DNA methylation analysis by Churros

6. DNA methylation analysis by Churros#

This page describes how to analyze Bisulfite sequencing data for DNA methylation analysis. Churros includes Bismark to handle Bisulfite sequencing data.

Note

This tutorial assumes using the Churros singularity image (churros.sif). Please add singularity exec churros.sif before each command below.
Example: singularity exec churros.sif download_genomedata.sh

6.1. Get data#

Here we use mouse DNA methylation data using PBAT from GSE203292. These are paired-end fastq files.

mkdir -p fastq
for id in SRR19268567
do
    fastq-dump --split-files --gzip $id -O fastq
done
Then download and generate the reference dataset including genome, gene annotation and index files. Chuross contains scripts for that: download_genomedata.sh and build-index.sh.
Here we specify mm39 for genome build. See Appendix for the detail of genome build.
mkdir -p log
build=mm39      # genome build
Ddir=Referencedata_$build   # output directory
ncore=12    # number of CPUs
# download the genome
download_genomedata.sh $build $Ddir 2>&1 | tee log/$Ddir
# make Bismark index
build-index.sh -p $ncore bismark $Ddir

6.2. Running Bismark#

Bismark.sh command executes all steps of Bismark as follows:

  • bismark (mapping)

  • deduplicate_bismark

  • bismark_methylation_extractor

  • bismark2report

  • bismark2summary

In addition, Bismark.sh executes MultiQC to make a summary of quality statistics.

Supply -m option to specify the mode of Bisulfite sequencing ([directional|non_directional|pbat|rrbs]). Because here we use a PBAT sample, -m pbat option is supplied. Paired-end fastq files should be quated, and supplied by -1 and -2.

index=Referencedata_mm39/bismark-indexes_genome
Bismark.sh -m pbat $index "-1 SRR19268567_1.fastq.gz -2 SRR19268567_2.fastq.gz"

The results are output in Bismarkdir/. If you want to specify the name of the output directory, use -d option.

  • Output

    • *_bismark_bt2.bam … Map file by bismark (BAM format)

    • [CpG|CHG|CHH]_context_*_bismark_bt2.txt.gz … Output of bismark_methylation_extractor. Context-dependent (CpG/CHG/CHH) methylation.

    • *_bismark_bt2.bedGraph.gz … Bedgraph-format methylation information

    • *_bismark_bt2.bismark.cov.gz … Coverage file including counts methylated and unmethylated residues

    • *_bismark_bt2_*_report.html … Output of bismark2report. Reports of Bismark alignment, deduplication and methylation extraction (splitting). Example

    • bismark_summary_report.html … Output of bismark2summary. Summary of multiple Bismark data. Example

    • multiqc_report.html … Output of MultiQC

See Bismark User Guide for more detail.