is applied by a chemical process.
  • ISFET ion-sensitive field-effect transistor
  • the FET may be a FET array.
  • an "array" is a planar arrangement of elements such as sensors or wells.
  • the array may be one or two dimensional.
  • a one dimensional array can be an array having one column (or row) of elements in the first dimension and a plurality of columns (or rows) in the second dimension. The number of columns (or rows) in the first and second dimensions may or may not be the same.
  • the FET or array can comprise 102, 103, 104, 105, 106, 107 or more FETs.
  • one or more microfluidic structures can be fabricated above the FET sensor array to provide for containment and/or confinement of a biological or chemical reaction.
  • the microfluidic structure(s) can be configured as one or more wells (or microwells, or reaction chambers, or reaction wells, as the terms are used interchangeably herein) disposed above one or more sensors of the array, such that the one or more sensors over which a given well is disposed detect and measure analyte presence, level, and/or concentration in the given well.
  • Microwells or reaction chambers are typically hollows or wells having well-defined shapes and volumes which can be manufactured into a substrate and can be fabricated using conventional microfabrication techniques, e.g. as disclosed in the following references: Doering and Nishi, Editors, Handbook of Semiconductor Manufacturing Technology, Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMS and Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al, Silicon Micromachining (Cambridge University Press, 2004); and the like. Examples of configurations (e.g. spacing, shape and volumes) of microwells or reaction chambers are disclosed in Rothberg et al, U.S. patent publication 2009/0127589 and Rothberg et al, U.K. patent application GB24611127 which are hereby incorporated by reference in their entireties.
  • the biological or chemical reaction can be performed in a solution or a reaction chamber that is in contact with, operatively coupled, or capacitively coupled to a FET such as a chemFET or an ISFET.
  • a FET such as a chemFET or an ISFET.
  • the FET (or chemFET or ISFET) and/or reaction chamber can be an array of FETs or reaction chambers, respectively.
  • a biological or chemical reaction can be carried out in a two- dimensional array of reaction chambers, wherein each reaction chamber can be coupled to a FET, and each reaction chamber is no greater than 10 ⁇ 3 (i.e., 1 pL) in volume. In some embodiments each reaction chamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pL in volume.
  • a reaction chamber can optionally be no greater than 2, 5, 10, 15, 22, 32, 42, 52, 62, 72, 82, 92, or 102 square microns in cross-sectional area at the top.
  • the array has at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 2 , or more reaction chambers.
  • at least one of the reaction chambers is operatively coupled to at least one of the FETs.
  • FET arrays as used in various embodiments according to the disclosure can be fabricated according to conventional CMOS fabrications techniques, as well as modified CMOS fabrication techniques and other semiconductor fabrication techniques beyond those conventionally employed in CMOS fabrication. Additionally, various lithography techniques can be employed as part of an array fabrication process.
  • Exemplary FET arrays suitable for use in the disclosed methods, as well as microwells and attendant fluidics, and methods for manufacturing them, are disclosed, for example, in U.S. Patent Publication No. 20100301398; U.S. Patent Publication No. 20100300895; U.S. Patent Publication No. 20100300559; U.S. Patent Publication No. 20100197507, U.S. Patent Publication No. 20100137143 ; U.S. Patent Publication No. 20090127589; and U.S. Patent Publication No. 20090026082, which are incorporated by reference in their entireties.
  • the disclosed methods, compositions, systems, apparatuses and kits can be used for carrying out label-free nucleic acid sequencing, and in particular, ion-based nucleic acid sequencing.
  • label-free detection of nucleotide incorporation has been described in the literature, including the following references that are incorporated by reference: Rothberg et al, U.S. patent publication 2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86 (2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103 : 6466-6470 (2006).
  • nucleotide incorporations are determined by measuring natural byproducts of polymerase-catalyzed extension reactions, including hydrogen ions, polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase).
  • examples of such ion-based nucleic acid sequencing methods and platforms include the Ion Torrent PGMTM or ProtonTM sequencer (Ion TorrentTM Systems, Life Technologies Corporation).
  • the disclosure relates generally to methods for sequencing target nucleic acids that have been amplified by the teachings provided herein.
  • the disclosure relates generally to a method for obtaining sequence information from polynucleotides, comprising: (a) amplifying target polynucleotides; and (b) performing template- dependent nucleic acid synthesis using at least one of the amplified target polynucleotides produced during step (a) as a template.
  • the amplifying can optionally be performed according to any of the multiplex amplification methods described herein.
  • the template-dependent synthesis includes incorporating one or more nucleotides in a template-dependent fashion into a newly synthesized nucleic acid strand.
  • the methods can further include producing one or more ionic byproducts of such nucleotide incorporation.
  • the methods can further include detecting the incorporation of the one or more nucleotides into the sequencing primer.
  • the detecting can include detecting the release of hydrogen ions.
  • the disclosure relates generally to a method for sequencing a nucleic acid, comprising: (a) amplifying target polynucleotides according to the methods disclosed herein; (b) disposing the amplified polynucleotides (e.g., amplicons or adapter-ligated amplified nucleic acids) into a plurality of reaction chambers, wherein one or more of the reaction chambers are in contact with a field effect transistor (FET).
  • FET field effect transistor
  • the method further includes contacting amplified nucleic acids which are disposed into one of the reaction chambers, with a polymerase thereby synthesizing a new nucleic acid strand by sequentially incorporating one or more nucleotides into a nucleic acid molecule.
  • the method further includes generating one or more hydrogen ions as a byproduct of such nucleotide incorporation.
  • the method further includes detecting the incorporation of the one or more nucleotides by detecting the generation of the one or more hydrogen ions using the FET.
  • the detecting includes detecting a change in voltage and/or current at the at least one FET within the array in response to the generation of the one or more hydrogen ions.
  • the FET can be selected from the group consisting of: ion- sensitive FET (isFET) and chemically-sensitive FET (chemFET).
  • One exemplary system involving sequencing via detection of ionic byproducts of nucleotide incorporation is the Ion Torrent PGMTM or ProtonTM sequencer (Life Technologies), which is an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions produced as a byproduct of nucleotide incorporation. Typically, hydrogen ions are released as byproducts of nucleotide incorporations occurring during template-dependent nucleic acid synthesis by a polymerase.
  • the Ion Torrent PGMTM or ProtonTM sequencer detects the nucleotide incorporations by detecting the hydrogen ion byproducts of the nucleotide incorporations.
  • the Ion Torrent PGMTM or ProtonTM sequencer can include a plurality of nucleic acid templates to be sequenced, each template disposed within a respective sequencing reaction well in an array.
  • the wells of the array can each be coupled to at least one ion sensor that can detect the release of H + ions or changes in solution pH produced as a byproduct of nucleotide incorporation.
  • the ion sensor comprises a field effect transistor (FET) coupled to an ion-sensitive detection layer that can sense the presence of H + ions or changes in solution pH.
  • FET field effect transistor
  • the ion sensor can provide output signals indicative of nucleotide incorporation which can be represented as voltage changes whose magnitude correlates with the H + ion concentration in a respective well or reaction chamber.
  • nucleotide types can be flowed serially into the reaction chamber, and can be incorporated by the polymerase into an extending primer (or polymerization site) in an order determined by the sequence of the template.
  • Each nucleotide incorporation can be accompanied by the release of H + ions in the reaction well, along with a concomitant change in the localized pH.
  • the release of H + ions can be registered by the FET of the sensor, which produces signals indicating the occurrence of the nucleotide incorporation.
  • Nucleotides that are not incorporated during a particular nucleotide flow may not produce signals.
  • the amplitude of the signals from the FET can also be correlated with the number of nucleotides of a particular type incorporated into the extending nucleic acid molecule thereby permitting
  • the plurality of amplicons that are produced by any of the present teachings can yield data (e.g., quantifying or counting data) which represents an approximation of the complexity and abundance of different transcripts that are present in a starting RNA or DNA sample.
  • the plurality of amplicons that are produced by any of the present teachings may not represent an absolutely accurate count of RNA or DNA sequences of interest in a sample.
  • the quantifying step can yield data that differs from the actual quantity of RNA or DNA sequences of interest present in a sample by about 0.01 - 0.1 %, or about 0.1 - 0.5%, or about 0.5 - 1 %, or about 1 - 2.5%, or about 2.5 - 5%, or about 5 - 7.5%, or about 7.5 - 10%, or about 10 - 25%, or more.
  • the plurality of target polynucleotides in the reaction mixture comprises sequences derived from RNA in the sample.
  • the plurality of target polynucleotides in the reaction mixture can include a plurality of cDNAs that individually correspond to one or more RNA sequences.
  • generating the plurality of polynucleotides comprises converting RNA to cDNA using any suitable means.
  • generating the plurality of polynucleotides comprises: conducting a reverse transcription reaction with RNA and plurality of primers to generate plurality of cDNA.
  • the plurality of primers used in the reverse transcription reaction comprises random sequence primers.
  • the reverse transcription reaction includes at least one enzyme having RNA-dependent DNA polymerase activity.
  • the enzyme having RNA-dependent DNA polymerase activity also has DNA-dependent DNA polymerase activity.
  • the plurality of cDNA includes polyA cDNA and non-polyA cDNA.
  • the plurality of cDNA includes a plurality of first strand cDNA, a plurality of second strand cDNA, or a plurality of first and second strand cDNA.
  • the plurality of target polynucleotides e.g., cDNA
  • the reverse transcribing can be conducted by directly ligating the RNA to a plurality of double-stranded RNA/DNA or DNA/DNA adaptors, heating to remove one strand of the double-stranded adaptors, and conducting a reverse transcription reaction with primers that hybridize at least one adaptor sequence.
  • the reverse transcribing can be conducted according to an RNA-Seq procedure described in U.S. patent No. 8, 192,941 , which is incorporated by reference in its entirety.
  • the plurality of target described in U.S. patent No. 8, 192,941 , which is incorporated by reference in its entirety.
