T-WGBS

T-WGBS

Tagmentation-based whole-genome bisulfite sequencing (T-WGBS) is a protocol that utilizes the Epicentre Tn5 transposome and bisulfite conversion to study 5mC . In this method, DNA is incubated with Tn5 transposome containing methylated primers, which fragments the DNA and ligates adapters. Tagged DNA first undergoes oligo displacement, followed by methylated oligo replacement and gap repair, assuring methylated adapter addition to tagmented DNA. DNA is then treated with sodium bisulfite, PCR-amplified, and sequenced. Deep sequencing provides single-base resolution of 5mC in the genome.

Pros:

• Can sequence samples with very limited starting material (~20 ng)
• Fast protocol with few steps
• Elimination of multiple steps prevents loss of DNA

Cons:

• 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

• T-WGBS: Wang Q., Gu L., Adey A., Radlwimmer B., Wang W., et al. (2013) Tagmentation-based whole-genome bisulfite sequencing. Nat Protoc 8: 2022-2032

1. Cabezas-Wallscheid N., Klimmeck D., Hansson J., Lipka D. B., Reyes A., et al. (2014) Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 15: 507-522
2. Wang Q., Gu L., Adey A., Radlwimmer B., Wang W., et al. (2013) Tagmentation-based whole-genome bisulfite sequencing. Nat Protoc 8: 2022-2032