Targeted bisulfite sequencing of the dynamic dna methylome. Tabspipe a pipeline to analyze the data obtained from targeted bisulfite sequencing through the iontorrent p. Dna methylation was the first discovered epigenetic mark, and remains the most studied. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of bismark to map the reads to a reference genome. Generating libraries is the first step in the workflow and consists of the ion torrent adapters flanking the dna fragments. However, very little is known about which genes are turned on or off, the role of epigenetics in heritability, or how these changes can lead to disease onset. Analysis and visualization tool for targeted amplicon bisulfite sequencing on ion torrent sequencers article pdf available in plos one 117. With ion torrent focused nextgeneration sequencing ngs technology, get the speed.
Consequently, bismark can handle bsseq data from both directional and nondirectional libraries. T his technology is based on the chemical conversion of unmethylated cytosine to uracil. Bisulfite sequencing is a method in which different regions of dna are analyzed using methylation. Analysis and visualization tool for targeted amplicon. Epigendx dna methylation and pyrosequencing service. In the current study, we developed a technique termed httrebs highthroughput targeted repeat element bisulfite sequencing.
Ion torrent sequencing uses a sequencing by synthesis approach, in which a new dna strand, complementary to the target strand, is synthesized one base at a time. Jul 28, 2016 analysis and visualization tool for targeted amplicon bisulfite sequencing on ion torrent sequencers article pdf available in plos one 117. Bisulfite sequencing services targeted nextgen or sanger. Although capillary electrophoresis offers many options for the detection of methylation in gdna figure 3, this troubleshooting guide will focus on improvements to bisulfitesequencing, which most methylation researchers depend on at. While whole genome bisulfite sequencing identifies methylated cytosines on a wholegenome scale, targeted bisulfite sequencing is an accurate, efficient and economical technology for dna methylation analysis of target regions, which may include a hybridizationbased step on platforms containing predesigned oligos that capture the cpg islands. Tabspipe a pipeline to analyze the data obtained from targeted bisulfite sequencing through the ion torrent p. 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. After which i performed sanger sequencing as the product size was around 300bp. Bisulfite sequencing an overview sciencedirect topics. Epigendx dna methylation and pyrosequencing service laboratory. After digestion and partial fillin dna fragments were ligated to barcoded adapters, bisulfite converted, sizeselected, and sequenced on the ion torrent personal. Tabsap is a pipeline to map bisulfite treated sequencing reads to a genome of interest and perform methylation calls in a single step and enables a researcher to analyze the methylation levels of their samples straight away. Bisulfite treatment results in a significant decrease in dna input and quality.
Methylation analysis by sequencing thermo fisher scientific tr. The kit provides the most comprehensive, uniform coverage of the methylome to support whole genome and targeted sequencing. Methylation sequencing methylseq or bisulfite sequencing is a powerful tool for understanding genomewide methylation with single nucleotide resolution and is considered a gold standard. In vertebrates, dna methylationmediated repression of retrotransposons is essential for the maintenance of genomic integrity. The bisulfite treated dna is then amplified by pcr, during which the 5methylcytosines are amplified as cytosines and the uracils are amplified as thymines. This generates a dna strand that is differentiable upon subsequent sequencing. Bisulfite amplicon sequencing bsas is a method for quantifying cytosine methylation in targeted genomic regions of interest. The ability to measure dna methylation precisely and efficiently continues to drive our understanding of this modification in development and disease.
Epigenetic modifications are a major determining factor in cellular differentiation, growth, disease progression and therapeutic response. Profiling dna methylation based on nextgeneration sequencing. Mar 17, 2016 the kit maximizes dna recovery of bisulfiteconverted samples and yields sequencing libraries that accurately represent base composition. Bisulfite sequencingbisseq bisulfite conversion dna. Bisulfite sequencing primers were designed to amplify a. One of the key steps in this process, bisulfite dna conversion, allows precise analysis of methylation in the target region by converting all non. Targeted ngs methylation analysis epigendx dna methylation. Bisulfite sequencing is the use of bisulfite treatment of dna to determine its pattern of methylation. Rapid and affordable genomewide bisulfite dna sequencing by. To analyze the sequencing output from this panel, a torrent suite. Hi, we have carried out targeted sequencing using ion torrent pgm of the pcr products amplified from bisulfite converted whole blood dna.