  • polynucleotides can be generated by and RNA-Seq method (see for example U.S. patent No. 8, 192,941 , incorporated by reference in its entirety), which can include: ligating double- stranded adaptors to both ends of single-stranded RNA, removing one of the strands of both double- stranded adaptors by denaturation to form an RNA molecule having single-stranded adaptors appended to both ends, hybridizing the RNA with an extendible primer that hybridizes to at least one of the single-stranded adaptors that is appended to the RNA, and conducting a reverse transcription reaction with an RNA-dependent DNA polymerase, and a plurality of nucleotides.
  • the double-stranded adaptors can include DNA/RNA or DNA/DNA.
  • the double-stranded adaptors are ligated to the RNA ends with an RNA ligase.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for providing a reverse transcription reaction mixture containing one or more RNA sequence.
  • the reverse transcription reaction mixture further includes any one or any combination of a plurality of primers, at least one enzyme having RNA- dependent DNA polymerase activity and/or a plurality of nucleotides.
  • the reverse transcription reaction mixture further includes
  • RNase H to degrade the RNA during or after the reverse transcription step.
  • the reverse transcription reaction mixture further includes any one or any combination of compounds: magnesium, manganese, formamide, DMSO, betaine, trehalose, spermidine, sulfones, sodium pyrophosphate, low molecular amides, single-stranded binding proteins and/or an archaeal accessory factor that enhances the activity of an RNA-dependent DNA polymerase or a DNA-dependent DNA polymerase.
  • the reverse transcription reaction mixture can be incubated under isothermal, thermal-cycling, or a combination of both temperature conditions.
  • a reverse transcription reaction further includes an external
  • the external control RNA comprises a known RNA sequence (e.g., beta-actin, glyceraldehydes-3-phosphate dehydrogenase, or rRNA) or a commercially-available ERCC Spike-In Control mix (AmbionTM ).
  • a known RNA sequence e.g., beta-actin, glyceraldehydes-3-phosphate dehydrogenase, or rRNA
  • ERCC Spike-In Control mix e.g., ERCC Spike-In Control mix
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for reverse transcribing RNA by contacting at least one RNA molecule with any one or any combination of a plurality of primers, an enzyme having RNA-dependent DNA polymerase activity and/or a plurality of nucleotides.
  • the at least one RNA molecule, the plurality of primers, the enzyme having RNA-dependent DNA polymerase activity and the plurality of nucleotides can be contacted together substantially simultaneously, or sequentially, in any combination and in any order.
  • the plurality of primers comprises a plurality of random sequence primers, target-specific primers, or polyT primers.
  • the contacting can be conducted in a single reaction mixture or in separate reaction mixtures.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for reverse transcribing RNA by hybridizing at least one RNA molecule with a plurality of primers to form at least one RNA/primer complex.
  • At least one of the plurality of primers can hybridize to at least a portion of one or more RNA molecules.
  • the hybridizing is conducted in a single reaction mixture (e.g., a reverse transcription reaction mixture).
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for converting RNA to cDNA by conducting a reverse transcription reaction.
  • the reverse transcription reaction generates a plurality of cDNA that represents a whole transcriptome, or represents a portion of RNA sequences in a whole
  • the reverse transcription reaction comprises at least one RNA molecule, a plurality of primers, an enzyme having RNA-dependent DNA polymerase activity, and a plurality of nucleotides.
  • the reverse transcription reaction includes hybridizing the RNA with the plurality of primers to form a plurality of RNA/primer complexes.
  • the reverse transcription reaction includes incorporating one nucleotide onto a primer that is part of the RNA/primer complex.
  • the nucleotide is incorporated onto the primer in a template-based manner, which can include complementary base pairing, including standard A-T or C-G base pairing, or optionally other forms of base-pairing interactions.
  • the primer extension reaction includes successively incorporating nucleotides onto a primer that is part of an RNA/primer complex.
  • the primer extension reaction can be conducted in a single reaction mixture.
  • the RNA can be naturally-occurring, recombinant or synthetically-prepared.
  • the RNA includes any one or any combination of total RNA, or RNA enriched for one or more RNA species, non-enriched RNA, coding RNA, non- coding RNA, polyA RNA or non-polyA RNA.
  • the RNA can be isolated from a single fresh or archived cell, fresh cells, fresh tissues, or archived cells or tissues that are formalin- treated and/or embedded in paraffin or plastic, or cells or tissues that are formalin fixed paraffin- embedded (FFPE).
  • FFPE formalin fixed paraffin- embedded
  • the RNA can be isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, and viruses; cells; tissues; normal or diseased cells or tissues or organs, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen;
  • organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, and viruses
  • cells tissues; normal or diseased cells or tissues or organs, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen;
  • the RNA can be unfragmented, or fragmented by mechanical force, chemical, enzyme or heat. In some embodiments, the RNA can be depleted of one or more species such as rRNA. In some
  • the RNA comprises any one or any combination of any type of RNA, including: total RNA, mRNA, polyA RNA, polysomal RNA, tRNA, ribosomal RNA, lincRNA, miRNA, piRNA, siRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, aRNA, crRNA, tasiRNA, rasiRNA and 7SKRNA.
  • RNA comprises any one or any combination of any type of RNA, including: total RNA, mRNA, polyA RNA, polysomal RNA, tRNA, ribosomal RNA, lincRNA, miRNA, piRNA, siRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, aRNA, crRNA, tasiRNA, rasiRNA and 7SKRNA.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprising conducting a reverse transcription reaction with about 200 pg - 10 ng of RNA, or about 10 ng - 100 ng of RNA, or about 100 ng - 500 ng of RNA, or about 500 ng - 1 ⁇ g, or more, of RNA.
  • the RNA can be isolated from an unfixed cells or tissues, or from an FFPE sample.
  • an external RNA control can be added to the RNA sample permit characterization of the starting RNA against defined performance criteria. For example, addition of an external RNA control can enable measurement of absolute abundance of an RNA sequence of interest.
  • the external control RNA comprises a known RNA sequence (e.g., beta-actin, glyceraldehydes-3 -phosphate dehydrogenase, or rRNA) or a known RNA sequence (e.g., beta-actin, glyceraldehydes-3 -phosphate dehydrogenase, or rRNA) or a known RNA sequence (e.g., beta-actin, glyceraldehydes-3 -phosphate dehydrogenase, or rRNA) or a known RNA sequence (e.g., beta-actin, glyceraldehydes-3 -phosphate dehydrogenase, or rRNA) or a known RNA sequence (e.g., beta-actin, gly
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, where the enzyme employed in the reverse transcription reaction comprises a polymerase.
  • the enzyme employed in the reverse transcription reaction comprises RNA-dependent DNA polymerase activity.
  • the enzyme employed in the reverse transcription reaction also has DNA-dependent DNA polymerase activity.
  • the enzyme having RNA-dependent DNA polymerase activity also has strand- displacement activity.
  • the enzyme employed in the reverse transcription reaction comprises a wild-type, mutant, or chimeric enzyme.
  • the enzyme employed in the reverse transcription reaction has RNase H activity, or lacks or exhibits reduced RNase H activity.
  • the enzyme employed in the reverse transcription reaction exhibits increased thermostability.
  • the enzyme employed in the reverse transcription reaction exhibits high fidelity.
  • the enzyme employed in the reverse transcription reaction comprises a reverse transcriptase enzyme.
  • the reverse transcription reaction can be conducted with any one or any combination of reverse transcriptases, including: Moloney murine leukemia virus (M- MLV) reverse transcriptase; human immunodeficiency virus (HIV) reverse transcriptase; rous sarcoma virus (RSV) reverse transcriptase; avian myeloblastosis virus (AMV) reverse transcriptase; rous associated virus (RAV) reverse transcriptase; myeloblastosis associated virus (MAV) reverse transcriptase or other avian sarcoma-leukosis virus (ASLV) reverse transcriptases.
  • M- MLV Moloney murine leukemia virus
  • HCV human immunodeficiency virus
  • RSV sarcoma virus
  • AMV avian myeloblastosis virus
  • RAV avian myeloblastosis virus
  • ASLV avian sarcoma-leukosis virus
  • the enzyme employed in the reverse transcription reaction comprises a mutant M-MLV reverse transcriptase that exhibits reduced Rnase H activity and is high fidelity (U.S. Patent No. 7,056,716 which is hereby incorporated by reference in its entirety).
  • the enzyme employed in the reverse transcription reaction comprises a mutant M-MLV reverse transcriptase that exhibits reduced terminal deoxynucleotidyl transferase activity (U.S. patent No. 8,541,219 which is hereby incorporated by reference in its entirety).
  • the enzyme employed in the reverse transcription reaction comprises a mutant M-MLV reverse transcriptase that exhibits reduced terminal deoxynucleotidyl transferase activity, or increased thermostability, or increased fidelity (U.S. patent No. 7,078,208 which is hereby incorporated by reference in its entirety).
  • the enzyme employed in the reverse transcription reaction comprises a mutant M-MLV reverse transcriptase that exhibits reduced terminal deoxynucleotidyl transferase activity, or increased thermostability, or increased fidelity (U.S. patent No. 8,753,845 which is hereby incorporated by reference in its entirety).
  • the enzyme employed in the reverse transcription reaction comprises a hyperactive reverse transcriptase having reduced RNase H activity (U.S. patent No. 8,361,754 which is hereby incorporated by reference in its entirety).
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, include a plurality of primers for conducting a reverse transcription reaction, where the primers comprise random-sequence primers.
  • a plurality of random-sequence primers can be used to generate cDNA in a reverse transcription reaction.
  • a random-sequence primer comprises DNA, RNA or DNA/RNA.
  • at least one of the random-sequence primers, in the plurality can hybridize to any portion of any type of RNA.
  • the random-sequence primers have extendible 3 ' ends.
  • a reverse transcription reaction can be conducted by contacting RNA with a plurality of random-sequence primers, an enzyme having RNA-dependent DNA polymerase activity, and a plurality of nucleotide (or analogs thereof).
  • the reverse transcription reaction can be conducted with a mixture of random-sequence primers and target- specific primers.
  • a random-sequence primer comprises an oligonucleotide that generally includes a sequence that is based on a statistical expectation, or an empirical observation, that the sequence of the random primer is hybridizable to one or more target sequences in a plurality of nucleic acids.
