Whole-Genome Bisulfite Sequencing (WGBS) is a powerful approach for genome-wide analysis of the methylome at single-base resolution. DNA samples undergo bisulphite treatment, leading to the conversion of unmethylated cytosines into uracils. Methylated cytosine residues, or 5-methylcytosines, are not affected. In this tutorial we will calculate methylation ratios on the Genestack platform, going from uploading raw data to visualising our results in a genome browser.
We will use Genestack applications to go through the following steps:
- Loading data from a public database
- Quality control and preprocessing of raw reads
- Mapping BS-Seq reads onto reference genome
- Determination of methylation rates
- Visualization of results in Genome Browser
The BS-Seq dataset we will use comes from Lyko, F. and Reik, W. 2013 and includes one Mus musculussample obtained from combining DNA from mice with a triple deletion (TKO) in three methylases (Dnmt1-/-, Dnmt3a-/- and Dnmt3b-/-). This library includes 100 bp paired end reads from Illumina technology. This dataset has been published as on GEO, the Gene Expression Omnibus database from NCBI, with accession number GSE42170.
This experiment is already available on Genestack Platform with accession number GSF000400. However, Genestack’s applications include several loading applications which allow us to import different kinds of datasets from several public datasets. In the next page we will import this experiment from the Gene Expression Omnibus (GEO) database.