On the basis of emulsion polymerase chain reaction pcr library amplification, ion torrent technology uses a novel method called semiconductor sequencing. Methylation sequencing methylseq roche sequencing solutions. Methylated cytosines are protected from this conversion allowing to determine dna methylation at the singe nucleotide level. In vivo, dna is methylated following dna replication and is involved in a number of biological processes including the regulation of imprinted genes, x chromosome inactivation. We provide library preps, enrichment, and bioinformatics as part of our comprehensive ngs services on the ion torrent platform. Methylseq harnesses the scope and throughput of next generation sequencing for analyses in epigenetics such as tissuespecific differentially methylated regions dmr, and individual cpg. Here, we present a novel tool, called tabsat, for analyzing targeted bisulfite sequencing data generated on ion torrent sequencers. Bisulfite methylation library production and analysis using. Comparison of sanger and targeted nextgen bisulfite sequencing data. Analysis and visualization tool for targeted amplicon bisulfite sequencing on ion torrent sequencers.
Dec 30, 2015 bisulfite sequencing is the use of bisulfite treatment of dna to determine its pattern of methylation. Ion torrent next gen sequencing fast turnaround with semiconductor sequencing. Since residual cytosines in the sequencing read are converted in silico into a fully bisulfiteconverted form before the alignment takes place, mapping performed in this manner handles partial methylation accurately and in an unbiased manner. Although capillary electrophoresis offers many options for the detection of methylation in gdna figure 3, this troubleshooting guide will focus on improvements to bisulfite sequencing, which most methylation researchers depend on at. However, at earlier time points of 2 and 3 dpi a large proportion of reads were truncated supplementary table s3, most likely due to the read terminating when it approached a homopolymer track quail et al. Bisulfite sequencing is a goldstandard technique that combines bisulfite treatment and high throughput sequencing rrbs, agilent sureselect human methylseq, nimblegen seqcap epi cpgiant, and wgbs for the detection of dna methylome. Dna methylation was the first discovered epigenetic mark and is the most widely studied topic in epigenetics. Cytosine methylation can significantly modify gene expression and chromatin remodeling. We have developed an ngsbased deep bisulfite sequencing protocol for the. The kit maximizes dna recovery of bisulfiteconverted samples and yields sequencing libraries that accurately represent base composition. A pipeline to analyze the data obtained from targeted bisulfite sequencing through the iontorrent platform. Bisulfite sequencing is the gold standard for validating methylation experiments, as only sequencing provides direct detection of methylation events as well as information across the entire region. Pcr products were purified using qiaquick pcr purification kit qiagen. Advantages targeted nextgen bisulfite sequencing is more economical and has a faster turnaround time than wholegenome methylation analysis wgbs or rrbs.
Next generation sequencing next generation sequencing ngs is a method of nonsangerbased high throughput dna sequencing in which millions or even billions of dna strands can be sequenced in parallel. Genome bisulfite sequencing for methylation analysis guide 15021861 support center wgbs for methylation analysis guide 15021861 instructions for the wgbs for methylation analysis. I gave bisulfite treatment to dna and did pcr using bisulfite sequencing primers. Leveraging the power of nextgeneration sequencing ngs, bisulfite sequencing can be done with targeted methods such as amplicon methylseq and target enrichment, to provide insight into methylation patterns at a single nucleotide level for specific regions of interest in the human.
Further your epigenetics research with our comprehensive ngs solution, which enables a complete, targeted 3day workflow for quantitative methylation analysis. A pipeline to analyze the data obtained from targeted bisulfite sequencing through the ion torrent platform. This method requires small amount of genomic dna and therefore seems to be very useful for the. Bisulfite methylation library production and analysis using the ion ampliseq kit for chef dl8 publication number man0017892 revision c. Dna methylation bisulfite sequencing epigenetics diagenode. Epigenetics is the study of functional modifications to the genome that do not change the dna sequence. Nextgeneration sequencing technology has enabled genomewide analysis of 5mc nucleotides at singlenucleotide resolution. Ballandstick models of the proposed bisulfite equilibrium.