  • the random sequence primer is not necessarily based on a particular or specific sequence of a nucleic acid.
  • a random-sequence primer comprises an oligonucleotide having a random sequence in which the nucleotides at any given position along the oligonucleotide can be any of the five deoxyribonucleotides (A, T, C, G or U) or analogs thereof.
  • the sequence of a random-sequence primer (or its complementary sequence) can be naturally-occurring, recombinant or synthesized by chemical synthesis methods.
  • the sequence of a random-sequence primer (or its complementary sequence) or may or may not be present in a plurality of nucleic acids.
  • a random-sequence primer comprises a randomly generated sequence.
  • the order of nucleotides in a random-sequence primer can be selected at random from two or more different nucleotides.
  • all possible sequence combinations of the nucleotides selected at random may be represented in a collection of random-sequence primers.
  • generation of one or more random primers does not include a step of excluding or selecting certain sequences or nucleotide combinations from the possible sequence combinations in the random portion of the one or more random primers.
  • a random-sequence primer can include a random sequence that is located in the 3 ' or 5' portion, or an internal portion of the random-sequence primer.
  • a random-sequence primer can include any homo-polymer sequence (e.g., polyA, polyG, polyC, polyT or polyU).
  • a plurality of random-sequence primers comprises two or more different sequences.
  • At least one random-sequence primer can hybridize to a region of an RNA molecule in a sample of a plurality of RNA molecules.
  • the 3' end of the random-sequence primers can hybridize to a portion of an RNA molecule.
  • the entire length of the random-sequence primers can hybridize to a portion of an RNA molecule.
  • a plurality of random-sequence primers contains a collection of random-sequence primers having the same or different sequences.
  • at least one of the random-sequence primers in the plurality can hybridize to at least one target sequence.
  • different random-sequence primers in the plurality can hybridize to different target sequences.
  • a random-sequence primer can hybridize to a plurality of different sites on a target nucleic acid.
  • one portion of a random-sequence primer includes a random sequence, and another portion of includes a defined sequence.
  • a random-sequence primer comprises a tailed primer.
  • a tailed primer includes a 3 '-region having a random sequence that hybridizes to a target nucleic acid molecule, and a 5 '-region that is a non-hybridizing sequence.
  • the non-hybridizing portion of a tailed primer includes non-random sequence.
  • the 3 '-region of a random-sequence primer includes a random sequence in
  • the 5' non- hybridizing portion of a tailed primer can be about 2-10, or about 10-20, or about 20-30, or about 30- 50 nucleotides in length.
  • the random-sequence primers are about 4 or 5 bases, or 6-10 bases, or about 10-15 bases, or about 15-20 bases, or about 20-25 bases, or about 25-30 bases in length, or longer. In some embodiments, the random-sequence primers can be up to about 100 bases in length.
  • the random-sequence primers comprise pentameric, hexameric, heptameric, octomeric, nonameric, decameric, or higher order lengths of oligonucleotide primers.
  • the reverse transcribing reactions employ single-stranded or double-stranded nucleic acid primers. In some embodiments, the reverse transcribing reactions employ DNA, RNA or DNA/RNA hybrid primers. [00375] In some embodiments, the reverse transcribing reactions produce a plurality of first strand cDNA products, a plurality of second strand cDNA products, and/or a plurality of first and second strand cDNA products.
  • the reverse transcribing reactions include RNA that is naturally-occurring, recombinant, synthetically-prepared, or any combination of these types of RNA.
  • the reverse transcribing reactions include RNA that comprises total RNA, RNA enriched for one or more RNA species, or non-enriched RNA.
  • the reverse transcribing reactions include a plurality of primers that comprise DNA, RNA or DNA/RNA hybrid oligonucleotides.
  • the plurality of primers comprises single-stranded or double-stranded primers.
  • at least one of the primers in the plurality of primers comprises a sequence that can hybridize to at least a portion of the one or more RNA.
  • plurality of primers comprises any one or any combination of: random-sequence primers, target-specific primers, homo-polymer primers (e.g., polyA, polyT, polyG, polyC or polyU primers), labeled primers, non-labeled primers, and/or non- extendible primers.
  • the non-extendible primers includes a 3 ' end linked to at least one blocking group that inhibits or blocks primer extension by a polymerase.
  • the reverse transcribing reactions comprise a plurality of random sequence primers and can generate a plurality of different cDNAs that correspond to polyA RNA and non-polyA RNA sequences.
  • the reverse transcribing reactions include an enzyme having
  • RNA-dependent DNA polymerase activity and RNase H activity or the enzyme has reduced or lacks RNase H activity.
  • the enzyme having RNA-dependent DNA polymerase activity can also include DNA-dependent DNA polymerase activity, or reduced or lack DNA- dependent DNA polymerase activity.
  • the enzyme having RNA-dependent DNA polymerase activity can be derived from a viral, retroviral, prokaryote or eukaryote source.
  • the enzyme having RNA-dependent DNA polymerase activity can be a heat- labile enzyme or can exhibit improved thermal-stability.
  • the reverse transcribing reactions include a plurality of nucleotides includes deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides or modified ribonucleotides.
  • the plurality of nucleotides comprises a purine and/or pyrimidine base, including adenine, guanine, cytosine, thymine or uracil.
  • the reverse transcribing reactions can be conducted at a temperature range of about 20 - 60 °C.
  • temperature ranges above approximately 42 °C are useful for reducing secondary structures that can form in RNA.
  • Temperature ranges of lower than about 42 °C are useful when employing random sequence primers.
  • a transcription step (e.g., in vitro transcription) can precede the reverse transcription step.
  • the reverse transcribing reaction and the multiplex nucleic acid amplification reaction can be conducted in a single reaction vessel.
  • the reverse- transcribing reaction can be conducted in a first reaction vessel, and the multiplex nucleic acid amplification reaction can be conducted in a second reaction vessel.
  • the reverse- transcribing reaction can be conducted in a first single reaction mixture.
  • the multiplex nucleic acid amplification reaction can be conducted in a second single reaction mixture.
  • the reverse transcribing reaction can be conducted in a first reaction mixture, and additional reagents can be added to the first reaction mixture to conduct the multiplex nucleic acid amplification reaction.
  • the disclosure relates generally to compositions, methods, systems, apparatuses and kits, comprising a kit which contains a plurality of target-specific primers.
  • the target-specific primers in a kit are complementary or identical to at least a portion of one or more target polynucleotides containing sequences derived from one or more expressed genes a single cell or from a plurality of cells.
  • the target-specific primers in a kit are complementary or identical to at least a portion of one or more target polynucleotides that contain sequences derived from one or more expressed genes in a non-diseased cell, a cancer cell, an ooctye, an embryo, a stem cell, or a cell exposed to a companion diagnostic compound.
  • the kit contains at least 1000, 2500, 5000, 7500, 10,000,
  • the kit contains at least 1000, 2500, 5000, 7500, 10,000,
  • At least one primer in the plurality of target-specific primers in the kit contains at least one cleavable group.
  • each of the plurality of target-specific primers in the kit contains at least one cleavable group.
  • the cleavable group can be 8-oxo-deoxyguanosine, deoxyuridine or bromodeoxyuridine.
  • kits further comprise a cleaving agent capable of cleaving the at least one cleavable group of the plurality of target specific primers.
  • the cleaving agent includes RNaseH, uracil DNA glycosylase, Fpg or alkali.
  • the cleaving agent includes uracil DNA glycosylase.
  • kits further comprise at least one polymerase.
  • the at least one DNA polymerase includes a thermostable or thermal labile DNA polymerase.
  • kits further comprise a plurality of nucleotides.
  • kits further comprise a ligase.
  • the ligase includes RNA or DNA ligase.
  • kits further comprise one or more adaptors.
  • the one or more adaptors in the kit are not complementary or identical to the 5' end of the plurality of target-specific primers.
  • the one or more adapters in the kit do not include a nucleic acid sequence that is complementary or identical to the terminal 10 nucleotides at the 5' end of the plurality of target-specific primers.
  • the one or more adapters in the kit comprise a universal priming sequence, a tag, or a unique identifier sequence (e.g., barcode sequence).
  • the universal priming sequence comprises an amplification priming sequence or a sequencing priming sequence.
  • At least one of the one or more adaptors in the kit is phosphorylated at the
  • a plurality of the one or more adaptors in the kit is single-stranded or double-stranded.
  • the kit further comprises reagents, which can include any one or any combination of: magnesium, manganese, calcium, potassium, dithiothreitol (DTT), glycerol, spermidine, and/or BSA (bovine serum albumin), formamide, DMSO, betaine, trehalose, sulfones, sodium pyrophosphate, low molecular amides, and/or single-stranded binding proteins.
  • reagents can include any one or any combination of: magnesium, manganese, calcium, potassium, dithiothreitol (DTT), glycerol, spermidine, and/or BSA (bovine serum albumin), formamide, DMSO, betaine, trehalose, sulfones, sodium pyrophosphate, low molecular amides, and/or single-stranded binding proteins.
  • each of the components of the kit can be provided in a separate container or vessel, or any combination of a mixture of different components can be
  • any one or more than one component of the kit can be provided in dry form, including in crystallized, freeze-dried, lyophilized form.
  • any one or more than one component in the kit can be provided in solution, including in an aqueous solution.
  • the components of the kit including any one or any combination of: primers (e.g., a plurality of target-specific primers and/or random sequence primers), polymerase, plurality of nucleotides, cleaving agent, reverse transcriptase, adaptors (e.g., DNA/DNA or RNA/DNA adaptors), RNA and/or RNA ligase, magnesium, manganese, calcium, potassium, dithiothreitol (DTT), glycerol, spermidine, and/or BSA (bovine serum albumin), formamide, DMSO, betaine, trehalose, sulfones, sodium pyrophosphate, low molecular amides, and/or single-stranded binding proteins.
  • the kit is provided to perform multiplex PCR in a single reaction chamber or vessel.
