WO2016040602A1 - Séquence au bisulfite à représentation réduite utilisant de l'uracile n-glycosylase (ung) et de l'endonucléase iv - Google Patents

Séquence au bisulfite à représentation réduite utilisant de l'uracile n-glycosylase (ung) et de l'endonucléase iv Download PDF

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WO2016040602A1
WO2016040602A1 PCT/US2015/049385 US2015049385W WO2016040602A1 WO 2016040602 A1 WO2016040602 A1 WO 2016040602A1 US 2015049385 W US2015049385 W US 2015049385W WO 2016040602 A1 WO2016040602 A1 WO 2016040602A1
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nucleic acid
dna
acid molecules
uracil
residues
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PCT/US2015/049385
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English (en)
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Joshua Burgess
Ramesh Vaidyanathan
Stephen Paul BRUINSMA
Haiying Li Grunenwald
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Epicentre Technologies Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • Embodiments provided herein relate to methods and compositions for next generation sequencing. Some embodiments relate to methods of preparing a library for reduced representation bisulfite sequencing (RRBS), followed by subsequent sequencing of the library.
  • RRBS reduced representation bisulfite sequencing
  • RRBS Reduced representation bisulfite sequencing
  • Current RRBS techniques combine restriction enzyme digestion, bisulfite conversion, and sequencing of the bisulfite-converted DNA in order to enrich and identify areas of the genome that have methylated CpG content.
  • embodiments disclosed herein provide methods for preparing a sample for sequencing, comprising: treating nucleic acid molecules in the sample to convert at least a portion of unmethylated cytosine residues into uracil residues; and cleaving the nucleic acid molecules at at least a portion of the uracil residues to obtain nucleic acid fragments. Further provided are populations of nucleic acid fragments resulting from a sample treated with the methods disclosed herein.
  • the sample is treated with bisulfite to convert at least a portion of unmethylated cytosine residues into uracil residues.
  • cleaving the nucleic acid molecules at the at least a portion of uracil residues comprises treating the nucleic acid molecules with a uracil DNA glycosylase resulting in a plurality of abasic residues in place of at least a portion of uracil residues.
  • the methods disclosed herein comprise treating the nucleic acid molecules with an endonuclease after treating with said uracil DNA glycosylase, wherein said endonuclease cleaves said nucleic acid molecules at at least a portion of the plurality of abasic residues.
  • said uracil DNA glycosylase and said endonuclease are present in a single reaction mixture.
  • the methods disclosed herein comprise treating the nucleic acid molecules with heat after treating with said uracil DNA glycosylase, wherein said nucleic acid molecules are broken at at least a portion of the plurality of abasic residues.
  • the treatments with said uracil DNA glycosylase and heat are conducted simultaneously.
  • alternative endonucleases may be used to cleave the genome.
  • the treatment with said uracil DNA glycosylase is conducted for about 1 minute to about 240 minutes.
  • the treatment with said uracil DNA glycosylase is conducted for about 1 minute to about 10 minutes.
  • the treatment with said uracil DNA glycosylase is conducted for about 1 minute to about 5 minutes.
  • the treatment with heat is conducted at 70 °C.
  • said uracil DNA glycosylase is human uracil-DNA glycosylase (UNG).
  • said UNG is available from Epicentre (Cat#UG13100; Madison, WI). In some embodiments, said UNG is at a concentration of about 0.02 U/1 ⁇ g of DNA to about 1 U/1 ⁇ g of DNA. In some embodiments, said UNG is at a concentration of about 0.04 U/1 ⁇ g of DNA to about 0.2 U/1 ⁇ g of DNA. In some embodiments, said UNG is at a concentration of about 0.04 U/1 ⁇ g of DNA to about 0.1 U/1 ⁇ g of DNA. In some embodiments, said endonuclease is endonuclease IV (Endo IV). In some embodiments, said Endo IV is at a concentration of about 2 U/1 ⁇ g of DNA.
  • Endo IV endonuclease IV
  • said Endo IV is at a concentration of about 0.2 U/1 ⁇ g of DNA. In some embodiments, said Endo IV is at a concentration of about 0.08 U/1 ⁇ g of DNA.
  • the methods disclosed herein comprise selecting the nucleic acid fragments based on their size. In some embodiments, said selecting the nucleic acid fragments based on their size comprises a bead- based method. In some embodiments, the portion of unmethylated cytosine residues is at least 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some embodiments, the portion of the uracil residues is at least 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
  • the portion of the plurality of abasic residues is at least 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
  • embodiments disclosed herein provide methods for sequencing nucleic acid fragments, comprising: obtaining the nucleic acid fragments from a sample treated with a method disclosed herein; and sequencing the nucleic acid fragments.
  • the methods disclosed herein comprise amplifying said nucleic acid fragments by PCR. In some embodiments, said PCR comprises about 10 cycles to about 15 cycles. In some embodiments, said sequencing is sequencing by synthesis (SBS). In some embodiments, the sequences of said nucleic acid fragments are compared to a reference sequence. In some embodiments, the methods disclosed herein comprise identifying methylated cytosines in said nucleic acid fragments. In some embodiments, the reference sequence comprises a methylome. In some embodiments, said methylated cytosines are identified using Bismark. In some embodiments, said methylated cytosines are analyzed using an integrated genome viewer.
  • said methylated cytosines comprise methylated CpG, methylated CHG and/or methylated CHH.
  • the methylation calls are significantly enriched in comparison to the methylation calls in a sample treated with bisulfite to convert unmethylated cytosine residues into uracil residues, but without cleaving the nucleic acid molecules at the uracil residues.
  • the enriched methylation calls comprise methylated CpG.
  • the enriched methylation calls comprise methylated CHG and/or methylated CHH.
  • the methylation calls comprise sites with a read depth of greater than about 10.
  • the cytosine sites are significantly enriched in comparison to the cytosine sites in a sample treated with bisulfite to convert unmethylated cytosine residues into uracil residues, but without cleaving the nucleic acid molecules at the uracil residues.
  • the cytosine sites comprise CpG.
  • the cytosine sites comprise CHG and/or CHH.
  • embodiments disclosed herein provide nucleic acid samples comprising fragmented nucleic acid molecules wherein substantially all cytosine residues in said fragmented nucleic acid molecules are methylated.
  • the first three residues of some of said fragmented nucleic acid molecules are not cytosine-guanine-guanine and/or the last four residues of some of said fragmented nucleic acid molecules are not thymine-cytosine-guanine-guanine.
  • the fragmented nucleic acid molecules comprise an apurinic/apyrimidinic site at the 5' or 3' end.
  • the fragmented nucleic acid molecules have a size from about 10 bp to about 2,000 bp.
  • the fragmented nucleic acid molecules have a size from about 50 bp to about 500 bp.
  • the fragmented nucleic acid molecules have a size from about 100 bp to about 200 bp. In some embodiments, the fragmented nucleic acid molecules have a mean size of about 140 bp to about 170 bp. In some embodiments, a portion of the CpG sites in said fragmented nucleic acid molecules are methylated. In some embodiments, at least about 0.1% of the CHG sites in said fragmented nucleic acid molecules are methylated. In some embodiments, at least about 0.1% of the CHH sites in said fragmented nucleic acid molecules are methylated.
  • kits comprising at least one container means, wherein the at least one container means comprises a reagent that cleaves a nucleic acid molecule at a uracil residue.
  • the reagent comprises UNG and endonuclease IV (Endo IV).
  • Figure 1 illustrates a flow diagram of an exemplary embodiment of a method for construction of a bisulfite -treated UNG/Endo IV methylation library for RRBS.
  • Figure 2 shows pictorially the steps of an exemplary embodiment of a method for construction of a bisulfite -treated UNG/Endo IV methylation library for RRBS.
  • Figure 3 shows a photograph of an agarose gel used to evaluate a
  • UNG/Endo IV digestion time-course of bisulfite -treated genomic DNA in an exemplary embodiment.
  • Figure 4 shows a panel of BioAnalyzer traces of the fragment size distribution in RRBS libraries generated using Hela DNA in an exemplary embodiment.
  • Figure 5A, Figure 5B, and Figure 5C show a screenshot of the Genome Analyzer set-up parameters, a screenshot of the status pane with quality metrics, and a screenshot of the read summary, read 1, and read 2 metrics, respectively, for the analysis of RRBS libraries from Hela or Coriell gDNA in an exemplary embodiment.
  • Figure 6A and Figure 6B show a series of insert size plots in RRBS libraries from Coriell gDNA treated for different amounts of time and with different enzyme dilutions, respectively, generated from the output sequencing data in an exemplary embodiment.
  • Figure 7 shows a data table of the Bismark methylation call results for the untreated and treated RRBS libraries from Hela or Coriell gDNA in an exemplary embodiment.
  • Figure 8 shows a screenshot of the IGV interface showing methylation patterns across a region of chromosome 8 in the untreated and 120-minute-treated libraries from Coriell gDNA in an exemplary embodiment.
  • Figure 9 shows a screenshot of the IGV interface showing methylation patterns across a region of chromosome 5 in RRBS libraries from Coriell gDNA treated with different enzyme dilutions in an exemplary embodiment.
  • Figure 10A and Figure 10B show a bar graph of Coriell CpG sites and a bar graph of Hela CpG sites in the RRBS libraries, respectively, in an exemplary embodiment.
  • Figure 11 A and Figure 1 IB show bar graphs of total number of CpG sites and CHH/CHG sites, respectively, at a read depth of >10 in RRBS libraries from Coriell gDNA treated with different enzyme dilutions in an exemplary embodiment.
