T-WGBS
Tagmentation-Based Whole-Genome Bisulfite Sequencing
T-WGBS uses the Tn5 transposome and bisulfite conversion to study 5mC (Wang et al., 2013). In this method, DNA is incubated with Tn5 transposome containing methylated primers, which fragments the DNA and ligates adapters. The tagged DNA first undergoes oligonucleotide displacement, followed by methylated oligonucleotide replacement and gap repair, ensuring the addition of methylated adapters to the tagmented DNA. Next, the DNA is treated with sodium bisulfite, PCR-amplified, and sequenced. Deep sequencing provides single-base resolution of 5mC in the genome.
Advantages:
- Can sequence samples with very limited starting material (~20 ng)
- Fast protocol with few steps
- Elimination of multiple steps minimizes loss of DNA
Disadvantages:
- Bisulfite converts unmethylated cytosines to thymidines, reducing sequence complexity, which can make it difficult to create alignments
- SNPs where a cytosine is converted to thymidine will be missed upon bisulfite conversion
- Bisulfite conversion does not distinguish between 5mC and 5hmC
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Tang J., Fang F., Miller D. F., et al. Global DNA methylation profiling technologies and the ovarian cancer methylome. Methods Mol Biol. 2015;1238:653-675
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661
Weichenhan D. and Plass C. The evolving epigenome. Hum Mol Genet. 2013;22:R1-6
References:
Wang Q., Gu L., Adey A., et al. Tagmentation-based whole-genome bisulfite sequencing. Nat Protoc. 2013;8:2022-2032
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History: T-WGBS
Revision by sbrumpton on 2017-06-21 07:50:20 - Show/Hide
Tagmentation-Based Whole-Genome Bisulfite Sequencing
T-WGBS uses the Tn5 transposome and bisulfite conversion to study 5mC (Wang et al., 2013). In this method, DNA is incubated with Tn5 transposome containing methylated primers, which fragments the DNA and ligates adapters. The tagged DNA first undergoes oligonucleotide displacement, followed by methylated oligonucleotide replacement and gap repair, ensuring the addition of methylated adapters to the tagmented DNA. Next, the DNA is treated with sodium bisulfite, PCR-amplified, and sequenced. Deep sequencing provides single-base resolution of 5mC in the genome.
Advantages:- Can sequence samples with very limited starting material (~20 ng)
- Fast protocol with few steps
- Elimination of multiple steps minimizes loss of DNA
Disadvantages:- Bisulfite converts unmethylated cytosines to thymidines, reducing sequence complexity, which can make it difficult to create alignments
- SNPs where a cytosine is converted to thymidine will be missed upon bisulfite conversion
- Bisulfite conversion does not distinguish between 5mC and 5hmC
Reagents:Illumina Library prep and Array Kit SelectorReviews:Tang J., Fang F., Miller D. F., et al. Global DNA methylation profiling technologies and the ovarian cancer methylome. Methods Mol Biol. 2015;1238:653-675Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661Weichenhan D. and Plass C. The evolving epigenome. Hum Mol Genet. 2013;22:R1-6References:Wang Q., Gu L., Adey A., et al. Tagmentation-based whole-genome bisulfite sequencing. Nat Protoc. 2013;8:2022-2032