  • the plurality of target polynucleotides within the reaction mixture includes, or is otherwise derived from, DNA or RNA isolated from a sample (e.g., a biological sample).
  • the biological sample optionally includes a single cell, a plurality of cells, a cell culture, cell lysate, a tissue, an organ, bodily fluid (including but not limited to urine, stool, saliva, blood, plasma, serum, lymph, cerebrospinal fluid, and cell or tissue exudate).
  • the plurality of polynucleotides can be extracted from DNA or RNA, cells or tumors circulating in any bodily fluid.
  • the bodily fluid includes blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen.
  • the plurality of target polynucleotides in the reaction mixture includes at least some polynucleotides synthesized in vitro (e.g., in vitro transcription).
  • the biological sample includes a single cell.
  • the biological sample includes fetal cells or fetal DNA extracted from maternal tissue or blood taken from a pregnant woman.
  • at least some of the plurality of target polynucleotides are extracted or otherwise derived from a biological sample containing at least one cell or bodily fluid.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits for amplifying one or more target polynucleotides derived from a single source, such as cDNA or RNA.
  • the one or more target polynucleotides are derived from RNA obtained from a single cell.
  • the one or more target polynucleotides derived from RNA are obtained from a population of cells.
  • the RNA derived from a population of cells is obtained from a cancer cell, oocyte, embryo, stem cell, or a cell exposed to a companion diagnostic compound.
  • the one or more target polynucleotides include cDNA that is reverse transcribed from a RNA transcriptome. In one embodiment, the one or more target polynucleotides include cDNA that is reverse transcribed from a RNA transcriptome and the plurality of target polynucleotides are representative or indicative of the level of mRNA expression of one or more active genes in the RNA transcriptome. In yet another embodiment, the one or more target polynucleotides include a cDNA population that represents mRNA expression in the RNA transcriptome.
  • the sample contains a single type of nucleic acid or a mixture of different types of nucleic acids. In some embodiments, the sample contains a plurality of nucleic acids having the same sequence or different sequences. In some embodiments, the sample contains single-stranded or double-stranded nucleic acids. In some embodiments, the sample contains RNA, cDNA or DNA. In some embodiments, the sample contains a plurality of nucleic acids that are naturally-occurring, recombinant or synthetically-prepared.
  • the sample contains nucleic acids that are isolated from a single fresh or archived cell, fresh cells, fresh tissues, or archived cells or tissues that are formalin-treated and/or embedded in paraffin or plastic, or cells or tissues that are formalin fixed paraffin-embedded (FFPE).
  • FFPE formalin fixed paraffin-embedded
  • the sample contains nucleic acids that are isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, and viruses; cells; tissues; normal or diseased cells or tissues or organs, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen; environmental samples; culture samples; or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods.
  • organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, and viruses
  • cells tissues; normal or diseased cells or tissues or organs, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, and semen
  • environmental samples culture samples
  • culture samples or synthesized nucleic acid molecules prepared using recombinant mole
  • the sample contains nucleic acids that are unfragmented, or fragmented by mechanical force, chemical, enzyme or heat. In some embodiments, the sample contains nucleic acids that are depleted of one or more nucleic acid species.
  • the sample includes polynucleotides derived from whole- genome amplification (WGA) of genomic DNA extracted from a single cell, multiple cells, whole tissue, blood or other bodily fluid.
  • WGA whole- genome amplification
  • the single cell is taken from a fertilized zygote, blastocyst or embryo, or is a fetal cell extracted from maternal tissue or blood, or is a tumor cell (e.g., a circulating tumor cell).
  • the plurality of target polynucleotides (e.g., in the single reaction mixture) comprises a plurality of single-stranded or double-stranded nucleic acids derived from one or more cells.
  • the plurality of target polynucleotides comprises RNA, DNA, or cDNA derived from one or more cells.
  • the DNA can be isolated from a naturally-occurring source, recombinant, or synthesize by a chemical synthesis procedure.
  • the cDNA can be derived from RNA.
  • the plurality of target polynucleotides comprises first strand cDNA, second strand cDNA, or both first and second strand cDNA.
  • the plurality of target polynucleotides comprises single-stranded or double-stranded cDNA.
  • the single-stranded or double-stranded cDNA can be generated from RNA.
  • the RNA can be isolated from one or more cells or the plurality of RNA can be generated by an in vitro transcription procedure. In some embodiments, any reverse transcription reaction can be used to generate the plurality of cDNA.
  • the plurality of target polynucleotides includes any one or any combination of wild-type sequences, mutant sequences, fusion sequences, splice isoforms, allelic variants, and/or single nucleotide variants.
  • the relative abundance of the different target polynucleotide sequences, in the plurality of target polynucleotides reflects the abundances of different polynucleotide sequences present in a whole transcriptome, or in a portion of a whole transcriptome.
  • the composition (as well as related methods, systems, apparatuses and kits) includes a plurality target polynucleotides where at least one of the target polynucleotides includes at least one mutational hotspot, single nucleotide polymorphism (SNP), short tandem repeat (STR), genetic variant, genetic rearrangement (such as a translocation, deletion, insertion, duplication, truncation, copy number variation), coding region, splice variant, RNA transcript, RNA transcript fusion, exon or gene.
  • SNP single nucleotide polymorphism
  • STR short tandem repeat
  • the composition (as well as related methods, systems, apparatuses and kits) includes a plurality of target polynucleotides where at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of the target polynucleotides include at least one mutational hotspot, single nucleotide polymorphism (SNP), short tandem repeat (STR), genetic variant, genetic rearrangement (such as a translocation, deletion, insertion, duplication, truncation, copy number variation), coding region, splice variant, RNA transcript, RNA transcript fusion, exon or gene.
  • SNP single nucleotide polymorphism
  • STR short tandem repeat
  • one or more of the target polynucleotides include a copy number variation. In another embodiment, one or more of the target polynucleotides include an RNA transcript or splice variant. In yet another embodiment, the plurality of target polynucleotides includes RNA transcripts. In yet another embodiment, the plurality of target polynucleotides can be derived from a cell population. In one embodiment, the plurality of target polynucleotides can be derived from RNA of a single cell. In another embodiment, the plurality of target polynucleotides can be formed by reverse transcription of RNA extracted from a cell population.
  • the plurality of target polynucleotides can include a plurality of cDNA formed via reverse transcription of total mRNA extracted from a cell population.
  • the total mRNA extracted from a cell population can include an aberrant transcript within the RNA transcriptome and the plurality of target polynucleotides includes a cDNA derived from the aberrant transcript.
  • the aberrant transcript can be associated with cancer.
  • the aberrant transcript can include a splice transcript within the RNA transcriptome and the plurality of target polynucleotides includes a cDNA derived from the aberrant splice transcript.
  • the aberrant splice transcript is associated with cancer.
  • the composition includes a plurality of target polynucleotides derived from RNA that are associated with at least one mutation associated with cancer, and where the mutation is located in at least one of the genes selected from: ABI1 ; ABL1 ; ABL2; ACSL3; ACSL6; AFF1 ; AFF3; AFF4;AKAP9; AKT1 ; AKT2; ALK; APC; ARHGAP26; ARHGEF12; ARID 1 A; ARNT; ASPSCR1 ; ASXL1 ; ATF1 ; ATIC; ATM; AXIN2; BAP1 ; BARD1 ; BCAR3; BCL10; BCL11A; BCL11B; BCL2; BCL3; BCL6; BCL7A;BCL9; BCR; BIRC3; BLM; BMPR1A; BRAF; BRCA1 ; BRCA2; BRD3; BRD4; BRIP1 ; BUB 1B; C
  • FGFRIOP FGFR2; FGFR3; FH; FIP1L1 ; FLCN; FLU ; FLT1 ; FLT3; FNBP1 ; FOXL2; FOXOl ; FOX03; FOX04; FOXP1 ; FUS; GAS7; GATA1 ; GATA2; GAT A3; GMPS; GNAQ; GNAS;
  • GOLGA5 GOPC; GPC3; GPHNGPR124; HIP1 ; HIST1H4I; HLF; HNF1A; HNRNPA2B 1 ;
  • HSP90AA1 HSP90AB 1 ; IDH1 ; IDH2; IKZF1 ; IL2; IL21R; IL6ST; IRF4; ITGA10; ITGA9; ITK; JAK1 ; JAK2; JAK3; KDM5A; KDM5C; KDM6A; KDR; KDSR; KIAA1549; KIT; KLF6; KLK2; KRAS; KTNl; LASP1; LCK; LCP1; LHFP; LIFR; LM02; LPP; MAF; MALT1; MAML2; MAP2K1; MAP2K4; MDM2; MDM4; MECOM; MEN1; MET; MITF; MKL1; MLH1; MLL; MLLT1; ML
  • the composition includes a plurality of target polynucleotides derived from RNA having at least one mutation associated with cancer, where the mutation associated with cancer is located in at least one of the genes selected from: ABL1; AKT1; ALK; APC; ATM; BRAF; CDH1; CDKN2A; CSF1R; CTNNB1; EGFR; ERBB2; ERBB4; FBXW7; FGFR1; FGFR2; FGFR3; FLT3; GNAS; HNF1A; HRAS; IDH1; JAK2; JAK3; KDR; KIT; KRAS; MET; MLH1; MPL; NOTCH1; NPM1; NRAS; PDGFRA; PIK3CA; PTEN; PTPN11; RBI; RET; SMAD4; SMARCB1; SMO; SRC; STK11; TP53; and VHL.
  • the mutation associated with cancer is located in at least one of the genes selected from:
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits comprising one or more primers.
  • the primers can be used in a reverse transcription reaction, or a multiplex nucleic acid amplification reaction.
  • the primers comprise single- or double-stranded DNA, RNA or DNA/RNA hybrid oligonucleotides.
  • a primer comprises an oligonucleotide, or a self -priming nucleic acid molecule, having a nucleotide sequence that can hybridize to a target nucleic acid, such as a target RNA, cDNA or DNA molecule. A portion or the entire length of a primer can hybridize to a target nucleic acid.
  • the primers can hybridize to a target nucleic acid molecule by hydrogen bond formation via Watson-Crick or Hoogstein binding to form a duplex nucleic acid structure.