  • Figure 12A and Figure 12B show correlation between Coriell CpG methylation calls in the RRBS libraries using the HiSeq method versus the UNG/Endo IV method with different enzyme dilutions, at a read depth of >10 and >50, respectively, in an exemplary embodiment.
  • Figure 13A and Figure 13B show a graph of total number of CpG sites and methylated CpG sites, respectively, containing Mspl recognition sequence in the RRBS libraries treated with different enzyme dilutions, compared directly to the CpG sites and methylated CpG sites called by the UNG/EndoIV method, in an exemplary embodiment.
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • nucleic acid molecule polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA").
  • DNA triple-, double- and single-stranded deoxyribonucleic acid
  • RNA triple-, double- and single-stranded ribonucleic acid
  • polynucleotide oligonucleotide
  • nucleic acid oligonucleotide
  • nucleic acid molecule include polydeoxyribonucleotides (containing 2-deoxy-D- ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C- glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (“PNAs”)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, OR., as NeuGene®) polyamide
  • PNAs peptide nucleic acids
  • these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' to P5' phosphoramidates, 2'-0-alkyl- substituted RNA, hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, "caps," substitution of one or more of the nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including enzymes (e.g., nucle
  • sequence identity or “identity” or “homology” in the context of two protein sequences (or nucleotide sequences) includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • the portion of the amino acid sequence or nucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acids are substituted for other amino acid residues with similar chemical properties (e.g.
  • sequences differ in conservative substitutions
  • the percentage sequence identity may be adjusted upwards to correct for the conservative nature of the substitutions. Sequences, which differ by such conservative substitutions are said to have "sequence similarity" or “similarity”. Means for making these adjustments are well known to persons skilled in the art. The percentage is calculated by determining the number of positions at which the identical amino acid or nucleic acid base residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially complementary or substantially matched means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, “substantially complementary or substantially matched” means that two nucleic acid sequences can hybridize under high stringency condition(s).
  • the stability of a hybrid is a function of the ion concentration and temperature.
  • a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency.
  • Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule.
  • the hybridized nucleic acid molecules generally have at least 60% identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity.
  • Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardfs solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS, at 42°C.
  • High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x Denhardfs solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in O.lx SSPE, and 0.1% SDS at 65°C.
  • Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5x Denhardfs solution, 6x SSPE, 0.2% SDS at 22°C, followed by washing in lx SSPE, 0.2% SDS, at 37°C.
  • Denhardfs solution contains 1% Ficoll, 1%) polyvinylpyrolidone, and 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • 20x SSPE sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)
  • EDTA ethylene diamide tetraacetic acid
  • Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art.
  • RRBS techniques combine restriction enzyme digestion, bisulfite conversion, and sequencing of the bisulfite-converted DNA in order to enrich and identify areas of the genome that have methylated CpG content.
  • spl-based methods typically employ the restriction enzyme Mspl to digest genomic DNA at C'CGG sites to enrich for CpG sites.
  • Mspl the restriction enzyme
  • any CpG sites not in proximity to the recognition sequence C'CGG are not recognized.
  • the current technique relies on recognition and restriction enzyme digestion of the 4 bp C'CGG sequence, the coverage of all CpG sites in the genome is limited and restricted to only CpG sites with flanking 5'-C and 3'-G nucleotides.
  • relatively high input levels of DNA e.g., about 1 ⁇ g to about 5 ⁇ g
  • Mspl-based methods also provide no CHH or CHG methylation context coverage.
  • a relatively high level of resolution and confidence in the first nucleotide of a read in a sequencing reaction is required for a methylation call.
  • embodiments of the methods presented herein do not rely on restriction endonucleases such as Mspl, and as such are capable of detecting_CpG, CHG, and CHH methylation contexts.
  • embodiments of the methods presented herein require much lowered input DNA with better data resolution and coverage, enriching for methylated regions of the genome on a context-neutral basis, and detecting a greater number of methylated sites and a wider scope of CpG sites than current RRBS methods.
  • Some embodiments disclosed herein provide methods for preparing a sample for sequencing, comprising: treating nucleic acid molecules in the sample to convert at least a portion of unmethylated cytosine residues into uracil residues. Typically, at least about 98% of unmethylated cytosine residues are converted into uracil residues. In some embodiments, the sample is treated with bisulfite to convert at least a portion of unmethylated cytosine residues into uracil residues.
  • the methods disclosed herein produce a population of nucleic acid molecules suitable for reduced representation bisulfite sequencing (RRBS).
  • RRBS reduced representation bisulfite sequencing
  • the methods provided herein may be used for construction of an RRBS library.
  • the nucleic acid molecules are genomic DNA.
  • the nucleic acid molecules may be treated with a uracil DNA glycosylase resulting in a plurality of abasic residues in place of uracil residues.
  • a uracil DNA glycosylase resulting in a plurality of abasic residues in place of uracil residues.
  • any suitable DNA glycosylase including but not limited to uracil DNA glycosylases, may be used to convert the uracil residues into abasic residues.
  • UNG human Uracil-DNA glycosylase
  • orthologs in organisms other than human may be used.
  • the methods disclosed herein could practically be applied to other sequencing methods, including but not limited to: 5mC detection (oxBS-Seq; Song et al, Nat. Biotech. 30: 1107-16 (2012)); 5hmC detection (TAB-Seq; Song et al, supra); 5caC detection (CAB-Seq; Liu et al, J. Am. Chem. Soc. 135: 9315-7 (2013)).
  • 5mC detection oxBS-Seq; Song et al, Nat. Biotech. 30: 1107-16 (2012)
  • 5hmC detection TAB-Seq; Song et al, supra
  • 5caC detection CAB-Seq; Liu et al, J. Am. Chem. Soc. 135: 9315-7 (2013).
  • some embodiments disclosed herein provide methods that investigate modified residues not converted by bisulfite from C to U.
  • DNA glycosylases such as a guanine DNA glycosylase, an adenine DNA glycosylase, a thymine DNA glycosylase, etc.
  • a target nucleotide residue may be converted to an abasic residue.
  • the methods further comprise cleaving the nucleic acid molecules at at least a portion of the uracil residues to obtain nucleic acid fragments.
  • the nucleic acid molecules having a plurality of abasic residues resulting from treatment with a uracil DNA glycosylase, e.g., UNG may be further treated to cleave the nucleic acid molecules at the abasic residues. Any suitable treatment that is effective in cleaving the nucleic acid molecules at the abasic residues can be used for the methods disclosed herein.
  • an endonuclease such as Endonuclease IV (Endo IV) may be used to cleave the nucleic acid molecules at the abasic residues.
  • heat treatment of the nucleic acid molecules can be used to cleave the molecules at the abasic residues.
  • the nucleic acid molecules having a plurality of abasic residues resulting from treatment with a uracil DNA glycosylase, e.g., UNG may be used in the absence of a cleaving step because most polymerases will not amplify at the abasic sites, generating various sized fragments during PCR amplification.
  • Figure 1 illustrates a flow diagram of an exemplary embodiment of a method 100 for construction of a bisulfite -treated nucleic acid methylation library for RRBS.
  • Figure 2 shows pictorially the steps of method 100 of Figure 1.
  • UNG and EndoIV are used in the diagrams as examples of a uracil DNA glycosylase and a treatment to cleave the nucleic acid molecules at abasic residues, and should not be interpreted to limit the presently disclosed methods. It would be readily appreciated by those skilled in the art that other suitable enzymes or treatments may be used in substitute but would generally achieve similar functions.
  • Method 100 may include, but is not limited to, the following steps.
  • a nucleic acid sample such as genomic DNA
  • a nucleic acid sample is purified through an accepted genomic DNA extraction protocol (e.g., MasterPureTM Complete DNA and RNA Purification Kit from Epicentre, Madison, WI).
  • an accepted genomic DNA extraction protocol e.g., MasterPureTM Complete DNA and RNA Purification Kit from Epicentre, Madison, WI.
  • a nucleic acid sample includes a double-stranded nucleic acid.
  • a nucleic acid sample includes genomic DNA, or cDNA.
  • mitochondrial or chloroplast DNA is used.
  • a nucleic acid sample includes RNA or derivatives thereof such as mRNA or cDNA.
  • Some embodiments described herein can utilize a plurality of different nucleic acid species (e.g., nucleic acid molecules having different nucleotide sequences being present in the plurality).
  • the nucleic acid sample may be prepared from nucleic acid molecules obtained from a single organism or from populations of nucleic acid molecules obtained from sources that include more than one organism.
  • a nucleic acid sample can be from a single cell; from multiple cells, tissue(s) or bodily fluids of a single organism; from cells, tissues or bodily fiuids of several organisms of the same species; or from multiple species, as with metagenomic samples, such as from environmental samples.
  • Sources of nucleic acid molecules include, but are not limited to, organelles, cells, tissues, organs, or organisms.
  • the methods disclosed herein enable the construction of RRBS library using less nucleic acid from the sample, e.g., genomic DNA than other methods known in the art.
  • nucleic acid e.g., genomic DNA
  • about 50 ng of nucleic acid e.g., genomic DNA
  • about 1 ⁇ g of input nucleic acid e.g., genomic DNA
  • less than 1 ⁇ g of input nucleic acid may be used for bisulfite conversion.
  • the amount of input nucleic acid (e.g., genomic DNA) used for the construction of an RRBS library is, is about, or is less than, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 500 ng, 1 ⁇ g, 10 ⁇ g, 100 ⁇ g of nucleic acid (e.g., genomic DNA), or is an amount of nucleic acid (e.g., genomic DNA) in a range defined by any two of these values, for example, 10 ⁇ g to 100 ⁇ g, 1 ⁇ g to 10 ⁇ g, 10 ng to 1 ⁇ g, 10 ng to 100 ng, 10 ng to 50 ng, 30 ng to 100 ng, etc.