Methylation often occurs in cytosineguanine rich regions. Dna sequencing can then be used to elucidate the methylation status of a region of interest or an entire genome at singlenucleotide resolution. We have developed tabsat, a tool for the analysis of targeted bisulfite sequencing data generated on ion torrent systems. Ion torrent deep sequencing libraries were constructed from bisulfiteconverted dna using the kapa library preparation kit kapa biosystems, quantified using the qiaxcel advanced system qiagen, and templated using the ion pgm template ot2 200 kit thermo fisher. Dna from two different cell lines was bisulfite converted using active motifs methyldetector bisulfite modification kit. The bisulfitetreated dna is then amplified by pcr, during which the 5methylcytosines are amplified as cytosines and the uracils are amplified as thymines. This technique is designed to measure the dna methylation levels of individual loci of any repeat families with nextgeneration sequencing. Whole genome bisulfite sequencing has the advantage of theoretically capturing all cytosines in the genome at singlenucleotide resolution, but it has a number of significant practical drawbacks that become amplified with increasing sample numbers. Ion torrent deep sequencing libraries were constructed from bisulfite converted dna using the kapa library preparation kit kapa biosystems, quantified using the qiaxcel advanced system qiagen, and templated using the ion pgm template ot2 200 kit thermo fisher. The accelngs methylseq dna library kit maximizes dna recovery of bisulfiteconverted samples, to support inputs down to single cell. Epigenetic researchers utilize ngs as an important tool to gain detailed and comprehensive insight into epigenetic modifications of genomes belonging to numerous species and from many. Elim is known for high quality, fast turnaround times, and costeffective ngs services. Active motif bisulfite sequencing services help your dna methylation research by performing all steps including primer design and optimization, bisulfite conversion, pcr, cloning, sequencing targeted nextgen or sanger and final analysis.
Loo keat wei, anthony au, in handbook of epigenetics second edition, 2017. Methylation is the process of adding a specific molecule, called a methyl group, to a nucleotide, in this case usually a cytosine. Bisulfite methylation library production and analysis. Methylation analysis by sanger sequencing thermo fisher. Inactive nucleotides are often methylated, so this method can be used for a variety of purposes, from determining active regions of a genome to identifying generich.
With ion torrent focused nextgeneration sequencing ngs technology, get the speed, scalability and precision you need to spend more time finding answers and less time looking for them. Analysis and visualization tool for targeted amplicon bisulfite. Tabsat targeted amplicon bisulfite sequencing analysis tool is a tool for analyzing targeted bisulfite sequencing data generated on an ion torrent pgm. Pdf analysis and visualization tool for targeted amplicon. Bisulfite modification of dna is the most commonly used, gold standard method for dna methylation studies providing single nucleotide resolution. This method allows precise analysis of methylation in a certain region by converting all nonmethylated cytosines into tymines, while methylated cytosines remain unchanged. 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. Bisulfite sequencing also known as bisulphite sequencing is the use of bisulfite treatment of dna before routine sequencing to determine the pattern of methylation. Consult the bisulfite methylation library construction protocol using the. Methylation analysis by sequencing thermo fisher scientific. Targeted dna methylation analysis by nextgeneration sequencing. Analysis of dna methylation using bisulphite sequencing. A semiconductor chip detects the hydrogen ions produced during dna polymerization. A dna sample is incubated with sodium bisulfite, a chemical compound that converts unmethylated c into u uracil, but leaves methylated c unmodified bisulfite conversion can be performed using commercial kits.
The impact of nextgeneration sequencing on the dna. Roche sequencing solutions offers a suite of target enrichment and library preparation options for methylseq. Dna is treated with bisulfite, which converts unmethylated cytosines to uracil but does not change methylated cytosines. We have aligned the reads and now analysing the reads against reference sequences using biq analyzer himod. Methylseq kits for library preparation and ngs contain compatible reagents for the genomewide analysis of cytosine methylation through bisulfite sequencing. Targeted dna methylation analysis by nextgeneration. Since residual cytosines in the sequencing read are converted in silico into a fully bisulfite converted form before the alignment takes place, mapping performed in this manner handles partial methylation accurately and in an unbiased manner. Sodium bisulfite converts unmethylated cytosines cs present. In combination with traditional methylation profiling strategies bisulfite. Tabsap is a pipeline to map bisulfite treated sequencing reads to a genome of interest and perform methylation calls in a single step and enables a researcher to analyze the methylation levels of. Next, the ion ampliseq library kit plus and ion ampliseq panels are used to prepare amplicon libraries of the bisulfitetreated dna for sequencing on ion torrent.
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