  • the hybridizing involves complementary base pairing, including standard A-T or C-G base pairing, or optionally other forms of base-pairing interactions.
  • a primer includes an extendible 3'-OH group.
  • a primer can promote nucleotide polymerization by a polymerase enzyme.
  • a primer includes a 3 ' end having a blocking group that inhibits or blocks primer extension.
  • the blocking group is removable with a chemical compound, enzyme, heat or electromagnetic energy.
  • a primer can be about 4 - 100 nts in length, or longer.
  • a primer used to generate cDNA can be about 5- 15 bases, or about 15-30 bases, or about 30-45 bases, or about 45-60 bases, or about 60-75 bases in length, or about 75- 100 bases in length, or longer.
  • a plurality of primers comprise any one or any mixture of target-specific primers, random-sequence primers, homo-polymer primers (e.g., polyA, polyT, polyG or polyC primers), primers labeled with a detectable moiety, non-labeled primers, and/or primers having the 3 ' end linked to at least one blocking group that inhibits or blocks primer extension.
  • homo-polymer primers e.g., polyA, polyT, polyG or polyC primers
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprising a plurality of target-specific primer pairs which include two or more different pairs of target-specific primers.
  • the multiplex nucleic acid amplification reaction includes a plurality of target-specific primer pairs that comprise DNA, RNA or DNA/RNA hybrid
  • the plurality of target-specific primer pairs comprises single-stranded or double-stranded primers.
  • the plurality of target-specific primer pairs comprises forward and reverse primers.
  • a pair of target-specific primers includes a forward and a reverse target-specific primer, or two forward target-specific primers, or two reverse target-specific primers.
  • each of the primers in the target-specific primer pairs comprises a sequence that can hybridize to at least one portion of a single target polynucleotide sequence, or comprises a sequence that can hybridize to a complementary sequence of at least one portion of a single target polynucleotide sequence.
  • a single pair of target-specific primers hybridizes to any given target polynucleotide.
  • a single pair of target-specific primers can hybridize to a single target polynucleotide.
  • a single pair of target-specific primers can hybridize to a single target sequence of cDNA, DNA or RNA.
  • a single pair of target-specific primers can be used to amplify a single target polynucleotide. In some embodiments, a single pair of target-specific primers can be used to amplify a single target sequence of cDNA, DNA or RNA. In some embodiments, more than one pair of target-specific primers can be used to amplify one cDNA.
  • At least one of the target-specific primer pairs has minimal cross-hybridization with any other pair of primers in the single reaction mixture.
  • each primer pair in the plurality of target-specific primer pairs is designed to hybridize to a different target polynucleotide sequence of interest. For example, if there are N different target polynucleotides sequences of interest, then the plurality of target-specific primer pairs will contain N different primer pairs.
  • the plurality of target-specific primer pairs includes 2-100, or about 100-500, or about 500-1,000, or about 1,000- 5,000, or about 5,000- 10,000, or about 10,000-15,000, or about 15,000-20,000, or about 20,000-25,000, or about 25,000- 50,000 or about 50,000-100,000, or more different target-specific primer pairs. In some embodiments, the plurality of target-specific primer pairs includes about 20,000 different target-specific primer pairs.
  • a target-specific primer can be about 5-15 bases, or about 15-
  • 30 bases or about 30-45 bases, or about 45-60 bases, or about 60-75 bases in length, or about 75-100 bases in length, or longer.
  • the two primers in a target-specific primer pair can be the same length or different lengths.
  • At least the 3' region of a target-specific primer can hybridize to a region of a cDNA molecule. In some embodiments, the entire length of a target-specific primer can hybridize to a region of a cDNA molecule.
  • At least one primer in a plurality of target specific primer pairs comprises a tailed primer.
  • the tailed primer includes a 3 '-region having a sequence that hybridizes to at least a portion of a target polynucleotide, and a 5 '-region that is a non- hybridizing sequence.
  • the non-hybridizing portion of a tailed primer includes non-random sequence.
  • the non-hybridizing portion of a tailed primer can be about 2-10, or about 10-20, or about 20-30, or about 30-50 nucleotides in length.
  • plurality of target-specific primer pairs comprises any one or any combination of: labeled primers, non-labeled primers, and/or non-extendible primers.
  • the non-extendible primers include a 3 ' end linked to at least one blocking group that inhibits or blocks primer extension by a polymerase.
  • At least one target-specific primer in a pair of primers can hybridize to an exon sequence, intron sequence, exon/intron junction sequence, or intron/exon junction sequence. In some embodiments, each pair of the target-specific primers can hybridize to a different exon sequence in a different target polynucleotide. In some embodiments, at least one of the primers of the plurality of different target-specific primer pairs can hybridize to a different exon sequence in a different target polynucleotide.
  • a primer in a plurality of target-specific primer pairs has minimal cross-hybridization with any other primer in the plurality.
  • At least one primer in a plurality of target-specific primer pairs comprises a nucleic acid sequence that is substantially non-complementary to one or more primers in the plurality.
  • At least one primer in a plurality of target-specific primer pairs comprises a nucleic acid sequence that is substantially non-self-complementary.
  • At least one primer of the plurality of target-specific primer pairs includes at least one cleavable group.
  • the cleavable group comprises: uracil, uridine, inosine, or 7,8-dihydro-8-oxoguanine (8-oxoG) nucleobases.
  • the cleavable group is cleavable with uracil DNA glycosylase (UDG, also referred to as UNG),
  • At least one primer of the plurality of target-specific primer pairs includes or lacks a protecting group that inhibits nucleic acid degradation or digestion.
  • At least one of the target-specific primer in the pair of primers includes a unique identifier sequence (e.g., a barcode sequence).
  • target-specific primers are designed to minimize the formation of primer- dimers, dimer-dimers or other non-specific amplification products.
  • target-specific primers are optimized to reduce GC bias and low melting temperatures (T m ) during the amplification reaction.
  • T m low melting temperatures
  • the target-specific primers are designed to possess a T m of about 55°C to about 72°C.
  • the target-specific primers of a target-specific primer pool can possess a T m of about 59°C to about 70°C, 60°C to about 68°C, or 60°C to about 65°C.
  • the target-specific primer pool can possess a T m that does not deviate by more than 5°C across the target-specific primer pool.
  • target-specific primers can be designed de novo using algorithms that generate oligonucleotide sequences according to specified design criteria.
  • the primers may be selected according to any one or more of the criteria specified herein.
  • one or more of the target-specific primers are selected or designed to satisfy any one or more of the following criteria: (1) inclusion of two or more modified nucleotides within the primer sequence, at least one of which is included near the 3' end or 5' end of the target-specific primer and at least one modified nucleotides is included at, or about the center nucleotide position of the target-specific primer sequence; (2) target-specific primer length of about 15 to about 50 bases in length; (3) T m of from about 60°C to about 70°C; (4) low cross-reactivity with non-target polynucleotides present in the sample of interest; (5) for each target-specific primer in a given reaction, the sequence of at least the first four nucleotides (going from 3' to 5
  • the target-specific primers include one or more target-specific primer pairs that amplify target polynucleotides from the sample that are about 100 base pairs to about 1,000 base pairs in length. In some embodiments, the target-specific primers include a plurality of target-specific primer pairs designed to amplify target polynucleotides, where the amplified target polynucleotides vary in length from each other by no more than 50%, typically no more than 25%, 10%, or 5%.
  • one target-specific primer pair is selected (or predicted) to amplify a product that is 100 nucleotides in length
  • other primer pairs are selected (or predicted) to amplify products that are between 50-150 nucleotides in length, typically between 75-125 nucleotides in length, 90-110 nucleotides, 95-105 nucleotides, or 99-101 nucleotides in length.
  • At least one primer pair in the amplification reaction is not designed de novo according to any predetermined selection criteria.
  • at least one primer pair can be an oligonucleotide sequence selected or generated at random, or previously selected or generated for other applications.
  • the amplification reaction can include at least one primer pair selected from the TaqMan® probe reagents (Roche Molecular Systems).
  • the TaqMan® reagents include labeled probes and can be useful, inter alia, for measuring the amount of target sequence present in the sample, optionally in real time.
  • a method comprising: (1) receiving one or more genomic regions or nucleic acid sequences of interest; (2) determining one or more target polynucleotides for the received one or more genomic regions or nucleic acid sequences of interest; (3) providing one or more target-specific primer pairs for each of the determined one or more target polynucleotides; (4) scoring the one or more target-specific primer pairs, wherein the scoring comprises a penalty based on the performance of in silico PCR for the one or more target- specific primer pairs, and optionally, wherein the scoring further comprises an analysis of SNP overlap for the one or more target-specific primer pairs; and (5) filtering the one or more target- specific primer pairs based on a plurality of factors, including at least the penalty and optionally, the analysis of SNP overlap, to identify a filtered set of target-specific primer pairs corresponding to one or more candidate amplicon sequences for the one or more genomic regions or nucleic acid sequences of interest.
  • receiving one or more genomic regions or nucleic acid sequences of interest may comprise receiving a list of one or more gene symbols, RNA transcripts or identifiers.
  • Receiving one or more genomic regions or nucleic acid sequences of interest may comprise receiving a list of one or more genomic coordinates or other genomic or transcriptome location identifiers.
  • the performance of in silico PCR may comprise performing in silico PCR against a reference or previously sequenced genome or RNA transcriptome, of any species.
  • the performance of in silico PCR may comprise performing in silico PCR against an hgl9 reference genome.
  • the performance of in silico PCR against a reference genome or RNA transcriptome may comprise determining a number of off-target hybridizations for each of the one or more target-specific primer pairs.
  • the performance of in silico PCR against a reference genome or RNA transcriptome may comprise determining a worst case attribute or score for each of the one or more target-specific primer pairs.
  • the performance of in silico PCR may comprise determining one or more genomic coordinates or transcriptome identifies for each of the one or more target-specific primer pairs.
  • the performance of in silico PCR may comprise determining one or more predicted amplicon sequences for each of the one or more target-specific primer pairs.