  • the purified sample nucleic acid e.g., genomic DNA
  • the purified sample nucleic acid is bisulfite converted.
  • sodium bisulfite treatment may be used to convert unmethylated cytosine into uracil, which is replaced by thymine after amplification (e.g., PCR), while 5-methylcytosine remains unchanged. This step is also shown pictorially in Figure 2.
  • the bisulfite conversion protocol is sufficient for conversion of >98% of the non-methylated cytosines.
  • the bisulfite-converted nucleic acid e.g., genomic DNA
  • UNG and Endo IV are co-digested with UNG and Endo IV.
  • UNG catalyzes the hydrolysis of the N-glycosydic bond between uracil and sugar in nucleic acid, leaving an apyrimidinic (abasic) site in uracil- containing nucleic acids (e.g., DNA).
  • the abasic sites formed in the nucleic acid by UNG may be subsequently cleaved by Endonuclease IV.
  • Endonuclease IV For efficient conduction of this protocol add 1U UNG/2U Endonuclease IV and incubate for 5 minutes at 37 °C.
  • Other embodiments of this procedure can use a 1 ⁇ 2, 1/5, 1/10, 1/25, and 1/50 dilution of this original concentration.
  • the exemplary embodiments herein are encompassed between 1/5 and 1/25 dilution, or otherwise understood to be 0.2U UNG/0.4U Endonuclease IV to 0.04U UNG/0.08U Endonuclease IV incubated for the time dictated above.
  • the digested DNA is purified over a Zymo Clean and Concentrator genomic DNA column (Zymo Research, Irvine, CA), after which library preparation using the EpiGnome Library Preparation Kit (Epicentre, Madison, WI) begins according to published protocols. Other embodiments of this procedure only require UNG for fragmentation of the genome. This step is also shown pictorially in Figure 2.
  • any suitable procedures may be used to cleave the abasic sites in the DNA treated with UNG or another DNA glycosylase.
  • treatment of the nucleic acid molecules with a physical condition, such as heat, sonication, etc. may be sufficient to cleave the nucleic acid molecules at the abasic residues.
  • the treatment to cleave the nucleic acid molecules may be conducted simultaneously or after the treatment of the nucleic acid molecules with a uracil DNA glycosylase.
  • UNG and Endo IV may be added to a reaction mixture at the same time in order to obtain nucleic acid fragments that are enriched in methylated cytosines.
  • the treatment conditions such as the treatment time, enzyme conditions, temperature, etc., may be varied in order to control the size distribution of the nucleic acid fragments and/or to enrich methylated cytosines in the nucleic acid fragments.
  • the bisulfite-treated nucleic acid molecules may be treated at a temperature that is, is about, is lower than, or is higher than, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C, or at a temperature that is in a range defined by any two of these values, for example, 40 °C to 100 °C, 50 °C to 90 °C, 60 °C to 80 °C, 70 °C to 80 °C, etc, to cleave the nucleic acid molecules at the abasic residues.
  • the bisulfite-treated nucleic acid molecules may be treated with a uracil DNA glycosylase for an amount of time that is, is about, is less than, or is more than, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 60 minutes, 120 minutes, 240 minutes, or an amount of time that is in a range defined by any two of these values, for example, 1 minute to 240 minutes, 5 minutes to 120 minutes, 10 minutes to 60 minutes, 20 minutes to 30 minutes, etc.
  • the bisulfite-treated nucleic acid molecules may be treated with a uracil DNA glycosylase for 1 minute to 5 minutes.
  • the nucleic acid molecules treated with a uracil DNA glycosylase such as UNG
  • the nucleic acid molecules treated with a uracil DNA glycosylase, such as UNG may be treated with Endo IV for 1 minute to 5 minutes.
  • the bisulfite-treated nucleic acid molecules may be treated with a uracil DNA glycosylase, such as UNG, at a concentration that is, is about, is less than, or is more than, 0.01 U/1 ⁇ g of DNA, 0.02 U/1 ⁇ g of DNA, 0.03 U/1 ⁇ g of DNA, 0.04 U/1 ⁇ g of DNA, 0.05 U/1 ⁇ g of DNA, 0.1 U/1 ⁇ g of DNA, 0.2 U/1 ⁇ g of DNA, 0.5 U/1 ⁇ g of DNA, 1 U/1 ⁇ g of DNA, 2 U/1 ⁇ g of DNA, 5 U/1 ⁇ g of DNA, or a concentration that is defined by any of these values, for example, 0.01 U/1 ⁇ g of DNA to 5 U/1 ⁇ g of DNA, 0.02 U/1 ⁇ g of DNA to 2 U/1 ⁇ g of DNA, 0.03 U/1 ⁇ g of DNA to 1 U/1 ⁇ g of DNA, 0.04 U/1 ⁇ g of DNA to 0.5 U/1
  • the nucleic acid molecules treated with a uracil DNA glycosylase, such as UNG may be treated with Endo IV at a concentration that is, is about, is less than, or is more than, 0.01 U/1 ⁇ g of DNA, 0.02 U/1 ⁇ g of DNA, 0.03 U/1 ⁇ g of DNA, 0.04 U/1 ⁇ g of DNA, 0.05 U/1 ⁇ g of DNA, 0.1 U/1 ⁇ g of DNA, 0.2 U/1 ⁇ g of DNA, 0.5 U/1 ⁇ g of DNA, 1 U/1 ⁇ g of DNA, 2 U/1 ⁇ g of DNA, 5 U/1 ⁇ g of DNA, or a concentration that is defined by any of these values, for example, 0.01 U/1 ⁇ g of DNA to 5 U/1 ⁇ g of DNA, 0.02 U/1 ⁇ g of DNA to 2 U/1 ⁇ g of DNA, 0.03 U/1 ⁇ g of DNA to 1 U/1 ⁇ g of DNA, 0.04 U/1 ⁇ g of DNA to 0.5 U/1 ⁇ g of DNA, 0.01 U/1
  • the highly methylated regions are captured in a DNA methylation library for R BS sequencing.
  • the EpiGnomeTM Methyl-Seq Kit (Epicentre, Madison, WI) may be used for the construction of a DNA methylation library.
  • the TruSeq® DNA Sample Prep Kit (Illumina Inc., San Diego, CA) may be used for the construction of a DNA methylation library.
  • the TruSeq® library preparation may be used to construct a library with fragment sizes of 500 bp or less.
  • EpiNext Post-Bisulfite DNA Library Preparation Kit Pico Methyl-Seq Library Prep Kit, Ovation Ultralow Mehtyl-Seq Library Kit, EpiTect Whole Bisulfitome Kit, NEXTflex Bisulfite-Seq Kit, or any other suitable kits may be used to construct a library. This step is also shown pictorially in Figure 2.
  • Some embodiments disclosed herein provide nucleic acid samples having fragmented nucleic acid molecules wherein substantially all cytosine residues in the fragmented nucleic acid molecules are methylated.
  • the fragmented nucleic acid molecules result from the methods for preparing a sample for sequencing disclosed herein.
  • cleaving the nucleic acid molecules at the previous location of a uracil residue, made abasic by UNG, and subsequently fragmented by, for example, using Endo IV or heat may result in an apurinic/apyrimidinic site at a 5' or 3' end of the nucleic acid molecule.
  • the Endonuclease IV cleaves at the phosphodiester bond 5 ' to the AP DNA lesion.
  • cytosine residues in the fragmented nucleic acid molecules may be methylated.
  • a portion of the CpG sites in the fragmented nucleic acid molecules are methylated. In some embodiments, at least about 0.1% of the CHG sites in the fragmented nucleic acid molecules are methylated. In some embodiments, at least about 0.1% of the CHH sites in the fragmented nucleic acid molecules are methylated.
  • the methylated cytosines in the nucleic acid samples having fragmented nucleic acid molecules are enriched in comparison to a reference sample or samples.
  • the reference sample may be a sample treated with bisulfite to convert unmethylated cytosine residues into uracil residues, but without cleaving the nucleic acid molecules at the uracil residues.
  • the reference sample may be a sample that is not treated with bisulfite to convert unmethylated cytosines residues into uracil residues, with or without being treated for cleaving at the uracil residues.
  • methylated cytosines may be located in a CpG island and/or a CpG shore (Pollard et al, Cell Stem Cell. 5(6): 571-2 (2009); Suzuki et al, Mol. Oncol. 6(6); 567-78 (2012)).
  • the treatment conditions such as bisulfite treatment, conversion of unmethylated cytosine to uracil, and/or cleavage of AP sites, may be optimized to enrich the methylated cytosines in the nucleic acid samples having fragmented nucleic acid molecules, and/or to enrich CpG coverage in the nucleic acid samples having fragmented nucleic acid molecules.
  • the treatment time, enzyme conditions, temperature, etc. may be varied in order to enrich methylated cytosines in the nucleic acid fragments. It will be appreciated by those skilled in the art that by reducing enzyme concentration, fragmentation of the nucleic acid molecules may be reduced, which leads to increased coverage of CpG sites.
  • one or more of the parameters of the treatment time, enzyme conditions, temperature, etc. may be varied in order to enrich methylated CpG, CHH, or CHG, or a combination thereof, in the nucleic acid fragments.
  • the bisulfite -treated nucleic acid molecules may be treated at temperature conditions disclosed elsewhere herein to cleave the nucleic acid molecules at the abasic residues.
