Using reduced representation bisulfite sequencing (RRBS) to characterize genomic DNA methylation

Background

• Reduced-representation bisulfite sequencing (RRBS) is an experimental ​protocol to measure and compare genomic methylation patterns. The experiments use one or multiple restriction enzymes on genomic DNA to produce sequence specific fragments that are subsequently treated with bisulfite and sequenced. The workflow below illustrates how to use ​bismark to analyze RRBS data on the resources at the Whitehead Institute.

Step by step analysis

• QC
  • Use ​Trim Galore or ​another read trimmer to apply quality filters and remove adapters.
  • See our ​QC and preprocessing guidelines for details on running Trim Galore.
  • A command for Trim Galore paired end reads using gzipped fastq input can look like:

bsub trim_galore --paired --rrbs --fastqc -o trimmedReads /path/to/raw/data/reads_1.fq.gz /path/to/raw/data/reads_2.fq.gz

• Quantification of methylation calls
  • ​Bismark produces BAM file(s) of aligned reads and methylation calls.

bsub /path/to/bismark/bismark --genome /nfs/genomes/mouse_mm10_dec_11_no_random/bowtie/ -1 trimmedReads_1.fq.gz -2 trimmedReads_2.fq.gz

• Bismark can (since version 0.6.beta1) use ​bowtie2 to map short reads to reference genome(s).
• In the command above, bismark will expect to find C-to-T and a G-to-A versions of the reference genome.

• Extract methylation calls
  • After running bismark...
  • For paired ended data...:

bsub /path/to/bismark/bismark_methylation_extractor -p --gzip --bedGraph trimmedReads_bismark_bt2_pe.bam 

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