  • the performance of in silico PCR may comprise querying an amplicon or other genomic or transcription sequence database for a presence therein of the one or more genomic regions or nucleic acid sequences of interest or of in silico PCR results for the one or more target specific primer pairs and information related thereto.
  • At least one of the target-specific primer pairs within the amplification reaction can be labeled, for example with an optically detectable label, to facilitate a particular application of interest.
  • labeling may facilitate quantification of target polynucleotide and/or amplification product, isolation of the target polynucleotide and/or amplification product, and the like.
  • the disclosure generally relates to compositions (as well as related kits, methods, systems and apparatuses using the disclosed compositions) for performing nucleic acid amplification and nucleic acid synthesis.
  • the compositions include a target-specific primer of about 15 to about 40 nucleotides in length having a uracil nucleotide located near the 3' or 5' end of the target-specific primer and a second uracil nucleotide located near a central nucleotide position of the target-specific primer.
  • compositions include a target-specific primer of about 15 to about 40 nucleotides in length having an inosine nucleotide located near the 3' end of the target-specific primer and at least a second inosine nucleotide located near a central nucleotide position of the target-specific primer.
  • the disclosure generally relates to compositions (as well as related kits, methods, systems and apparatuses using the disclosed compositions) for performing nucleic acid amplification and nucleic acid synthesis.
  • one or more of the compositions disclosed herein (as well as related methods, kits, systems and apparatuses) can include at least one target-specific primer and/or at least one adapter.
  • the compositions disclosed herein can include at least one target-specific primer and/or at least one adapter.
  • compositions include a plurality of target-specific primers (or primer pairs) and adapters that are about 15 to about 40 nucleotides in length.
  • the compositions include one or more target-specific primers (or primer pairs) or adapters that include one or more cleavable groups.
  • one or more types of cleavable groups can be incorporated into a target- specific primer (or one or more primer pairs) or an adapter.
  • a cleavable group can be located at, or near, the 3' end of a target-specific primer or adapter.
  • a cleavable group can be located at a terminal nucleotide, a penultimate nucleotide, or any location that corresponds to less than 50% of the total nucleotide length of the target-specific primer or adapter.
  • a cleavable group can be incorporated at, or near, the central nucleotide of the target-specific primer or the adapter.
  • a target specific primer of 40 bases can include a cleavage group at nucleotide positions 15-25.
  • a target-specific primer or an adapter can include a plurality of cleavable groups within its 3' end, its 5' end, or about the central nucleotide position.
  • the 5' end of a target-specific primer includes only non-cleavable nucleotides.
  • the cleavable group can include a modified nucleobase or modified nucleotide.
  • the cleavable group can include a nucleotide or nucleobase that is not naturally occurring in the corresponding nucleic acid.
  • a DNA nucleic acid can include a RNA nucleotide or nucleobase.
  • a DNA based nucleic acid can include uracil or uridine.
  • a DNA based nucleic acid can include inosine.
  • the cleavable group can include a moiety that can be cleaved from the target- specific primer or adapter by enzymatic, chemical or thermal means.
  • a uracil or uridine moiety can be cleaved from a target-specific primer or adapter using a uracil DNA glycosylase.
  • an inosine moiety can be cleaved from a target-specific primer or adapter using hAAG or EndoV.
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprise nucleic acid hybridization, include hybridizing primers to RNA, cDNA or target polynucleotides.
  • any of the reverse transcription or multiplex PCR reactions can include conditions that are suitable for hybridizing primers to nucleic acids.
  • a plurality of random sequence primers can be hybridized to a plurality of RNA under suitable hybridization conditions to form a primer/RNA complex.
  • a plurality of target-specific primer pairs can be hybridized to a plurality of target polynucleotides under suitable hybridization conditions to form a primer/polynucleotide complex.
  • hybridizing involves hydrogen bond formation via Watson-
  • the hybridizing involves complementary base pairing, including standard A-T or C-G base pairing, or optionally other forms of base-pairing interactions.
  • the suitable hybridizing conditions include hybridizing primers to nucleic acids at a temperature that is close to a calculated or empirically-derived melting temperature.
  • Methods for nucleic acid hybridization are well known in the art.
  • a thermal melting temperature is calculated for the primers, target template and product.
  • thermal melting temperature (T m ) for nucleic acids can be a temperature at which half of the nucleic acid strands are double-stranded and half are single-stranded under a defined condition.
  • a defined condition can include ionic strength and pH in an aqueous reaction condition.
  • a defined condition can be modulated by altering the concentration of salts (e.g., sodium), magnesium, temperature, pH, buffers, and/or formamide.
  • the calculated thermal melting temperature can be at about 5-30° C below the T m , or about 5-25° C below the T m , or about 5-20° C below the T m , or about 5-15° C below the T m , or about 5-10° C below the T m .
  • T m for hybridizing or denaturing nucleic acids
  • OligoAnalyze from Integrated DNA Technologies
  • Primer3 distributed by the Whitehead Institute for Biomedical Research
  • the disclosure relates generally to compositions, as well as related, systems, methods, kits and apparatuses, comprising one or more nucleotides.
  • the compositions includes one type, or a mixture of different types of nucleotides.
  • a nucleotide comprises any compound that can bind selectively to, or can be polymerized by, a polymerase. Typically, but not necessarily, selective binding of the nucleotide to the polymerase is followed by polymerization of the nucleotide into a nucleic acid strand by the polymerase.
  • nucleotides include not only naturally occurring nucleotides but also any analogs, regardless of their structure, that can bind selectively to, or can be polymerized by, a polymerase. While naturally occurring nucleotides typically comprise base, sugar and phosphate moieties, the nucleotides of the present disclosure can include compounds lacking any one, some or all of such moieties. In some embodiments, the nucleotide can optionally include a chain of phosphorus atoms comprising three, four, five, six, seven, eight, nine, ten or more phosphorus atoms. In some embodiments, the phosphorus chain can be attached to any carbon of a sugar ring, such as the 5' carbon.
  • the phosphorus chain can be linked to the sugar with an intervening O or S.
  • one or more phosphorus atoms in the chain can be part of a phosphate group having P and O.
  • the phosphorus atoms in the chain can be linked together with intervening O, NH, S, methylene, substituted methylene, ethylene, substituted ethylene, CNH 2 , C(O), C(CH 2 ), CH 2 CH 2 , or C(OH)CH 2 R (where R can be a 4-pyridine or 1- imidazole).
  • the phosphorus atoms in the chain can have side groups having O, BH 3 , or S.
  • a phosphorus atom with a side group other than O can be a substituted phosphate group.
  • phosphorus atoms with an intervening atom other than O can be a substituted phosphate group.
  • nucleotides that can be used in the disclosed compositions (and related methods, systems, kits and apparatuses) include, but are not limited to, ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotide polyphosphates, modified ribonucleotide polyphosphates, modified deoxyribonucleotide polyphosphates, peptide nucleotides, modified peptide nucleotides, metallonucleosides, phosphonate nucleosides, and modified phosphate-sugar backbone nucleotides, analogs, derivatives, or variants of the foregoing compounds, and the like.
  • the nucleotide can comprise non-oxygen moieties such as, for example, thio- or borano- moieties, in place of the oxygen moiety bridging the alpha phosphate and the sugar of the nucleotide, or the alpha and beta phosphates of the nucleotide, or the beta and gamma phosphates of the nucleotide, or between any other two phosphates of the nucleotide, or any combination thereof.
  • a nucleotide can include a purine or pyrimidine base, including adenine, guanine, cytosine, thymine or uracil.
  • a nucleotide includes dATP, dGTP, dCTP, dTTP and dUTP.
  • the nucleotide is unlabeled.
  • the nucleotide comprises a label and referred to herein as a "labeled nucleotide".
  • the label can be in the form of a fluorescent dye attached to any portion of a nucleotide including a base, sugar or any intervening phosphate group or a terminal phosphate group, i.e., the phosphate group most distal from the sugar.
  • the disclosure relates generally to compositions, as well as related, systems, methods, kits and apparatuses, comprising any one or any combination of capture primers, reverse primers, fusion primers, target nucleic acids and/or nucleotides that are non-labeled or attached to at least one label.
  • the label comprises a detectable moiety.
  • the label can generate, or cause to generate, a detectable signal.
  • the detectable signal can be generated from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events).
  • a proximity event can include two reporter moieties approaching each other, or associating with each other, or binding each other.
  • the detectable signal can be detected optically, electrically, chemically, enzymatic ally, thermally, or via mass spectroscopy or Raman spectroscopy.
  • the label can include compounds that are luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent or
  • the label can include compounds that are fluorophores, chromophores, radioisotopes, haptens, affinity tags, atoms or enzymes.
  • the label comprises a moiety not typically present in naturally occurring nucleotides.
  • the label can include fluorescent, luminescent or radioactive moieties.
  • the disclosure relates generally to compositions, as well as related, systems, methods, kits and apparatuses, comprising cleaved amplified nucleic acids linked to one or more adaptors to generate adapter-ligated amplified nucleic acids.
  • one or more adaptors can be joined to the cleaved amplified nucleic acid by ligation.
  • a tailed amplification primer can be used in a PCR reaction to append one or more adaptors to an amplicon or a cleaved amplified nucleic acid, where the tailed amplification primer includes the sequence of one or more adaptors.
  • the adaptor comprises a nucleic acid, including DNA, RNA,
  • the adaptor can include one or more deoxyribonucleoside or ribonucleoside residues.
  • the adaptor can be single-stranded or double-stranded nucleic acids, or can include single-stranded and/or double- stranded portions.
  • the adaptor can have any structure, including linear, hairpin, forked (Y-shaped), or stem-loop.
  • the adaptor can have any length, including fewer than 10 bases in length, or about 10-20 bases in length, or about 20-50 bases in length, or about 50-100 bases in length, or longer.
  • the adaptor can have any combination of blunt end(s) and/or sticky end(s).
  • at least one end of the adaptor can be compatible with at least one end of a cleaved amplified nucleic acid.
  • a compatible end of the adaptor can be joined to a compatible end of an amplified nucleic acid.