  • the bisulfite -treated nucleic acid molecules may be treated with a uracil DNA glycosylase for an amount of time that is, is about, is less than, or is more than, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 60 minutes, 120 minutes, 240 minutes, or an amount of time that is in a range defined by any two of these values, for example, 1 minute to 240 minutes, 5 minutes to 120 minutes, 10 minutes to 60 minutes, 20 minutes to 30 minutes, etc.
  • the bisulfite- treated nucleic acid molecules may be treated with a uracil DNA glycosylase for 1 minute to 5 minutes.
  • the nucleic acid molecules treated with a uracil DNA glycosylase such as UNG
  • the nucleic acid molecules treated with a uracil DNA glycosylase, such as UNG may be treated with Endo IV for 1 minute to 5 minutes.
  • the bisulfite-treated nucleic acid molecules may be treated with a uracil DNA glycosylase, such as UNG, at a concentration that is, is about, is less than, or is more than, 0.01 U/1 ⁇ g of DNA, 0.02 U/1 ⁇ g of DNA, 0.03 U/1 ⁇ g of DNA, 0.04 U/1 ⁇ g of DNA, 0.05 U/1 ⁇ g of DNA, 0.1 U/1 ⁇ g of DNA, 0.2 U/1 ⁇ g of DNA, 0.5 U/1 ⁇ g of DNA, 1 U/1 ⁇ g of DNA, 2 U/1 ⁇ g of DNA, 5 U/1 ⁇ g of DNA, or a concentration that is defined by any of these values, for example, 0.01 U/1 ⁇ g of DNA to 5 U/1 ⁇ g of DNA, 0.02 U/1 ⁇ g of DNA to 2 U/1 ⁇ g of DNA, 0.03 U/1 ⁇ g of DNA to 1 U/1 ⁇ g of DNA, 0.04 U/1 ⁇ g of DNA to 0.5 U/1
  • the bisulfite-treated nucleic acid molecules may be treated with UNG at a concentration of 0.02 U/1 ⁇ g of DNA to 0.1 U/1 ⁇ g of DNA.
  • the nucleic acid molecules treated with a uracil DNA glycosylase, such as UNG may be treated with Endo IV at a concentration that is, is about, is less than, or is more than, 0.01 U/1 ⁇ g of DNA, 0.02 U/1 ⁇ g of DNA, 0.03 U/1 ⁇ g of DNA, 0.04 U/1 ⁇ g of DNA, 0.05 U/1 ⁇ g of DNA, 0.1 U/1 ⁇ g of DNA, 0.2 U/1 ⁇ g of DNA, 0.5 U/1 ⁇ g of DNA, 1 U/1 ⁇ g of DNA, 2 U/1 ⁇ g of DNA, 5 U/1 ⁇ g of DNA, or a range of concentration that is defined by any of these values, for example, 0.01 U/1 ⁇ g of DNA to 5 U/1 ⁇ g of DNA, 0.02 U
  • fragmented nucleic acid molecules may have a size distribution that is suitable for capture in the subsequent library prep and sequencing reaction.
  • the size distribution of the fragmented nucleic acid molecules may be adjusted by a variety of reaction conditions, such as the time of the treatment, enzyme concentration, temperature, etc.
  • a large portion of fragmented nucleotides may not be large enough for capture, as a result of the uracil site cleavage. This loss will eventually lead to the methylation call and CpG, CHG, and CHH site enrichment of the methylated residues containing cytosines.
  • the size distribution of the fragmented nucleic acid molecules may be optimized in order to achieve an enrichment of methylation calls, including CpG methylation, CHG methylation, and/or CHH methylation.
  • the fragmented nucleic acid molecules have a size that is, is about, is less than, or is more than, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 100 bp, or 2,000 bp, or a size that is a range between any of these values, for example, 10 bp to 2000 bp, 20 bp to 1000 bp, 50 bp to 500 bp, 100 bp to 200 bp, etc.
  • the fragmented nucleic acid molecules have a size of 50 bp to 500 bp. In some embodiments, the fragmented nucleic acid molecules have a size of 100 bp to 200 bp. In some embodiments, the fragmented nucleic acid molecules have a mean size that is, is about, is less than, or is more than, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, or a size that is a range between any of these values, for example, 50 bp to 200 bp, 60 bp to 190 bp, 70 bp to 180 bp, 800 bp to 170 bp, etc. In some embodiments, the fragmented nucleic acid molecules have a mean size
  • Embodiments disclosed herein further provide methods for sequencing nucleic acid fragments, which comprises obtaining the nucleic acid fragments from a sample treated with a method disclosed herein, and sequencing the nucleic acid fragments.
  • the methods disclosed herein may be optimized for constructing a library for RRBS sequencing.
  • the library construction protocol may also include a fragment size selection step.
  • the fragment size selection step may be a bead-based protocol that is used to select fragments within a desired base pair range.
  • SPRIselect beads (Beckman Coulter) may be used to select fragments in a certain base pair range.
  • Agencourt AmPure® XP system (Beckman Coulter) may be used to select fragments in a certain base pair range.
  • the library of fragmented nucleic acid molecules is sequenced by sequencing by synthesis (SBS). In some embodiments, the sequences of the nucleic acid fragments are compared to a reference sequence.
  • methylated cytosines in the nucleic acid fragments are identified.
  • the reference sequence comprises a methylome.
  • the methylated cytosines are identified using Bismark.
  • the methylated cytosines are analyzed using an integrated genome viewer.
  • the method of the invention provides for construction of an RRBS library with reduced bias for CpG methylation regions.
  • Methylation of cytosine at CpG sites is considered to be the most important methylation site physiologically.
  • methylation of cytosine also occurs in other sequence contexts such as CHG and CHH.
  • the methods disclosed herein provide for enrichment of CHG methylated regions.
  • the methods disclosed herein provide for enrichment of CHH methylated regions. Enrichment of CHG and CHH regions may provide a mechanism for analyzing the physiological role of cytosine methylation at alternative sites in the genome, e.g., cytosine methylation in the CHG and CHH context.
  • the fragmented nucleic acid molecules may be constructed by the methods for preparing a sample for sequencing methods disclosed herein.
  • the fragmented nucleic acid molecules are subjected to amplification for preparing a sample for sequencing. Any suitable amplification methodology known in the art can be used.
  • nucleic acid fragments are amplified in or on a substrate.
  • the nucleic acid fragments are amplified using bridge amplification methodologies as exemplified by the disclosures of U.S. Pat. No. 5,641,658; U.S. Patent Publ. No. 2002/0055100; U.S. Pat. No. 7,115,400; U.S. Patent Publ.
  • Bridge amplification methods allow amplification products to be immobilized in or on a substrate in order to form arrays comprised of clusters (or "colonies") of immobilized nucleic acid molecules.
  • Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands.
  • the arrays so-formed can be referred to herein as "clustered arrays".
  • bridged structures when formed by annealed pairs of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on the solid support at the 5' end, preferably via a covalent attachment.
  • Bridge amplification methodologies are examples of methods wherein an immobilized nucleic acid template is used to produce immobilized amplicons.
  • Other suitable methodologies can also be used to produce immobilized amplicons from immobilized nucleic acid fragments produced according to the methods provided herein. For example one or more clusters or colonies can be formed via solid-phase PCR, solid-phase MDA, solid-phase RCA etc. whether one or both primers of each pair of amplification primers are immobilized.
  • amplification methodologies described herein or generally known in the art can be utilized with universal or target- specific primers to amplify immobilized DNA fragments.
  • Suitable methods for amplification include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence based amplification (NASBA), for example, as described in U.S. Patent No. 8,003,354, which is incorporated herein by reference in its entirety.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • TMA transcription mediated amplification
  • NASBA nucleic acid sequence based amplification
  • the above amplification methods can be employed to amplify one or more nucleic acids of interest.
  • PCR multiplex PCR
  • SDA single-stranded DNA
  • TMA RNA amplification reaction
  • NASBA NASBA
  • primers directed specifically to the nucleic acid of interest are included in the amplification reaction.
  • oligonucleotide extension and ligation can include rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), which is incorporated herein by reference in its entirety) and oligonucleotide ligation assay (OLA) (See e.g., U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524 and 5,573,907; EP 0320308; EP 0336731; EP 0439182; WO 90101069; WO 89/12696; and WO 89109835, each of which is incorporated herein by reference in its entirety).
  • RCA rolling circle amplification
  • OVA oligonucleotide ligation assay
  • the amplification method can include ligation probe amplification or oligonucleotide ligation assay (OLA) reactions that contain primers directed specifically to the nucleic acid of interest.
  • the amplification method can include a primer extension- ligation reaction that contains primers directed specifically to the nucleic acid of interest.
  • the amplification can include primers used for the GoldenGate ® assay (Illumina, Inc., San Diego, CA) or one or more assay set forth in U.S. Pat. No. 7,582,420 and 7,611,869, each of which is incorporated herein by reference in its entirety.
  • An isothermal amplification technique can be used in a method of the present disclosure.
  • Exemplary isothermal amplification methods include, but are not limited to, Multiple Displacement Amplification (MDA) as exemplified by, for example, Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002) or isothermal strand displacement nucleic acid amplification as exemplified by, for example U.S. Pat. No. 6,214,587, each of which is incorporated herein by reference in its entirety.
