Genome Spot

Bismark is currently the de facto standard for primary analysis of high throughput bisulfite sequencing data. Bismark can align the reads to the genome and perform methylation calling. In this post, I'll go through Illumina whole genome bisulfite sequence (WGBS) alignment and methylation calling using Bismark. First I want to mention that this post is just a summary, not meant to be a user manual or thorough troubleshooting guide. Fortunately, Bismark has some of the best documentation for any bioinformatics suite and is mandatory reading. The Bismark crew are very proactive with responding to user queries on various forums as well.

First step in getting Bismark to work is to index the genome, in this case with Bowtie2:

bismark_genome_preparation --bowtie2 /pathto/refgenome/

Conventionally, multiplexed libraries will be sequenced over a number of lanes. Resist concatenating or merging the smaller fastq files for each patient/sample until after the alignment, as the concatenated fil…

I've been a fan of GNU parallel for a long time. Initially I was sceptical about using it, preferring to write huge for loops but over time I've grown to love it. The beauty of GNU parallel is that it spawns a specified number of jobs in parallel and then submits more jobs as others are completed. This means that you get maximum usage out of the CPUs without overloading the system. There are many excuses for not using it, but perhaps the only valid one is that you have Sun Grid Engine or another job scheduler or manager in place.

GNU parallel is particularly useful when used with functions. Functions are subroutines that may be repeated many times to complete a piece of work. In bash, here is a simple example, which declares a function consisting of a chain of piped commands, and then executes 4 jobs in parallel, until all of *files.txt have been processed.

#!/bin/bash
my_func2() {
INPUT=$1
VAR1=bar
cmd1 $INPUT $VAR1 | cmd2 | cmd3 > ${1}.out
}
export -f my_func
parallel -j4…