Whole genome shotgun bisulfite sequencing (WGSBS) is every bit as fierce as its name suggests. Shotgun sequencing is a processes for sequencing a lot DNA, such as a whole genome. The DNA is broken up into short fragments which are then sequenced in parallel. The many short fragments (or “reads”) are then aligned in a computer program to recreate the entire sequence (Anderson, 1981).
Most next-gen sequencing technologies rely on this principle. Next-gen bisulfite-seg techniques have dropped the “shotgun” terminology, however almost all use this method. In WGSBS, the genomic DNA is treated with sodium bisulfite. Adapters are then ligated to the ends of the fragments to allow amplification and sequencing. This is especially necessary for WGSBS since bisulfite essentially reduces the genome from 4 bases to 3, so primer design becomes impossible to capture the whole-genome (Li and Tollefsbol, 2011).
The library is then size selected and then sequenced using a next-gen platform. WGSBS offers the highest levels of resolution and scale of 5mC investigation. On the downside, WGSBS is very expensive per sample, over ten times the cost of RRBS per sample (Gu et al., 2011). It also requires a great deal of sample DNA for each run since bisulfite fragments DNA so greatly.
WGSBS Additional Reading
This review examines many of the challenges of interpreting whole-genome BS-seq data. The authors cover the basics of BS-seq, how different sequencing technologies compare, and the challenges at each level of analysis.
Bock, C. (2012). Analysing and interpreting DNA methylation data. Nat. Rev. Genet. 13, 705-719.
This review focuses on the analysis of WGSBS data. Workflow, available software, alignment procedures, quality control and visualization are all discussed at length.
Reference List
- Anderson, S. (1981). Shotgun DNA sequencing using cloned DNase I-generated fragments. Nucleic Acids Res. 9, 3015-3027.
- Gu, H., Smith, Z.D., Bock, C., Boyle, P., Gnirke, A., and Meissner, A. (2011). Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nat. Protoc. 6, 468-481.
- Li, Y., and Tollefsbol, T.O. (2011). DNA methylation detection: bisulfite genomic sequencing analysis. Methods Mol. Biol. 791, 11-21.