  • MDA Multiple Displacement Amplification
  • Non-PCR-based methods include, for example, strand displacement amplification (SDA) which is described in, for example Walker et al, Molecular Methods for Virus Detection, Academic Press, Inc., 1995; U.S. Pat. Nos. 5,455,166, and 5,130,238, and Walker et al, Nucl. Acids Res. 20: 1691-96 (1992) or hyperbranched strand displacement amplification which is described in, for example Nurse et al, Genome Research 13:294-307 (2003), each of which is incorporated herein by reference in its entirety.
  • SDA strand displacement amplification
  • modified nucleic acid fragments can be captured at locations within a region of a surface, replicated on one or more cycles of an amplification process, the original fragments and/or amplicons thereof can be released from the locations, the released nucleic acids can be captured at other locations in the same region, and the newly captured nucleic acids can be amplified.
  • a single cycle of bridge amplification can be carried out for a fragment that was seeded on a surface and instead of washing away the original template fragment upon release from the surface, the template fragment can re-seed the surface at a new location that is proximal to the location where it had originally seeded.
  • nucleic acid sequencing techniques can be used in conjunction with a variety of nucleic acid sequencing techniques. Particularly applicable techniques are those wherein nucleic acids are attached at fixed locations in an array such that their relative positions do not change and wherein the array is repeatedly imaged. Embodiments in which images are obtained in different color channels, for example, coinciding with different labels used to distinguish one nucleotide base type from another are particularly applicable.
  • the process to determine the nucleotide sequence of a target nucleic acid can be an automated process. Preferred embodiments include sequencing-by-synthesis ("SBS”) techniques.
  • SBS sequencing-by-synthesis
  • SBS sequencing-by-synthesis
  • a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery.
  • more than one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.
  • SBS can utilize nucleotide monomers that have a terminator moiety or those that lack any terminator moieties.
  • Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using ⁇ -phosphate-labeled nucleotides, as set forth in further detail below.
  • the number of nucleotides added in each cycle is generally variable and dependent upon the template sequence and the mode of nucleotide delivery.
  • the terminator can be effectively irreversible under the sequencing conditions used as is the case for traditional Sanger sequencing which utilizes dideoxynucleotides, or the terminator can be reversible as is the case for sequencing methods developed by Solexa (now Illumina, Inc.).
  • SBS techniques can utilize nucleotide monomers that have a label moiety or those that lack a label moiety. Accordingly, incorporation events can be detected based on a characteristic of the label, such as fluorescence of the label; a characteristic of the nucleotide monomer such as molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of pyrophosphate; or the like.
  • a characteristic of the label such as fluorescence of the label
  • a characteristic of the nucleotide monomer such as molecular weight or charge
  • a byproduct of incorporation of the nucleotide such as release of pyrophosphate
  • the different nucleotides can be distinguishable from each other, or alternatively, the two or more different labels can be the indistinguishable under the detection techniques being used.
  • the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by the sequencing methods developed by
  • Preferred embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P.
  • PPi inorganic pyrophosphate
  • the nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminscent signals that are produced due to incorporation of a nucleotides at the features of the array.
  • An image can be obtained after the array is treated with a particular nucleotide type (e.g., A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images.
  • the images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.
  • cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in International Patent Pub. No. WO 04/018497 and U.S. Patent 7,057,026, the disclosures of which are incorporated herein by reference in their entireties.
  • This approach is being commercialized by Solexa (now Illumina Inc.), and is also described in International Patent Pub. No. WO 91/06678 and International Patent Pub. No. WO 07/123,744, the disclosures of which are incorporated herein by reference in their entireties.
  • the labels do not substantially inhibit extension under SBS reaction conditions.
  • the detection labels can be removable, for example, by cleavage or degradation. Images can be captured following incorporation of labels into arrayed nucleic acid features.
  • each cycle involves simultaneous delivery of four different nucleotide types to the array and each nucleotide type has a spectrally distinct label. Four images can then be obtained, each using a detection channel that is selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially and an image of the array can be obtained between each addition step.
  • each image will show nucleic acid features that have incorporated nucleotides of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature. However, the relative position of the features will remain unchanged in the images. Images obtained from such reversible terminator-SBS methods can be stored, processed and analyzed as set forth herein. Following the image capture step, labels can be removed and reversible terminator moieties can be removed for subsequent cycles of nucleotide addition and detection. Removal of the labels after they have been detected in a particular cycle and prior to a subsequent cycle can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labels and removal methods are set forth below.
  • nucleotide monomers can include reversible terminators.
  • reversible terminators/cleavable fluors can include fluor linked to the ribose moiety via a 3' ester linkage (Metzker, Genome Res. 15: 1767-1776 (2005), which is incorporated herein by reference in its entirety).
  • Other approaches have separated the terminator chemistry from the cleavage of the fluorescence label (Ruparel et al, Proc Natl Acad Sci USA 102: 5932-7 (2005), which is incorporated herein by reference in its entirety).
  • Ruparel et al described the development of reversible terminators that used a small 3' allyl group to block extension, but could easily be deblocked by a short treatment with a palladium catalyst.
  • the fluorophore was attached to the base via a photocleavable linker that could easily be cleaved by a 30 second exposure to long wavelength UV light.
  • disulfide reduction or photocleavage can be used as a cleavable linker.
  • Another approach to reversible termination is the use of natural termination that ensues after placement of a bulky dye on a dNTP.
  • the presence of a charged bulky dye on the dNTP can act as an effective terminator through steric and/or electrostatic hindrance.
  • Some embodiments can utilize detection of four different nucleotides using fewer than four different labels.
  • SBS can be performed utilizing methods and systems described in U.S. Patent Pub. No. 2013/0079232, which is incorporated herein by reference in its entirety.
  • a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g., via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair.
  • nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc). Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal.
  • one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected in no more than one of the channels.
  • An exemplary embodiment that combines all three examples is a fluorescent- based SBS method that uses a first nucleotide type that is detected in a first channel (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and the second channel (e.g., dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide type that lacks a label that is not, or minimally, detected in either channel (e.g., dGTP having no label).
  • a first nucleotide type that is detected in a first channel e.g., dATP having a label that is detected in the first channel when excited
  • sequencing data can be obtained using a single channel.
  • the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated.
  • the third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.
  • Some embodiments can utilize sequencing by ligation (SBL) techniques.
  • SBL sequencing by ligation
  • Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides.
  • the oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize.
  • images can be obtained following treatment of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images.
  • Some embodiments can utilize nanopore sequencing (Deamer, D. W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, “Characterization of nucleic acids by nanopore analysis”. Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, "DNA molecules and configurations in a solid- state nanopore microscope” Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties).
  • the target nucleic acid passes through a nanopore.
  • the nanopore can be a synthetic pore or biological membrane protein, such as a-hemolysin.
  • each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore.
  • Some embodiments can utilize methods involving the real-time monitoring of DNA polymerase activity.
  • Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and ⁇ -phosphate-labeled nucleotides as described, for example, in U.S. Patent 7,329,492 and U.S. Patent 7,211,414 (each of which is incorporated herein by reference in its entirety) or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S.
  • FRET fluorescence resonance energy transfer
  • Patent 7,315,019 which is incorporated herein by reference in its entirety
  • fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Patent 7,405,281 and U.S. Patent Pub. No. 2008/0108082 (each of which is incorporated herein by reference in its entirety).
  • the illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M. J. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations.” Science 299, 682-686 (2003); Lundquist, P. M.
  • Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product.
  • sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in U.S. Patent Pub. No. 2009/0026082; U.S. Patent Pub. No. 2009/0127589; U.S. Patent Pub. No. 2010/0137143; or U.S. Patent Pub. No. 2010/0282617, each of which is incorporated herein by reference in its entirety.
  • Methods set forth herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons.
  • the above SBS methods can be advantageously carried out in multiplex formats such that multiple different target nucleic acids are manipulated simultaneously.
  • different target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner.
  • the target nucleic acids can be in an array format. In an array format, the target nucleic acids can be typically bound to a surface in a spatially distinguishable manner.
  • the target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface.
  • the array can include a single copy of a target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PCR as described in further detail below.
  • the methods set forth herein can use arrays having features at a density that is, is about, is less than, or is more than, 10 features/cm 2 , 100 features/cm 2 , 500 features/cm 2 , 1,000 features/cm 2 , 5,000 features/cm 2 , 10,000 features/cm 2 , 50,000 features/cm 2 , 100,000 features/cm 2 , 1,000,000 features/cm 2 , 5,000,000 features/cm 2 , or a density that is a range between any of these values, for example, 10 features/cm 2 to 5,000,000 features/cm 2 , 100 features/cm 2 to 1,000,000 features/cm 2 , 500 features/cm 2 to 100,000 features/cm 2 , 1,000 features/cm 2 to 50,000 features/cm 2 , 5,000 features/cm 2 to 10,000 features/cm 2 , etc.
  • an advantage of the methods set forth herein is that they provide for rapid and efficient detection of a plurality of target nucleic acid in parallel. Accordingly the present disclosure provides integrated systems capable of preparing and detecting nucleic acids using techniques known in the art such as those exemplified above.
  • an integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, the system comprising components such as pumps, valves, reservoirs, fluidic lines and the like.
  • a flow cell can be configured and/or used in an integrated system for detection of target nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent Pub. No. 2010/0111768 Al and U.S.
  • one or more of the fluidic components of an integrated system can be used for an amplification method and for a detection method.
  • one or more of the fluidic components of an integrated system can be used for an amplification method set forth herein and for the delivery of sequencing reagents in a sequencing method such as those exemplified above.
  • an integrated system can include separate fluidic systems to carry out amplification methods and to carry out detection methods.
