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SulfitesDNA MethylationCpG IslandsSequence Analysis, DNADNAGene ConversionCytosineAzacitidineSulfur AcidsEpigenesis, GeneticPromoter Regions, Genetic5-MethylcytosineThiosulfatesGene SilencingBase SequenceLong Interspersed Nucleotide ElementsDNA Modification MethylasesDNA (Cytosine-5-)-MethyltransferasePolymerase Chain ReactionConversion DisorderMolecular Sequence DataDesulfovibrioDeamination
Sodium bisulfiteEpiTect Plus Bisulfite KitsMethylation analysisTraditional bisulfiteOxidativeZymo ResearchWorkflowAmplificationGenome-wideGenomicAssayLigationUracilFragmentationWGBSDigestionProcedureEfficiencyMethodsBeadChipNegligibleReactionDownstreamClinicalKitsMethylomesSequenceUniformProductsSequencing technologyDetectionTreatmentLibraryDataApproachEntireCellsHighly
Sodium bisulfite7
  • Whole genome bisulfite sequencing is a next-generation sequencing technology used to determine the DNA methylation status of single cytosines by treating the DNA with sodium bisulfite before high-throughput DNA sequencing. (wikipedia.org)
  • In 1970, a breakthrough occurred when it was discovered that treating DNA with sodium bisulfite deaminated cytosine residues into uracil. (wikipedia.org)
  • In the following decade, this discovery led to the revelation that unmethylated cytosine reacted much faster to sodium bisulfite treatment than did 5-methylcytosine. (wikipedia.org)
  • Size selection occurs before the DNA is treated with sodium bisulfite. (wikipedia.org)
  • The adapter-ligated DNA sample is treated with sodium bisulfite, a chemical compound that converts unmethylated cytosines into uracil, at low pH and high temperatures. (wikipedia.org)
  • These bisulfite conversion kits are based on the three-step reaction that takes place between cytosine and sodium bisulfite where cytosine is converted into uracil. (zymoresearch.com)
  • According to the authors, the protocol for the Illumina Infinium HumanMethylation450K BeadChip relies on sodium bisulfite conversion of DNA, which discriminates between methylated and unmethylated cytosine bases within the genome, but cannot differentiate between 5hmC and 5-methylcytosine, the epigenetic modification traditionally surveyed using the array. (genomeweb.com)
EpiTect Plus Bisulfite Kits1
  • Best results are obtained when used in combination with EpiTect Plus Bisulfite Kits, which contain a uniquely formulated DNA Protect system. (qiagen.com)
Methylation analysis3
  • This technique was developed in 2009 along with reduced representation bisulfite sequencing after bisulfite sequencing became the gold standard for DNA methylation analysis. (wikipedia.org)
  • Prior to the development of whole genome bisulfite sequencing, genome methylation analysis relied heavily on early non-specific and differential methods such as paper chromatography, high-performance liquid chromatography, and thin-layer chromatography to analyze methylation profiles. (wikipedia.org)
  • 1992), the bisulfite conversion technique has become a preferred tool for methylation analysis. (thermofisher.com)
Traditional bisulfite1
  • Though traditional bisulfite conversion leads to the potential overestimation of 5mC levels in a sample because of the presence of 5hmC, the researchers' oxBS-450K method generates a more accurate 5mC profile by removing 5hmC. (genomeweb.com)
Oxidative3
  • The resulting 5fC behaves similarly to unmodified cytosine under bisulfite conditions, "allowing only true 5mC positions to be detected as cytosine after oxidative bisulfite conversion and PCR amplification of the DNA sample," they wrote. (genomeweb.com)
  • Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. (biomedcentral.com)
  • Intended applications include whole genome (WGBS), oxidative (oxBS-seq), and other bisulfite-next generation sequencing. (epigentek.com)
Zymo Research2
  • These kits are the original bisulfite conversion kit from Zymo Research. (zymoresearch.com)
  • The JAX OncoMethyl™ Array uses genomic DNA extracted from FFPE tissues (≥70% neoplastic content) that is followed by bisulfite conversion (Zymo Research). (jax.org)
Workflow2
  • The following steps are derived from one potential workflow of conventional whole genome bisulfite sequencing: target DNA extraction, bisulfite conversion, library amplification, and bioinformatics analysis. (wikipedia.org)
  • Workflow of the EpiNext™ High-Sensitivity Bisulfite-Seq Kit (Illumina). (epigentek.com)
Amplification5
  • Library preparation protocols undergo DNA fragmentation, end repair, dA-tailing, and adapter ligation prior to bisulfite treatment and library amplification. (wikipedia.org)