  • the adaptor can have a 5' or 3' overhang end.
  • the adaptor can have a 5' or 3' overhang tail.
  • the tail can be any length, including 1-50 or more nucleotides in length.
  • the adapter overhang includes a homopolymeric stretch of at least 5, 10, 20 or 25 identical contiguous nucleotide residues.
  • the adaptor can include an internal nick.
  • the adaptor can have at least one strand that lacks a terminal 5' phosphate residue.
  • the adaptor lacking a terminal 5' phosphate residue can be joined to a cleaved amplified nucleic acid to introduce a nick at the junction between the adaptor and the cleaved amplified nucleic acid.
  • the adaptor can include a nucleotide sequence that is identical or complementary to any portion of a capture primer, fusion primer, reverse primer, amplification primer, or a sequencing primer.
  • the adaptor can include identification sequences, such as for example, a uniquely identifiable sequence (e.g., barcode sequence).
  • a barcoded adaptor can be used for constructing a multiplex library of amplified nucleic acids.
  • the barcoded adaptors can be appended to a cleaved amplified nucleic acid and used for sorting or tracking the source of the target polynucleotide.
  • one or more barcode sequences can allow identification of a particular adaptor among a mixture of different adaptors having different barcodes sequences.
  • a mixture can include 2, 3, 4, 5, 6, 7-10, 10-50, 50- 100, 100-200, 200-500, 500- 1000, or more different adaptors having unique barcode sequences.
  • the adaptor can include degenerate sequences. In some embodiments, the adaptor can include one or more inosine residues.
  • the adaptor can include at least one scissile linkage.
  • the scissile linkage can be susceptible to cleavage or degradation by an enzyme or chemical compound.
  • the adaptor can include at least one phosphorothiolate, phosphorothioate, and/or phosphoramidate linkage.
  • the adaptor can include any type of restriction enzyme recognition sequence, including type I, type II, type lis, type IIB, type III, type IV restriction enzyme recognition sequences, or recognition sequences having palindromic or non-palindromic recognition sequences.
  • the adaptor can include a cell regulation sequences, including a promoter (inducible or constitutive), enhancers, transcription or translation initiation sequence, transcription or translation termination sequence, secretion signals, Kozak sequence, cellular protein binding sequence, and the like.
  • any target polynucleotide, amplicon or adaptor-ligated amplified nucleic acid can include a mutant sequence (e.g., aberrant sequence).
  • the mutant sequence includes any sequence that differs from a wild-type or normal sequence.
  • the mutant sequence includes any one or any combination of nucleotide: deletions, insertions, or substitutions or one or more nucleotides; inversions; rearrangements; truncations; and/or variant or abnormal splice junction sequences.
  • the reverse transcription or the multiplex nucleic acid amplification step can include a nucleic acid digestion step.
  • the digestion step can be conducted before or after any step of the disclosed methods.
  • the digestion step can be conducted enzymatically, chemically, with light or with heat.
  • the disclosure relates generally to compositions, and related methods, systems, kits and apparatuses, comprising a reverse transcription reaction, or nucleic acid amplification reaction that can be conducted under thermocycling or isothermal conditions, or a combination of both types of conditions.
  • a reaction mixture for conducting a reverse transcription reaction or a nucleic acid amplification reaction that is subjected to a temperature variation which is constrained within a limited range during at least some portion of the reverse transcription or amplification, including for example a temperature variation is within about 20 °C, or about 10 °C, or about 5 °C, or about 1-5 °C, or about 0.1- 1 °C, or less than about 0.1 °C.
  • an isothermal nucleic acid amplification reaction can be conducted for about 2, 5, 10, 15, 20, 30, 40, 50, 60 or 120 minutes, or longer.
  • an isothermal nucleic acid amplification reaction can be conducted at about 15-30 °C, or about 30-45 °C, or about 45-60 °C, or about 60-75 °C, or about 75-
  • the multiplex amplification reactions is conducted under temperature-cycling conditions (U.S. patent Nos. 4,683,202, 4,683, 195, 4,889,818, hereby incorporated by reference in their entireties).
  • the disclosure relates generally to methods, compositions, systems, apparatuses and kits, comprising a reaction vessel which includes a tube (e.g., Eppendorf TM tube), inner wall of a tube, well, reaction chamber, groove, channel reservoir, or flowcell.
  • a tube e.g., Eppendorf TM tube
  • inner wall of a tube e.g., well, reaction chamber, groove, channel reservoir, or flowcell.
  • two or more reaction vessels can be two or more reaction chambers arranged in an array.
  • the array can include one or more reaction chambers on a solid support.
  • a reaction chamber can have walls that define width and depth. The dimensions of a reaction chamber can be sufficient to permit deposition of reagents or for conducting reactions.
  • a reaction chamber can have any shape including cylindrical, polygonal or a combination of different shapes. Any wall of a reaction chamber can have a smooth or irregular surface.
  • a reaction chamber can have a bottom with a planar, concave or convex surface. The bottom and side walls of a reaction chamber can comprise the same or different material and/or can be coated with a chemical group that can react with a biomolecule such as nucleic acids, proteins or enzymes.
  • the reaction chamber can be one of multiple reaction chambers arranged in a grid or array.
  • An array can include two or more reaction chambers. Multiple reaction chambers can be arranged randomly or in an ordered array.
  • An ordered array can include reaction chambers arranged in a row, or in a two-dimensional grid with rows and columns.
  • An array can include any number of reaction chambers for depositing reagents and conducting numerous individual reactions.
  • an array can include at least 256 reaction chambers, or at least 256,000, or at least 1-3 million, or at least 3-5 million, or at least 5-7 million, or at least 7-9 million, at least 9- 1 1 million, at least 1 1-13 million reaction chambers, or even high density including 13-700 million reaction chambers or more.
  • Reaction chambers arranged in a grid can have a center-to-center distance between adjacent reaction chambers (e.g., pitch) of less than about 10 microns, or less than about 5 microns, or less than about 1 microns, or less than about 0.5 microns.
  • An array can include reaction chambers having any width and depth dimensions.
  • a reaction chamber can have dimensions to accommodate a single microparticle (e.g., microbead) or multiple microparticles.
  • a reaction chamber can hold 0.001 - 100 picoliters of aqueous volume.
  • At least one reaction vessel can be coupled to one or more sensors or can be fabricated above one or more sensors.
  • a reaction chamber that is coupled to a sensor can provide confinement of reagents deposited therein so that products from a reaction can be detected by the sensor.
  • a sensor can detect changes in products from any type of reaction, including any nucleic acid reaction such as primer extension, amplification or nucleotide incorporation reactions, within the reaction vessel.
  • a sensor can detect changes in ions (e.g., hydrogen ions), protons, phosphate groups such as pyrophosphate groups.
  • a sensor can detect at least one by product of nucleotide incorporation, including pyrophosphate, hydrogen ions, charge transfer, or heat.
  • at least one reaction chamber can be coupled to one or more field effect transistor (FET), including for example an ion sensitive field effect transistor (ISFET).
  • FET field effect transistor
  • ISFET ion sensitive field effect transistor
  • Examples of an array of reaction chambers coupled to ISFET sensors can be found at U.S. Patent No. 7,948,015, and U.S. serial No. 12/002,781 , hereby incorporated by reference in their entireties.
  • Other examples of sensors that detect byproducts of a nucleotide incorporation reaction can be found, for example, in Pourmand et al, Proc. Natl. Acad.
  • any of the methods for characterizing RNA can be conducted manually or by automation.
  • the steps of amplifying, analyzing, comparing, reverse transcribing, nucleic acid amplification, cleaving, adapter- ligating, characterizing, and/or sequencing can be conducted manually or by automation.
  • any reagents for conducting any of these steps can be deposited into, or removed from, a reaction vessel via manual or automated modes.
  • the methods of the disclosure can be performed as “addition- only” processes.
  • the “addition-only” process excludes the removal of all, or a portion of the first reaction mixture including the amplifying compositions, for further manipulation during the amplification steps, ligation and/or digestion steps.
  • the "addition- only” process can be automated for example for use in high-throughput processing.
  • the disclosed methods, compositions, systems, apparatuses and kits for characterizing RNA or DNA offers advantages over conventional methods.
  • one embodiment of the disclosed methods, compositions, systems, apparatuses and kits employs random-sequence primers in the reverse transcription step.
  • the random-sequence primers are designed to hybridize to many different types of RNA, including polyA and non-polyA RNA, which permits analysis of total RNA samples. Use of the random-sequence primers also obviates the requirement for a priori knowledge of the RNA sequences.
  • conducting the reverse transcription step with random-sequence primers generates a population of cDNA with improved representation of the original RNA population present in the starting sample. Preparing RNA samples having reduced sequence representation bias is important for RNA abundance analyses. Additionally, conducting the reverse transcription step with random-sequence primers reduces 3 ' sequence bias, which is prevalent when using polyT primers for priming polyA RNA.
  • the disclosed methods, compositions, systems, apparatuses and kits can be used to characterize RNA or DNA from any type of sample, including those from fresh or archived samples, or total RNA or pre-enriched RNA samples.
  • the methods do not require pre-enrichment procedures, the results can be optimized by removal of rRNA, or other abundant RNA species, using procedures such as RNA depletion, polyA selection, size selection, size modification, or RNA-protein complex selection.
  • the disclosed methods, compositions, systems, apparatuses and kits for characterizing RNA or DNA can be conducted in a single reaction vessel, which eliminates centrifugation steps, and transfer of the reagents to a fresh tube. This simplified the workflow requires fewer steps that would cause loss of nucleic acid material, and enables amplification of a sequence-of-interest from a sample containing as little as 500 pg of RNA (unfixed samples) or 5 ng RNA from FFPE samples.
  • the disclosed methods, compositions, systems, apparatuses and kits for characterizing RNA or DNA includes a multiplex amplification reaction performed in a single reaction mixture using hundreds, thousands, tens-of-thousands or even hundreds of thousands of different target-specific primer pairs that enable substantially simultaneous amplification of many- thousands of different target polynucleotide sequences-of-interest, to more accurately reflect the complexity and abundance of the RNA or DNA sequences of interest in the sample.