  • Examples of integrated sequencing systems that are capable of creating amplified nucleic acids and also determining the sequence of the nucleic acids include, without limitation, the MiSeqTM platform (Illumina, Inc., San Diego, CA) and devices described in U.S. Patent App. No. 13/273,666, which is incorporated herein by reference in its entirety.
  • kits comprising at least one container means, wherein the at least one container means comprises a reagent that cleaves a nucleic acid molecule at a uracil residue.
  • the reagent is UNG and/or endonuclease IV (Endo IV).
  • the container means may be a tube, a well, a microtiter plate, etc.
  • the UNG/Endo IV library construction method 100 of Figure 1 was evaluated using two different genomic DNA sources, i.e., Coriell 18507 and Hela CpG hypermethylated DNA.
  • the whole genome methylation profile (methylome) for Coriell 18507 is known and may be used as a reference for determining percent coverage in subsequent RRBS of the UNG/Endo IV generated R BS libraries.
  • the Hela genome is hypermethylated (i.e., every CpG site is methylated) and may be used as a reference for percent coverage of CpG sites in subsequent RRBS of the UNG/Endo IV generated libraries.
  • all references to Hela refer specifically to the Hela CpG hypermethylated genome.
  • FIG. 3 shows a photograph of an agarose gel used to evaluate a UNG/Endo IV digestion time-course of bisulfite-treated genomic DNAs.
  • T2 DNA which is fully hydroxymethylated, was used as a negative control for UNG/Endo IV digestion of bisulfite-treated DNA.
  • the enzyme treatment (UNG/Endo IV), bisulfite treatment, and lane designation on the agarose gel are shown in Table 1.
  • a DNA size ladder (M) was loaded as shown in the Figure 3.
  • the arrow indicates a DNA ladder fragment size of about 500 bp.
  • the data show that UNG and Endo IV co-digest and fragment bisulfite- treated DNA appropriately when compared to T2 DNA (negative control).
  • T2 DNA negative control
  • the DNA is digested to 100-200 bp. Excessive fragmentation of the genomic DNA may, for example, be reduced by reducing the digestion time period.
  • bisulfite treatment i.e., Coriell lanes 6 and 7; Hela lanes 6 and 7
  • the DNA was not digested by UNG/Endo IV treatment.
  • the bisulfite-treated Coriell and Hela DNAs were used to construct RRBS libraries using the EpiGnomeTM Methyl-Seq Kit (Epicentre). Briefly, the bisulfite-treated and UNG/Endo IV DNA is random primed using a polymerase able to read uracil nucleotides to synthesize DNA strands containing a specific sequence tag. The 3 '-ends of the newly synthesized DNA strands are then selectively tagged with a second specific sequence tag resulting in di-tagged DNA molecules with known sequence tags at the 5'- and 3 '-ends. The di-tagged DNA is PCR amplified and ready for sequencing.
  • Figure 4 shows a panel of Bio Analyzer traces of the fragment size distribution in RRBS libraries from Hela gDNA.
  • the libraries were prepared using 50 ng input of each untreated (no UNG/Endo IV digestion) and treated (UNG/Endo IV for 30, 120, and 240 minutes) bisulfite-converted DNA and 10 (untreated), 12 (UNG/Endo IV 30 minutes), or 15 (UNG/Endo IV 30, 120, and 240 minutes) cycles of PCR amplification.
  • the untreated (no UNG/Endo IV) Hela DNA and the 30 minute treated Hela DNAs generated libraries with good size distribution and yield.
  • the 120 and 240 minute treated Hela DNAs generated libraries with reduced yield which may be due to over fragmentation.
  • Figure 5 A, 5B, and 5C show a screenshot of the Genome Analyzer Sequencer (Illumina Inc., San Diego, CA) set-up parameters, a screenshot of the status pane with quality metrics, and a screenshot of the read summary, read 1, and read 2 metrics, respectively, for the analysis of RRBS libraries from Hela or Coriell gDNA.
  • screenshot 500 shows a summary of the sequencing run parameters. For example, the sequencing run was paired-end (no index read) with 76 cycles/read and 24 tiles per lane. The sample ID shows the lane designation, library type, insert size, and concentration for each sample analyzed.
  • screenshot shows the status pane of the sequencing analysis viewer showing quality metrics for the run.
  • the quality metrics demonstrate a high Q30 quality score, averaging 91.4% for Read 1 and Read 2.
  • Typical acceptable Q30 is considered >80%.
  • the data by cycle demonstrates the % Cytosine is depleted as one would expect after bisulfite conversion; however, the cytosine is slightly higher than the expected 1% remaining due to the effects of cytosine concentration in the UNG/Endo IV method.
  • screenshot shows a run summary and the sequencing metrics for reads 1 and 2.
  • the percent clusters that pass filter (Cluster PF (%)) is about 93% to about 95% for all libraries. Typical acceptable PF is >80%.
  • the reads that pass filter (Reads PF (M)) were about 10 million for all libraries.
  • Example 4 Effects of treatment time and enzyme dilution on fragment size.
  • Figures 6 A and 6B show a series of plots of the insert size in the Coriell RRBS libraries treated with enzyme for different times and a series of plots 650 of the insert size in the Coriell RRBS libraries treated with enzyme dilutions for 5 minutes, respectively, generated from the output sequencing data.
  • the mean insert size in the untreated Coriell library is about 163 bp, which is within the expected range.
  • the mean insert size reads as a mean output of 141 bp for the 30 minute library, 138 bp for the 120 and 240 minute libraries, but the actual mean insert size for the treated libraries is potentially about 50 bp to about 60 bp, if extrapolated. Similar results were obtained for the Hela RRBS libraries.
  • the mean insert size in the untreated Coriell library is about 164 bp, which is within the expected range.
  • the mean insert size reads as a mean output of 143 bp for the lx enzyme library, 155 bp for the 1/1 Ox enzyme library and 153 bp for the l/25x enzyme library (lx enzyme being 1U UNG and 2U Endonuclease IV per ⁇ g DNA).
  • FIG. 7 shows a data table of the Bismark methylation call results for the untreated and treated Hela and Coriell RRBS libraries.
  • Bismark is an open source program that may be used to analyze methylated DNA.
  • CpG methylation is about 95%
  • CHG methylation is 0.60%
  • CHH methylation is 0.60%
  • the CpG methylation is about 93.2% and 95%, respectively
  • CHG methylation is 70% and 87%, respectively
  • CHH methylation is 90.2% and 95.8%, respectively.
  • FASTQC analysis of the GC content of the 30 minute treated Hela library indicated that the GC content (data not shown) suggested that the library may be contaminated with a second genome.
  • CpG methylation is 50%, CHG methylation is 0.50%, and CHH methylation is 0.60%%, as would be typically expected in Whole Genome Bisfulfite-converted samples.
  • CpG methylation is 78.3% CHG methylation is 71.2%, and CHH methylation is 91.3%.
  • the CpG methylation is about 77.4% and 81.1%, respectively, CHG methylation is 64.8% and 74.7%, respectively, and CHH methylation is 88.3% and 93.4%, respectively.
  • UNG/Endo IV treatment of bisulfite-treated DNA significantly increases enrichment of CpG, CHG, and CHH methylated regions.
  • CpG methylation in the untreated (no UNG/Endo IV digestion) Coriell library is 52% compared to about 80% in treated Coriell libraries.
  • CHG methylation in the untreated (no UNG/Endo IV digestion) Coriell library is 0.5%> compared to about 71 > to about 75%> in treated Coriell libraries.
  • CHH methylation in the untreated (no UNG/Endo IV digestion) Coriell library is 0.6% compared to about 88% to about 93% in treated Coriell libraries.
  • the method of the invention provides a mechanism to enrich for CHG and CHH methylated regions of a genome.
  • the method of the invention also provides a mechanism to further enrich (compared to Mspl digestion) CpG methylated regions of a genome.
  • the enrichment of methylation content of the RRBS libraries may also be assessed using an antibody specific for cytosine methylation (data not shown).
  • Figure 8 shows a screenshot 800 of the IGV interface showing methylation patterns across a region of chromosome 8 in the untreated and 120 minute treated Coriell libraries.
  • This region of chromosome 8 includes CpG islands.
  • a track 810 shows the captured reads across the region of chromosome 8 for the 120 minute treated Coriell library.
  • a track 815 shows the captured reads across the region of chromosome 8 for the untreated (no UNG/Endo IV digestion) Coriell library.
  • one read was captured in this region of chromosome 8 for the treated Coriell library (track 810).
  • the failure to detect methylation in this region may be due to over-fragmentation of the DNA by UNG/Endo IV digestion.
  • For the untreated Coriell library (track 815) reads were detected, but they did not necessarily correlate with known CpG islands.
  • Figure 9 shows a screenshot of the IGV interface showing reads captured across a region of chromosome 5 in the Coriell libraries treated with different enzyme dilutions at 5 minute incubation times.
  • the IGV data demonstrate that increasing enzyme concentration directly correlates with lower coverage of the genome, as expected. Enzyme digestion should enrich only methylated portions of the genome. Concentration of the enzyme may be optimized for maximizing methylation call detection and CpG, CHH, and CHG site enrichment. Example 6 Enrichment in CpG sites in UNG/Endo IV treated libraries.
  • Figures 10A and 10B show a bar graph of Coriell CpG sites and a bar graph of Hela CpG sites in the RRBS libraries, respectively. Bar graphs and show the number of CpG sites with greater than 10 reads within each library (untreated (no UNG/Endo IV digestion), 30, 120, and 240 minute treated). For both the Coriell and the Hela untreated libraries the number of CpG sites (> 10 reads) is about 2000 to about 3000. In the 30 minute treated Coriell library, the number of CpG sites (> 10 reads) is increased about 8 to 10 fold.
