Bismark

Bismark performs bisulfite mapping alignments and methylation calls in whole genome bisulfite sequencing and reduced representation bisulfite sequencing. The reads are mapped using Bowtie2, a tool for aligning reads to a reference genome. After alignment, the software outputs several plaintext files that contain summaries of the percentage methylation of cytosines in CG, CHG, and CHH context. The software also provides a method for visualizing these outputs using plots and graphs in Plotly.
See the user guide and paper.

Download

Download Bismark through the Babraham Bioinformatics website. Images can also be retrieved through Quay.io.

Genome Preparation

# Template
bismark_genome_preparation [path_to_ref_genome_directory]

# Example
bismark_genome_preparation --path_to_aligner /my_path/bowtie2-2.5.1-linux-x86_64/ /my_path/Genome/Human_Genome_GRCh38.p14/ncbi_dataset/data/GCF_000001405.40_RefSeq/ 

Common error

If the file extension of the reference genome is in .fna, change file extension to .fa:

mv <file>.fna <file>.fa

Alignment

# Template
bismark [path_to_ref_genome_directory] -1 [read_1] -2 [read_2]

# Example
bismark /my_path/Genome/d_rerio/ --path_to_bowtie2 /my_path/bowtie2-2.5.1-linux-x86_64 -1 /my_sequences/read_1.fq -2 /my_sequences/read_2.fq

Deduplication

This is an optional step and is best for WGBS reads only.

# Template
deduplicate_bismark --paired --bam [BAM_file]

# Example
deduplicate_bismark --paired --bam /my_path/myseq_bismark_bt2_pe.bam

Methylation Extraction

# Template
bismark_methylation_extractor --comprehensive --bedGraph --cytosine_report --genome_folder [path_to_ref_genome_directory] [BAM_file]

# Example
bismark_methylation_extractor --comprehensive --bedGraph --cytosine_report --genome_folder /my_path/Genome/d_rerio/ myseq_bismark_bt2_pe.deduplicated.bam

Processing Report

bismark2report

Summary Report

bismark2summary