  • This ultra-plexy amplification reaction eliminates the requirement to perform separate amplification reactions and pooling, which simplifies the workflow, and reduces variations in amplification efficiency that arise in separate reaction vessels.
  • the disclosed methods, compositions, systems, apparatuses and kits for characterizing RNA or DNA includes a multiplex amplification reaction, where each target-specific primer pair is designed to hybridize to a single target polynucleotide sequence of interest.
  • the multiplex amplification reactions of the present teachings can yield data that more accurately measures transcript abundance because each target-specific primer pair has approximately a one-to-one correspondence with a single target polynucleotide sequence.
  • the number of amplicons containing the same (or substantially the same) sequence that are formed in the multiplex amplification step more directly reflects the abundance of a sequence-of- interest from which the amplicons were derived.
  • the number of amplicons identified as containing a first target sequence of interest can be determined to obtain a first number.
  • the first number can be used to calculate a first abundance value for the first target sequence.
  • the number of amplicons identified as containing a second target sequence of interest can also be determined to obtain a second number, optionally in the same sequencing assay or in a different and separate sequencing assay.
  • the methods can include determining a second abundance value for the second target sequence using the second number.
  • the relative abundances of the first and second target sequences can be compared, optionally as a ratio of the first and second numbers, or as a percentage (e.g., percentage of total sequence reads containing the first and/or second target sequence), or using any other suitable calculation method.
  • RNA or DNA includes target-specific primers that enable a streamlined library prep workflow, because they include at least one cleavable group which is used to create nucleic acid fragment ends that are ready for adapter-joining and sequencing.
  • characterizing RNA or DNA includes substantially reduced sequence read assembly, or sequence read assembly is not performed, because a single pair of target-specific primers is configured to generate a single sequence for each target polynucleotide.
  • RNA RNA styrene-maleic anhydride copolymer
  • the disclosed methods, compositions, systems, apparatuses and kits for characterizing RNA offers a one -pot amplification reaction that requires very small amounts of starting RNA, does not require pre-enrichment, yields amplified nucleic acids with improved transcript sequence representation that are sequence-ready with fewer steps.
  • a tube of diluted ERCC Spike-In Mix was prepared. 9 uL of nuclease-free water was distributed into each of two 0.5 mL tubes. The tubes were labeled 1: 10, 1: 100 and 1: 1,000. One uL of ERCC Spike-In Control Mix was added to the 1: 10 tube and mixed by vortexing, and spun down. One uL of the 1: 10 diluted ERCC was transferred to the 1 : 100 tube and mixed by vortexing, and spun down. One uL of the 1: 100 diluted ERCC was transferred to the 1: 1,000 tube and mixed by vortexing, and spun down.
  • RNA used for this experiment was from one of three different sources, including: a commercially-available mixture of RNA from different multiple tissue types and individuals (Universal Human Reference, from Agilent, catalog No. 740000); or a mixture of RNA from multiple individuals (Human Brain Reference, from Ambion, catalog No. AM6050), or RNA extracted from FFPE samples that originated from various tissues and individuals.
  • the starting concentration of one RNA sample was 100 ng/L.
  • One uL of the 100 ng/uL RNA was mixed with 1 uL of the 1: 100 diluted ERCC tube.
  • the final concentration of this RNA was about 50 ng/uL.
  • the starting concentration of another RNA sample was 10 ng/uL.
  • One uL of the 10 ng/uL RNA was mixed with 1 uL of the 1: 1 ,000 diluted ERCC tube.
  • the final concentration of this RNA was about 5 ng/uL.
  • the ERCC Spike-In Control Mix was not added to FFPE RNA samples.
  • a reverse transcription reaction was set up in a 96-well plate.
  • the VILO RT The VILO RT
  • a reverse transcription reaction having a total volume of 5 uL contained: 2 uL of RNA (the 5 ng/uL or 50 ng/uL RNA sample), 1 uL of 5X VILO RT Reaction Mix (which contains random-sequence hexamer primers), 0.5 uL of 10X Superscript III Enzyme Mix, and 1.5 uL of nuc lease-free water.
  • the reaction was gently vortexed and spun briefly.
  • the reverse transcription reaction was incubated at 42 °C for 30 minutes, and inactivated at 85 °C for 5 minutes.
  • the PCR amplification reaction mixture was set up in a 0.5 mL or 1.5 mL tube.
  • the 5X Ion AmpliSeq HiFi Master Mix was obtained from an Ion AmpliSeq Library Kit Plus (Life Technologies, catalog No. 448890AB).
  • the total volume of the PCR amplification reaction mixture was 15 uL and contained: 4 uL of 5X Ion AmpliSeq HiFi Master Mix (red cap), 8 uL of 21K Primer Panel, 1 uL of 20X 10 ERCC primer panel, and 2 uL of nuclease-free water.
  • the 21K Primer Panel contained roughly 20,000 different pairs of target-specific primers, and each pair was designed to hybridize to one target polynucleotide sequence having an exon sequence using proprietary primer design parameters and algorithms, including those described in further detail in U.S. Patent No. 8,673,560.
  • the amplification reaction mixture was gently vortexed and spun down. 15 uL of the amplification reaction mixture was added to the reverse transcription reaction in the 96-well plate. The plate was sealed, gently vortexed to mix, and spun down. The plate was loaded into a thermo-cycler and run according to the following conditions.
  • the primer-derived sequences that were appended to the ends of the amplified target nucleic acids were partially digested (cleaved) by adding to the PCR amplification reaction mixture, 2 uL of FuPa Reagent (brown cap) which was obtained from the Ion AmpliSeq Library Kit Plus (Life).
  • the mixture was mixed by pipetting up and down 5 times or by gentle vortexing.
  • the plate was sealed and loaded into a thermo-cycler, and run according to the following conditions.
  • Adapters were ligated to the partially digested (cleaved) samples in a ligation reaction.
  • the Switch Solution and DNA ligase were obtained from an Ion AmpliSeq Library Kit Plus (Life Technologies, catalog No. 448890AB).
  • a diluted adaptor mix was prepared by mixing together: 2 uL of Ion PI adaptor (violet cap), 2 uL of Ion AmpliSeq Barcode X (white cap), and 4 uL nuclease-free water.
  • 2 uL of Ion PI adaptor violet cap
  • 2 uL of Ion AmpliSeq Barcode X white cap
  • 4 uL nuclease-free water To each well containing the partially digested samples (22 uL), add: 4 uL of Switch solution (yellow cap), 2 uL of the diluted adaptor mix. The plate was sealed, and mixed by gentle vortexing and spun down.
  • 2 uL of DNA Ligase blue cap
  • the plate was loaded into a thermo-cycler and run according to the following conditions.
  • the adaptor-ligated library was purified using Ampure XP beads. For each sample, a mixture was prepared containing 230 uL of freshly prepared 70% ethanol and 100 uL of nuclease- free water. To each sample in the 96-well plate, 45 uL of Ageneourt® AMPure® XP Reagent ( 1.5X sample volume) was added, and mixed by pipetting up and down five times, and incubated at room temperature for 5 minutes. The plate was placed in a magnetic stand and incubated for 2 minutes, or until the solution turned clear. The supernatant was carefully removed without disturbing the pellet.
  • the beads were washed by adding 150 uL of the freshly prepared ethanol mixture, and the plate was moved side-to-side in the two positions of the magnetic stand. The supernatant was carefully removed without disturbing the pellet. The beads were re-washed using the same wash procedure. The supernatant was carefully removed without disturbing the pellet, and all the ethanol droplets were removed from the wells. The beads were air-dried for about 2 minutes at room temperature. The plate was removed from the magnetic stand. The beads were dispersed by adding 50 uL of Low TE to the pellet to disperse the beads. The plate was sealed, and vortexed thoroughly, and spun down to collect the droplets. The plate was placed on a magnetic stand for at least 2 minutes. About 45 uL of the supernatant was transferred to new wells (e.g., on the same plate).
  • the adaptor-ligated library was quantified. About 1 pM of the library was used in a bead templating workflow using an Ion PITM Template OT 200 Kit (Life Technologies, catalog No. 4488318) according to the manufacturer's instructions, and the templated beads were used for sequencing on an Ion TorrentTM ProtonTM I chip on an Ion TorrentTM ProtonTM instrument according to the manufacturer-provided protocols (Ion ProtonTM System, catalog No. 4476610, and Ion PITM Sequencing 200 Kit v3, catalog No. 4488315).
  • the different target polynucleotide sequences contained in the adaptor-ligated library were mapped against a list of different target sequences of interest, where each target sequence of interest correlated with a single target-specific primer pair.
  • the number of reads corresponding to each of the different target polynucleotide sequences was binned and counted.
  • Figure 1 shows a graph of a tally of the number of different amplicon sequences that yielded less than 10, 10- 100, 100- 1000, 1000- 10,000, or more than 10,000 reads. More than 1 1 million reads were mapped (TABLE 1), which contained over 20,000 different amplicon sequences.
  • transcripts having the AARS sequence are approximately 1 1 times (e.g., 11 -fold) more abundant compared to transcripts having the ABCB 10 sequence.
  • RNA samples were converted into cDNA and amplified using a pool of roughly 20,000 different target-specific primer pairs (referring to herein as the "Transcriptome Primer Panel") as described for Example 1 above.
  • the transcriptome primer panel was designed to include one single set of target-specific primers for each of the different transcripts present in a typical human transcriptome.
  • the resulting amplicons were adapted via attachment of Ion Torrent standard adapters including one of 6 different barcodes (named
  • IonXpress_002 through “IonXpress_008" to generate 6 different transcriptome libraries, each attached to a different identifying barcode.
  • the resulting libraries were pooled, subjected to emulsion PCR according to the Ion PITM Template OT2 200 Kit v3 (catalog No. 4488318) protocol, and sequenced on the Ion Torrent Proton System, essentially as described for Example 1.
  • each sequencing read provides the sequence a single instance of a particular adapted amplicon within the transcriptome library:
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