  • the number of CpG sites (> 10 reads) is further increased in the 120 minute (about 24,000) and 240 minute (about 32,000) treated Coriell libraries. Similarly, the number of CpG sites (> 10 reads) is increased in the 120 minute (about 32,000) and 240 minute (45,000) treated Hela libraries. The 30 minute treated Hela library is potentially contaminated and the data invalid.
  • Example 7 CpG and CHH/CHG enrichment related to enzyme dilution.
  • Figure 11 shows results on CpG and CHH (white bar)/CHG (black bar) enrichment related to enzyme dilution.
  • the results show that optimizing for dilution demonstrates up to 20-fold enrichment of CpG sites and CHH/CHG sites (not based on methylation) due to enzymatic cleavage and improved unique alignment. Therefore, for best data, the genome may be partially digested for optimal unique alignment, and enrichment of non-cleaved regions that contain methylated residues.
  • Example 8 Methylation Call Correlation of Coriell 18507 HiSeq versus UNG/Endo IV.
  • Figure 12 shows correlation of methylation calls between libraries prepped with UNG/Endo IV and EpiGnome vs. EpiGnome-only.
  • High enzyme concentration is excellent for correlating 100% methylated reads.
  • lower concentration enzyme provides more nuanced, better correlation output to previous EpiGnome runs.
  • the lOx read depth provides adequate correlation in the original analysis. With higher read depth (50x), the methylation call correlation becomes outstanding.
  • Selecting the correct dilution relies upon using a balanced approach. After examining all of the crucial indicators from sequencing, the best data becomes available at either the 1/1 Ox or l/25x dilution of UNG and Endonuclease IV with a 5 minute incubation. This dilution provides:
  • FIG. 13 shows that the sites detected by the UNG/Endo IV method are almost completely different than those detected by Mspl RRBS. This is a tremendous expansion of the sites detected by the new method, when compared to the currently available strategy.
  • Use of UNG/Endo IV RRBS demonstrates significant expansion of CpG sites not containing a Mspl recognition sequence. 2.5M CpG sites, encompassing 1.4M methylated CpG sites, would have been missed by Mspl RRBS that are detected with UNG/Endo IV.
  • the results show that the scope of Mspl RRBS and UNG/Endo IV method are entirely different.

Abstract

Dans un premier aspect, les formes de réalisation exposées dans l'invention fournissent des procédés de préparation d'un échantillon pour séquençage, comprenant : le traitement de molécules d'acides nucléiques dans l'échantillon pour convertir en résidus d'uracile au moins une portion des résidus de cytosine non-méthylée ; et le clivage des molécules d'acides nucléiques au niveau d'au moins une portion des résidus d'uracile, pour obtenir des fragments d'acides nucléiques. L'invention concerne en outre des populations de fragments d'acides nucléiques résultant d'un échantillon traité avec les procédés exposés dans l'invention.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018031760A1 (fr) 2016-08-10 2018-02-15 Grail, Inc. Procédés de préparation de bibliothèques d'adn à double indexation pour le séquençage avec conversion au bisulfite
WO2023082240A1 (fr) * 2021-11-15 2023-05-19 深圳华大智造科技股份有限公司 Procédé de détection de modification de méthylation d'adn à l'échelle du génome

Citations (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0320308A2 (fr) 1987-12-11 1989-06-14 Abbott Laboratories Procédé pour détecter une séquence cible d'acide nucléique
EP0336731A2 (fr) 1988-04-06 1989-10-11 City Of Hope Procédé pour l'amplification et détection des séquences d'acides nucléiques
WO1989009835A1 (fr) 1988-04-08 1989-10-19 The Salk Institute For Biological Studies Procede d'amplification a base de ligase
WO1989012696A1 (fr) 1988-06-24 1989-12-28 Amgen Inc. Procede et reactifs de detection de sequences d'acides nucleiques
WO1990001069A1 (fr) 1988-07-20 1990-02-08 Segev Diagnostics, Inc. Procede d'amplification et de detection de sequences d'acide nucleique
WO1991006678A1 (fr) 1989-10-26 1991-05-16 Sri International Sequençage d'adn
EP0439182A2 (fr) 1990-01-26 1991-07-31 Abbott Laboratories Procédé amélioré pour amplifier d'acides nucléiques cibles applicable à la réaction en chaîne de polymérase et ligase
US5130238A (en) 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
US5185243A (en) 1988-08-25 1993-02-09 Syntex (U.S.A.) Inc. Method for detection of specific nucleic acid sequences
US5223414A (en) 1990-05-07 1993-06-29 Sri International Process for nucleic acid hybridization and amplification
US5455166A (en) 1991-01-31 1995-10-03 Becton, Dickinson And Company Strand displacement amplification
US5573907A (en) 1990-01-26 1996-11-12 Abbott Laboratories Detecting and amplifying target nucleic acids using exonucleolytic activity
US5641658A (en) 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
US5679524A (en) 1994-02-07 1997-10-21 Molecular Tool, Inc. Ligase/polymerase mediated genetic bit analysis of single nucleotide polymorphisms and its use in genetic analysis
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6214587B1 (en) 1994-03-16 2001-04-10 Gen-Probe Incorporated Isothermal strand displacement nucleic acid amplification
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US20020055100A1 (en) 1997-04-01 2002-05-09 Kawashima Eric H. Method of nucleic acid sequencing
US20040002090A1 (en) 2002-03-05 2004-01-01 Pascal Mayer Methods for detecting genome-wide sequence variations associated with a phenotype
WO2004018497A2 (fr) 2002-08-23 2004-03-04 Solexa Limited Nucleotides modifies
US20040096853A1 (en) 2000-12-08 2004-05-20 Pascal Mayer Isothermal amplification of nucleic acids on a solid support
US20050100900A1 (en) 1997-04-01 2005-05-12 Manteia Sa Method of nucleic acid amplification
US20050112590A1 (en) * 2002-11-27 2005-05-26 Boom Dirk V.D. Fragmentation-based methods and systems for sequence variation detection and discovery
WO2005065814A1 (fr) 2004-01-07 2005-07-21 Solexa Limited Arrangements moleculaires modifies
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
US7001792B2 (en) 2000-04-24 2006-02-21 Eagle Research & Development, Llc Ultra-fast nucleic acid sequencing device and a method for making and using the same
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
WO2006064199A1 (fr) 2004-12-13 2006-06-22 Solexa Limited Procede ameliore de detection de nucleotides
US7115400B1 (en) 1998-09-30 2006-10-03 Solexa Ltd. Methods of nucleic acid amplification and sequencing
US20060240439A1 (en) 2003-09-11 2006-10-26 Smith Geoffrey P Modified polymerases for improved incorporation of nucleotide analogues
US20060281109A1 (en) 2005-05-10 2006-12-14 Barr Ost Tobias W Polymerases
WO2007010251A2 (fr) 2005-07-20 2007-01-25 Solexa Limited Preparation de matrices pour sequencage d'acides nucleiques
US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US20070128624A1 (en) 2005-11-01 2007-06-07 Gormley Niall A Method of preparing libraries of template polynucleotides
WO2007123744A2 (fr) 2006-03-31 2007-11-01 Solexa, Inc. Systèmes et procédés pour analyse de séquençage par synthèse
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US20080009420A1 (en) 2006-03-17 2008-01-10 Schroth Gary P Isothermal methods for creating clonal single molecule arrays
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US20080108082A1 (en) 2006-10-23 2008-05-08 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing
US7399590B2 (en) 2002-02-21 2008-07-15 Asm Scientific, Inc. Recombinase polymerase amplification
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US7582420B2 (en) 2001-07-12 2009-09-01 Illumina, Inc. Multiplex nucleic acid reactions
US20090233804A1 (en) * 2003-12-29 2009-09-17 Nurith Kurn Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids
US7611869B2 (en) 2000-02-07 2009-11-03 Illumina, Inc. Multiplexed methylation detection methods
US7670810B2 (en) 2003-06-20 2010-03-02 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100282617A1 (en) 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US8003354B2 (en) 2000-02-07 2011-08-23 Illumina, Inc. Multiplex nucleic acid reactions
US20120171691A1 (en) * 2009-09-23 2012-07-05 Koninklijke Philips Electronics N.V. Method for monitoring the bisulfite-mediated conversion of dna
US20120270305A1 (en) 2011-01-10 2012-10-25 Illumina Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
US20130079232A1 (en) 2011-09-23 2013-03-28 Illumina, Inc. Methods and compositions for nucleic acid sequencing
US20130260372A1 (en) 2012-04-03 2013-10-03 Illumina, Inc. Integrated optoelectronic read head and fluidic cartridge useful for nucleic acid sequencing

Patent Citations (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0320308A2 (fr) 1987-12-11 1989-06-14 Abbott Laboratories Procédé pour détecter une séquence cible d'acide nucléique
