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

=== Background ===

    * Reduced-representation bisulfite sequencing (RRBS) is an experimental [https://academic.oup.com/nar/article/33/18/5868/2401288 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 [https://academic.oup.com/bioinformatics/article/27/11/1571/216956 bismark] to analyze RRBS data on the resources at the Whitehead Institute. 

=== Step by step analysis ===

  * **QC**
    * Use [https://www.bioinformatics.babraham.ac.uk/projects/trim_galore Trim Galore] or [http://barcwiki.wi.mit.edu/wiki/SOPs/qc_shortReads another] read trimmer to apply quality filters and remove adapters.
    * See our [http://barcwiki.wi.mit.edu/wiki/SOPs/qc_shortReads 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**
    * [https://www.bioinformatics.babraham.ac.uk/projects/bismark/ 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 [https://www.nature.com/articles/nmeth.1923 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 
}}}
    * Comment 1
    * Comment 2