Route: rrbs/wgbs

Alignment and methylation calling for reduced representation or whole genome bisulfite sequencing data.


  • For WGBS, trim adapters and low quality bases (Trimmomatic).
  • For RRBS, trim adapters, low quality bases, and the cytosine artificially introduced in the end-repair step during the library preparation (Trim Galore).
  • Align to the bisulfite converted reference genome (Bismark and Bowtie2).
  • For WGBS, remove duplicate reads (Bismark).
  • Extract methylation calls (Bismark).
  • Generate graphical report for alignment, deduplication, and methylation extraction (Bismark).
  • Generate methylation calls genome browser tracks.


Set up a new analysis (common across all routes).

cd <project dir>
git clone --depth 1
sns/generate-settings <genome>
sns/gather-fastqs <fastq dir>

Run rrbs or wgbs route.

sns/run rrbs

Check for potential problems.

grep "ERROR:" logs-sbatch/*


Primary results:

  • BAM-Bismark: Alignment files.
  • BIGWIG-Bismark: Methylation ratio bigWig files. Can be used for visual inspection as genome browser tracks.
  • meth-Bismark-*: Methylation calls.

Run metrics:

  • summary-combined.rrbs.csv: Summary table that includes the number of reads, alignment rate, fraction of PCR duplicates, number of covered Cs, and Cs methylated in different contexts.
  • Bismark-report: Graphical report for Bismark steps (alignment, deduplication, and methylation extraction).