EP0336731A2 (fr) 1988-04-06 1989-10-11 City Of Hope Procédé pour l'amplification et détection des séquences d'acides nucléiques
WO1989009835A1 (fr) 1988-04-08 1989-10-19 The Salk Institute For Biological Studies Procede d'amplification a base de ligase
WO1989012696A1 (fr) 1988-06-24 1989-12-28 Amgen Inc. Procede et reactifs de detection de sequences d'acides nucleiques
US5130238A (en) 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
WO1990001069A1 (fr) 1988-07-20 1990-02-08 Segev Diagnostics, Inc. Procede d'amplification et de detection de sequences d'acide nucleique
US5185243A (en) 1988-08-25 1993-02-09 Syntex (U.S.A.) Inc. Method for detection of specific nucleic acid sequences
WO1991006678A1 (fr) 1989-10-26 1991-05-16 Sri International Sequençage d'adn
EP0439182A2 (fr) 1990-01-26 1991-07-31 Abbott Laboratories Procédé amélioré pour amplifier d'acides nucléiques cibles applicable à la réaction en chaîne de polymérase et ligase
US5573907A (en) 1990-01-26 1996-11-12 Abbott Laboratories Detecting and amplifying target nucleic acids using exonucleolytic activity
US5223414A (en) 1990-05-07 1993-06-29 Sri International Process for nucleic acid hybridization and amplification
US5455166A (en) 1991-01-31 1995-10-03 Becton, Dickinson And Company Strand displacement amplification
US5679524A (en) 1994-02-07 1997-10-21 Molecular Tool, Inc. Ligase/polymerase mediated genetic bit analysis of single nucleotide polymorphisms and its use in genetic analysis
US6214587B1 (en) 1994-03-16 2001-04-10 Gen-Probe Incorporated Isothermal strand displacement nucleic acid amplification
US5641658A (en) 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US20050100900A1 (en) 1997-04-01 2005-05-12 Manteia Sa Method of nucleic acid amplification
US20020055100A1 (en) 1997-04-01 2002-05-09 Kawashima Eric H. Method of nucleic acid sequencing
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
US7115400B1 (en) 1998-09-30 2006-10-03 Solexa Ltd. Methods of nucleic acid amplification and sequencing
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US7611869B2 (en) 2000-02-07 2009-11-03 Illumina, Inc. Multiplexed methylation detection methods
US8003354B2 (en) 2000-02-07 2011-08-23 Illumina, Inc. Multiplex nucleic acid reactions
US7001792B2 (en) 2000-04-24 2006-02-21 Eagle Research & Development, Llc Ultra-fast nucleic acid sequencing device and a method for making and using the same
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
US20040096853A1 (en) 2000-12-08 2004-05-20 Pascal Mayer Isothermal amplification of nucleic acids on a solid support
US7582420B2 (en) 2001-07-12 2009-09-01 Illumina, Inc. Multiplex nucleic acid reactions
US20060188901A1 (en) 2001-12-04 2006-08-24 Solexa Limited Labelled nucleotides
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
US7427673B2 (en) 2001-12-04 2008-09-23 Illumina Cambridge Limited Labelled nucleotides
US7399590B2 (en) 2002-02-21 2008-07-15 Asm Scientific, Inc. Recombinase polymerase amplification
US20040002090A1 (en) 2002-03-05 2004-01-01 Pascal Mayer Methods for detecting genome-wide sequence variations associated with a phenotype
WO2004018497A2 (fr) 2002-08-23 2004-03-04 Solexa Limited Nucleotides modifies
US20070166705A1 (en) 2002-08-23 2007-07-19 John Milton Modified nucleotides
US20050112590A1 (en) * 2002-11-27 2005-05-26 Boom Dirk V.D. Fragmentation-based methods and systems for sequence variation detection and discovery
US7670810B2 (en) 2003-06-20 2010-03-02 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US20060240439A1 (en) 2003-09-11 2006-10-26 Smith Geoffrey P Modified polymerases for improved incorporation of nucleotide analogues
US20090233804A1 (en) * 2003-12-29 2009-09-17 Nurith Kurn Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids
WO2005065814A1 (fr) 2004-01-07 2005-07-21 Solexa Limited Arrangements moleculaires modifies
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
WO2006064199A1 (fr) 2004-12-13 2006-06-22 Solexa Limited Procede ameliore de detection de nucleotides
US20060281109A1 (en) 2005-05-10 2006-12-14 Barr Ost Tobias W Polymerases
WO2007010251A2 (fr) 2005-07-20 2007-01-25 Solexa Limited Preparation de matrices pour sequencage d'acides nucleiques
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US20070128624A1 (en) 2005-11-01 2007-06-07 Gormley Niall A Method of preparing libraries of template polynucleotides
US20080009420A1 (en) 2006-03-17 2008-01-10 Schroth Gary P Isothermal methods for creating clonal single molecule arrays
WO2007123744A2 (fr) 2006-03-31 2007-11-01 Solexa, Inc. Systèmes et procédés pour analyse de séquençage par synthèse
US20100111768A1 (en) 2006-03-31 2010-05-06 Solexa, Inc. Systems and devices for sequence by synthesis analysis
US20080108082A1 (en) 2006-10-23 2008-05-08 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20100282617A1 (en) 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20120171691A1 (en) * 2009-09-23 2012-07-05 Koninklijke Philips Electronics N.V. Method for monitoring the bisulfite-mediated conversion of dna
US20120270305A1 (en) 2011-01-10 2012-10-25 Illumina Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
US20130079232A1 (en) 2011-09-23 2013-03-28 Illumina, Inc. Methods and compositions for nucleic acid sequencing
US20130260372A1 (en) 2012-04-03 2013-10-03 Illumina, Inc. Integrated optoelectronic read head and fluidic cartridge useful for nucleic acid sequencing

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Uracil-DNA Excision Mix - Cat No UEM04100", 1 October 2012 (2012-10-01), XP055232599, Retrieved from the Internet <URL:http://www.epibio.com/docs/default-source/protocols/uracil-dna-excision-mix.pdf?sfvrsn=8> [retrieved on 20151201] *
COCKROFT, S. L.; CHU, J.; AMORIN, M.; GHADIRI, M. R.: "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution", J. AM. CHEM. SOC., vol. 130, 2008, pages 818 - 820
DEAMER, D. W.; AKESON, M.: "Nanopores and nucleic acids: prospects for ultrarapid sequencing", TRENDS BIOTECHNOL., vol. 18, 2000, pages 147 - 151
DEAMER, D.; D. BRANTON: "Characterization of nucleic acids by nanopore analysis", ACC. CHEM. RES., vol. 35, 2002, pages 817 - 825
DEAN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 99, 2002, pages 5261 - 66
HEALY, K.: "Nanopore-based single-molecule DNA analysis", NANOMED., vol. 2, 2007, pages 459 - 481
HUANG RONG ET AL: "A novel combined bisulfite UDG assay for selective 5-methylcytosine detection", TALANTA, vol. 117, 15 December 2013 (2013-12-15), pages 445 - 448, XP028767799, ISSN: 0039-9140, DOI: 10.1016/J.TALANTA.2013.09.026 *
JIN ET AL., PLOS GENET., vol. 9, no. 6, 2013, pages E1003515
KORLACH, J. ET AL.: "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures", PROC. NATL. ACAD. SCI. USA, vol. 105, 2008, pages 1176 - 1181
LAGE ET AL., GENOME RESEARCH, vol. 13, 2003, pages 294 - 307
LEE ET AL., BIOL. PROCED. ONLINE, vol. 16, no. 1, 2014, pages 1
LEVENE, M. J. ET AL.: "Zero-mode waveguides for single-molecule analysis at high concentrations", SCIENCE, vol. 299, 2003, pages 682 - 686
LI, J.; M. GERSHOW; D. STEIN; E. BRANDIN; J. A. GOLOVCHENKO: "DNA molecules and configurations in a solid- state nanopore microscope", NAT. MATER., vol. 2, 2003, pages 611 - 615
LIU ET AL., J. AM. CHEM. SOC., vol. 135, 2013, pages 9315 - 7
LIZARDI ET AL., NAT. GENET., vol. 19, 1998, pages 225 - 232
LUNDQUIST, P. M. ET AL.: "Parallel confocal detection of single molecules in real time", OPT. LETT., vol. 33, 2008, pages 1026 - 1028
METZKER, GENOME RES., vol. 15, 2005, pages 1767 - 1776
MEYERS; MILLER, COMPUTER APPLIC. BIOL. SCI., vol. 4, 1998, pages 11 - 17
POLLARD ET AL., CELL STEM CELL, vol. 5, no. 6, 2009, pages 571 - 2
RONAGHI, M.: "Pyrosequencing sheds light on DNA sequencing", GENOME RES., vol. 11, no. 1, 2001, pages 3 - 11
RONAGHI, M.; KARAMOHAMED, S.; PETTERSSON, B.; UHLEN, M.; NYREN, P.: "Real-time DNA sequencing using detection of pyrophosphate release", ANALYTICAL BIOCHEMISTRY, vol. 242, no. 1, 1996, pages 84 - 9
RONAGHI, M.; UHLEN, M.; NYREN, P.: "A sequencing method based on real-time pyrophosphate", SCIENCE, vol. 281, no. 5375, 1998, pages 363
RUPAREL ET AL., PROC NATL ACAD SCI USA, vol. 102, 2005, pages 5932 - 7
SONG ET AL., NAT. BIOTECH., vol. 30, 2012, pages 1107 - 16
SONI, G. V.; MELLER: "A. Progress toward ultrafast DNA sequencing using solid-state nanopores", CLIN. CHEM., vol. 53, 2007, pages 1996 - 2001
SUZUKI ET AL., MOL. ONCOL., vol. 6, no. 6, 2012, pages 567 - 78
VARLEY ET AL., GENOME RES., vol. 23, no. 3, 2013, pages 555 - 67
WALKER ET AL., NUCL. ACIDS RES., vol. 20, 1992, pages 1691 - 96
WALKER ET AL.: "Molecular Methods for Virus Detection", 1995, ACADEMIC PRESS, INC.
XU ET AL., EMBO REP, vol. 5, 2004, pages 795 - 800

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