  • The EpiTect Whole Bisulfitome Kit provides DNA polymerase, buffers, and reagents for amplification of the entire bisulfite converted genome - the bisulfitome - using multiple displacement amplification technology (MDA). (qiagen.com)
  • The EpiTect Whole Bisulfitome Kit has been specially developed for the amplification of DNA after conversion with EpiTect Bisulfite Kits. (qiagen.com)
  • This whole bisulfitome amplification (WBA) procedure provides high yields of bisulfite converted DNA with accurate locus representation, when used in conjunction with EpiTect Bisulfite Kits. (qiagen.com)
  • Extremely convenient - The kit contains all required components for each step of DNA library preparation, which are sufficient for bisulfite conversion, ligation, clean-up, size selection and library amplification, thereby allowing the bisulfite DNA library preparation to be streamlined for the most reliable and consistent results. (epigentek.com)
Genome-wide3
  • Whole genome bisulfite sequencing measures single-cytosine methylation levels genome-wide and directly estimates the ratio of molecules methylated rather than enrichment levels. (wikipedia.org)
  • 1: Chatterjee A, Stockwell P A, Rodger E J, and Morison I M, Comparison of alignment software for genome-wide bisulphite sequence data , Nucleic Acids Research Volume 40, Issue 10Pp. (otago.ac.nz)
  • Benchmarking genome-wide nanopore 5mC detection against whole-genome, short-read bisulfite sequencing, the traditional method of 5mC detection, has shown nanopore sequencing to enable gold-standard 5mC calling (Figure 1). (nanoporetech.com)
Genomic1
  • According to the authors, oxBS involves a selective oxidation step prior to bisulfite conversion of genomic DNA that converts 5hmC residues to 5-formylcytosine. (genomeweb.com)
Assay1
  • Sodium bisulphite conversion of 500 ng of each sample was performed using the EZDNA Methylation-Gold Kit according to the manufacturer's recommendation for Illumina Infinium Assay. (nih.gov)
Ligation1
  • High sensitivity and efficiency - Innovative adaptor ligation of bisulfite DNA eliminates loss of fragments and selection bias, which enables input DNA to be as low as 0.2 ng, making it ideal for methylation profiling of precious, limited samples. (epigentek.com)
Uracil1
  • This enables methylation detection by distinguishing the methylated cytosines (5-methylcytosine), which resist bisulfite treatment, from uracil. (wikipedia.org)
Fragmentation1
  • Innovative method - Allows for simultaneous bisulfite conversion and size-appropriate DNA fragmentation. (epigentek.com)
WGBS2
  • Whole-genome bisulfite sequencing (WGBS) is increasingly used for fundamental and clinical research of CpG methylation. (biomedcentral.com)
  • The bisulfite DNA can be directly ligated to adaptors thereby eliminating the possibility of breaking adaptor-ligated fragments, which often occurs with currently used WGBS. (epigentek.com)
Digestion1
Procedure2
  • The EZ DNA Methylation Kits are bisulfite conversion kits that feature a simplified procedure that streamlines bisulfite treatment of DNA. (zymoresearch.com)
  • Fast and streamlined procedure - The procedure from DNA bisulfite treatment to ready-to-use library DNA can be completed within 6 hours and 30 minutes. (epigentek.com)
Efficiency1
  • Since its development, many various protocols of whole genome bisulfite sequencing have been developed aiming to improve the efficiency and efficacy of its single-base mapping. (wikipedia.org)
Methods1
  • With the improvement of library preparation methods and next-generation sequencing technology over the past decade, whole genome bisulfite sequencing has become an increasingly widespread and informative method for analyzing DNA methylation in epigenomic-wide studies. (wikipedia.org)
BeadChip1
  • 4 ul of bisulfite converted DNA were hybridized on Infinium HumanMethylation 450 BeadChip, following manufacturer's instructions. (nih.gov)
Negligible1
  • 99%) with negligible inappropriate- or error-conversions of methylcytosine to thymine. (epigentek.com)
Reaction1
  • Typical DNA yields from the EpiTect Whole Bisulfitome Kit are 1-3 μg per reaction, sufficient for up to 300 PCR analyses from just a single EpiTect bisulfite reaction. (qiagen.com)
Downstream1
  • The EpiTect Whole Bisulfitome Kit is the ideal tool to provide sufficient DNA for all downstream methylation analyses, whenever bisulfite converted DNA is limited. (qiagen.com)
Clinical1
  • As the costs of next-generation sequencing have decreased, whole genome bisulfite sequencing has become more widely used in clinical and experimental research. (wikipedia.org)
Kits5
  • All bisulfite conversion kits are available in spin-column, 96-well, and MagBead formats. (zymoresearch.com)
