We have adapted transposasebased in vitro shotgun library construction tagmentation for wholegenome bisulfite sequencing. This method uses bisulfite conversion paired with pcr amplification of target regions prior to nextgeneration sequencing to produce absolute quantitation of dna methylation at a basespecific level. Tagmentationbased wholegenome bisulfite sequencing qi wang 1,6, lei gu 1,2,6, andrew adey 3, bernhard radlwimmer 4, wei wang 4, volker hovestadt 4, marion bahr 2. Macromolecular crowding allows bluntend ligation by dna ligases from rat liver or escherichia coli. Bisulfite amplicon sequencing bsas is a method for quantifying cytosine methylation in targeted genomic regions of interest. Tagmentationbased wholegenome bisulfite sequencing twgbs is a protocol that utilizes the epicentre tn5 transposome and bisulfite conversion to study 5mc. Tagmentationbased wholegenome bisulfite sequencing article pdf available in nature protocol 810. Bisulfite sequencing was first demonstrated by frommer et al.
Wholegenome bisulfite sequencing wgbs is the current gold standard of. Tagmentationbased wholegenome bisulfite sequencing md. Whole genome bisulfite sequencing and dna methylation. Moreover, recent advancements in sequencing technology allow researchers to perform the parallel analysis between dna methylation and histone modification by chip with bisulfite sequencing chipbsseq and bisulfite sequencing of chromatinimmunoprecipitated dna bischipseq. Major challenges for whole genome bisulfite sequencing are the limited sequencing. So far, analyses of the complete methylome by wholegenome bisulfite sequencing.
Postbisulfite adaptor tagging for pcrfree wholegenome. Wholegenome bisulfite sequencing for the analysis of. This method, tn5mcseq, enables a 100fold reduction in starting material relative to conventional protocols, such that we generate highly complex bisulfite sequencing libraries from as little as 10 ng of input dna, and ample useful sequences from 1 ng of input dna. This epigenetic variation biases progenitors toward specific islet. This protocol describes whole genome bisulfitesequencing library preparation from plant tissue and subsequent data analysis. Improved quality of wholegenome bisulfite sequencing wgbs with. Bisulfite sequencing an overview sciencedirect topics. Software and webbased tools for bisulfite sequencing analysis. Postbisulfite adaptor tagging pbat is a highly efficient procedure to construct libraries for wholegenome bisulfite sequencing wgbs. The mechanisms driving this divergence are unknown. Single nucleotide analysis of cytosine methylation by. Wholegenome bisulfite sequencing for the analysis of genomewide dna methylation and hydroxymethylation patterns at singlenucleotide resolution. Ctcf mchip enables analysis of dna methylation of distinct cpgs in the ctcf motif.
Tagmentationbased wholegenome bisulfite sequencing. Wholegenome bisulfite sequencing with improved accuracy and cost. Previous methods for wholegenome bisulfite sequencing wgbs yielded reduced genomic representation due to required dna shearing, ligation of methylated sequencing adapters, and bisulfite conversion of. Highly efficient singlestranded dna ligation technique improves. As described in this unit, this method allows for the quantitative measurement of methylation at each cytosine base by treatment of genomic dna with sodium bisulfite followed by sequencing and alignment of the. Parallel analysis of rna ends sequencing pareseq or 27 genomewide mapping of uncapped transcripts gmuct 27 transcript isoform sequencing tifseq or 29. Whole genome bisulphite sequencing using the illumina. Consequently, it enables pcrfree wgbs from nanogram quantities of mammalian dna, thereby. Dna fragments were analyzed on denaturing polyacrylamide gel. Wholegenome bisulfite sequencing sperm wgbs libraries were prepared according to a modified protocol based on the tagmentationbased method described by wang et al. The dna was treated with sodium bisulfite and then amplified by pcr using two sets of strandspecific primers to yield amplified fragments in which thymine took. During embryogenesis, seemingly equivalent progenitors produce distinct cell types. Wholegenome bisulfite sequencing wgbs covers all genomic.
Dna methylation and nucleosome positioning by nucleosome occupancy. This method, tn5mcseq, enables a 100fold reduction in starting material relative to conventional protocols, such that we generate highly complex bisulfite sequencing libraries from as little as 10 ng of input dna, and ample useful sequences from 1 ng of. Bisulfite treatment of dna followed by highthroughput sequencing bisulfiteseq is an important method for studying dna methylation and epigenetic gene regulation, yet current software tools do not adequately address single nucleotide polymorphisms snps. Whole genome bisulfite sequencing wgbs is the only analytical method covering the complete methylome. Until recently, bisulfite sequencing was mainly used for locusspecific analysis and several software packages were developed to address this issue. Briefly, 10ng sperm dna supplemented with 1% unmethylated lambdadna promega, d152a were incubated in a 50. Method ultralowinput, tagmentationbased wholegenome. In this method, dna is incubated with tn5 transposome containing methylated primers, which fragments the dna and ligates adapters. Based on this idea, we developed a highly efficient ssdna ligation method, termed.
Pdf tagmentationbased wholegenome bisulfite sequencing. Tagmentationbased library preparation for low dna input. Adey a, shendure j 2012 ultralowinput, tagmentationbased wholegenome bisulfite sequencing. Twgbs illumina sequencing and arraybased solutions. Dieter weichenhan, qi wang, andrew adey, stephan wolf, jay shendure, roland eils, christoph plass.
Ultralowinput, tagmentationbased wholegenome bisulfite sequencing. Targeted dna methylation analysis by nextgeneration. Allelespecific methylation analysis and genomewide identification of differentially methylated regions are additional features of the analysis procedure. Improved tagmentationbased wholegenome bisulfite sequencing for input dna. Illumina provides the truseq dna methylation kit for wgbs, which uses. Pbat attaches adaptors to bisulfiteconverted genomic dna to circumvent bisulfiteinduced degradation of library dna inherent to conventional wgbs protocols. For example, tn5mcseq used tn5 transposasemediated tagmentation. Unlike other methods to assess methylation, wholegenome shotgun bisulfite sequencing is used to generate quantitative genomewide methylation profiles at singlenucleotide resolution. Nextgeneration sequencing technology has enabled genomewide analysis of 5mc nucleotides at singlenucleotide resolution. Ultralowinput, tagmentationbased wholegenome bisulfite sequencing andrew adey and jay shendure1 department of genome sciences, university of washington, seattle, washington 98195, usa we have adapted transposasebased in vitro shotgun library construction tagmentation for wholegenome bisulfite sequencing. Tagmentationbased library preparation for low dna input whole genome bisulfite sequencing. Identification of epigenetically deregulated transcripts in cll the promoter dna methylation status is widely used.
However, as the cost of the wholegenome sequencing has become more affordable, many laboratories are able to produce a snapshot of the entire methylome at nucleotide resolution. Tagmentationbased whole genome bisulfite sequencing twgbs 72 oxidative bisulfite sequencing oxbsseq 73. So far, analyses of the complete methylome by wholegenome bisulfite. Initial analysis of sperm dna methylome in holstein bulls. Tagmentationbased whole genome bisulfite sequencing twgbs.