What are the requirements of species for WGBS?
1) Is the species with low methylation rate? Six species reported with low methylation rate are drosophila, flour beetle, Saccharomyces cerevisiae, Schizosaccharomyces pomonella, nematode and Aspergillus flavus.
2) The completion of this species genome affects bisulfite-seq alignment. The genome is assembled at least in scaffold level with more annotation.
3) Whether there are complex factors in genome: high GC content, high heterozygosity, transposons, repeat regions, etc.
4) The species is eukaryote.
What is the CT conversion rate of unmethylated C after bisulfite treatment?
With abundant WGBS projects experience and professional experimental process, it can be ensured that the CT conversion rate of unmethylated C after bisulfite treatment is above 99.5%. If there is no control sample without methylation (e.g. Arabidopsis chloroplast DNA is not methylated), the transformation rate of bisulfite will be verified by mixing control DNA into the samples.
How many sequence depths is suggested for WGBS? Does WGBS need biological repeats?
Generally, the sequence depth of WGBS is recommended at 30x, with two biological repeats. If the same kind of cells are studied, it needs 5-15x sequencing depth. If the short DMR is interested to study, it needs more than 15x sequencing depth. If the longer DMR is interested to analyze, it needs 1-2x sequencing depth. It is suggested that at least two biological repeats for analyzing DMR.