  • These innovative bisulfite conversion kits feature streamlined workflows, ≥ 99% conversion rates, automation friendly options, and low elution volumes for concentrated, bisulfite-converted DNA. (zymoresearch.com)
  • Designed to reduce template degradation, these bisulfite conversion kits minimize DNA loss during treatment and cleanup, while ensuring complete conversion of the DNA. (zymoresearch.com)
  • These bisulfite conversion kits are recommended with Illumina's GoldenGate® and Infinium® Assays. (zymoresearch.com)
  • We further sequenced these cell lines using long-read technology on an Oxford Nanopore PromethION and here compare its performance alongside more common chemical/enzymatic conversion kits and short-read sequencing. (biomedcentral.com)
Methylomes2
  • DMAP components can filter and process aligned bisulphite sequenced data to generate comprehensive reference methylomes in different units for any genome. (otago.ac.nz)
  • However, capturing the true diversity of a population requires the ability to observe each individual in that group and for methylomes that requires innovations in single-cell bisulfite sequencing . (epigenie.com)
Sequence2
  • It uses proven REPLI-g technology and has been adapted to the specific requirements of bisulfite converted DNA (smaller DNA fragment size and altered nucleotide composition due to bisulfite conversion), while maintaining the converted sequence representation. (qiagen.com)
  • The EpiTect Whole Bisulfitome Kit has been specially developed using proven REPLI-g technology and has been adapted to the special requirements of bisulfite converted DNA (smaller DNA fragment size and changed nucleotide composition due to bisulfite conversion), while maintaining the converted sequence representation. (qiagen.com)
Uniform1
  • Nanopore read depth is more uniform than the bisulfite data (Fig. 1b), maps completely to the genome (Fig. 1c) and requires far less analysis time than bisulfite data (Fig. 1d). (nanoporetech.com)
Products1
  • Bisulfite-converted products are directly processed for second-strand synthesis in the presence of RNase H and dUTP. (elifesciences.org)
Sequencing technology1
  • Whole genome bisulfite sequencing was derived as a combination of this bisulfite treatment and next-generation sequencing technology, such as shotgun sequencing. (wikipedia.org)
Detection1
  • As PCR removes base modifications, their detection via traditional sequencing technologies requires the use of special library preparation steps (e.g. bisulfite conversion) which damage nucleic acids, resulting in very short sequencing reads. (nanoporetech.com)
Treatment1
  • B ) Correlation plots of normalized read densities between forward and reverse strands in BisMapR, MapR, BisMapR samples treated with RNase A, and MapR samples without bisulfite treatment and with second-strand synthesis (NBSS). (elifesciences.org)
Library3
  • The EpiNext™ High-Sensitivity Bisulfite-Seq Kit (Illumina) was used to bisulfite convert DNA and prepare a library to be sequencing on an Illumina HiSeq 2500. (epigentek.com)
  • The EpiNext™ High-Sensitivity Bisulfite-Seq Kit (Illumina) is designed to bisulfite-convert DNA and prepare an Illumina-based library for bisulfite sequencing, all in one kit. (epigentek.com)
  • The optimized protocol and components of the kit allow subnanogram amounts of DNA to be bisulfite converted and fragmented simultaneously followed by quick non-barcoded (singleplexed) and barcoded (multiplexed) library construction in less than 7 hours. (epigentek.com)
Data3
  • Nanopore methylation data was compared to publicly available bisulfite datasets. (nanoporetech.com)
  • For the first time, we determine the correlation of in silico telomere content quantification from whole genome sequencing and whole genome bisulfite sequencing data derived from the same tumor sample (r = 0.78). (biomedcentral.com)
  • Sulfur dioxide, sulfites, bisulfites, metabisulfites, and hydrochloric acid could not be classified on the basis of currently available data. (who.int)
Approach1
Entire1
  • The whole genome sequencing technique was first applied to the DNA methylation mapping at single nucleotide resolution to Arabidopsis thaliana in 2008, and shortly after in 2009, the first single-base-resolution DNA methylation map of the entire human genome was created using whole genome bisulfite sequencing. (wikipedia.org)
Cells1
  • R-loops are released from cells using MapR and subjected to non-denaturing bisulfite conversion. (elifesciences.org)
Highly1
  • Nanopore 5mC calls correlate well with bisulfite (0.94) and are highly reproducible (0.95) (Fig. 1f). (nanoporetech.com)