Bismark Alignment

Prepare Genome Index

One-time genome preparation (creates bisulfite-converted index)

bismark_genome_preparation --bowtie2 /path/to/genome_folder/

Genome folder should contain FASTA files (e.g., hg38.fa, chr1.fa, etc.)

Creates Bisulfite_Genome/ subdirectory with CT and GA converted indices

Basic Single-End Alignment

bismark --genome /path/to/genome_folder/ reads.fastq.gz -o output_dir/

Paired-End Alignment

bismark --genome /path/to/genome_folder/
-1 reads_R1.fastq.gz
-2 reads_R2.fastq.gz
-o output_dir/

Common Options

bismark --genome /path/to/genome_folder/
--bowtie2 \ # Use bowtie2 (default) --parallel 4 \ # Number of parallel instances --temp_dir /tmp/ \ # Temporary directory --non_directional \ # For non-directional libraries --nucleotide_coverage \ # Generate nucleotide coverage report -o output_dir/
reads.fastq.gz

RRBS Mode

Reduced Representation Bisulfite Sequencing

bismark --genome /path/to/genome_folder/
--pbat \ # For PBAT libraries (post-bisulfite adapter tagging) reads.fastq.gz

MspI digestion (RRBS standard)

Bismark handles MspI-digested libraries automatically

PBAT Libraries

Post-Bisulfite Adapter Tagging (e.g., scBS-seq)

bismark --genome /path/to/genome_folder/ --pbat reads.fastq.gz

Non-Directional Libraries

For libraries where all 4 strands are present

bismark --genome /path/to/genome_folder/ --non_directional reads.fastq.gz

With Quality/Adapter Trimming (Pre-alignment)

Trim adapters first with Trim Galore (recommended)

trim_galore --illumina --paired reads_R1.fastq.gz reads_R2.fastq.gz

Then align

bismark --genome /path/to/genome_folder/
-1 reads_R1_val_1.fq.gz
-2 reads_R2_val_2.fq.gz

Multicore Processing

--parallel sets instances per alignment direction

Total threads = parallel * 2 (for directional) or parallel * 4 (non-directional)

bismark --genome /path/to/genome_folder/
--parallel 4
reads.fastq.gz

Output Files

Bismark produces:

- reads_bismark_bt2.bam # Aligned reads

- reads_bismark_bt2_SE_report.txt # Alignment report

View alignment report

cat output_dir/reads_bismark_bt2_SE_report.txt

Sort and Index BAM

Bismark output is unsorted

samtools sort output.bam -o output.sorted.bam samtools index output.sorted.bam

Deduplicate (Optional)

Remove PCR duplicates (recommended for WGBS, not RRBS)

deduplicate_bismark --bam output_bismark_bt2.bam

For paired-end

deduplicate_bismark --paired --bam output_bismark_bt2_pe.bam

Check Alignment Statistics

Bismark generates detailed report

cat *_SE_report.txt

Key metrics:

- Sequences analyzed

- Unique alignments

- Mapping efficiency

- C methylated in CpG context

Genome Preparation with HISAT2 (Recommended for Large Genomes)

HISAT2 is faster and uses less memory for large mammalian genomes

bismark_genome_preparation --hisat2 /path/to/genome_folder/

Align with HISAT2

bismark --genome /path/to/genome_folder/ --hisat2 reads.fastq.gz

HISAT2 paired-end

bismark --genome /path/to/genome_folder/ --hisat2
-1 reads_R1.fastq.gz
-2 reads_R2.fastq.gz

Key Parameters

Parameter Description

--genome Path to genome folder

--bowtie2 Use Bowtie2 aligner (default)

--hisat2 Use HISAT2 aligner

--parallel Parallel alignment instances

--non_directional Non-directional library

--pbat PBAT library protocol

-o Output directory

--temp_dir Temporary file directory

--nucleotide_coverage Generate nuc coverage report

-N Mismatches in seed (0 or 1, default 0)

-L Seed length (default 20)

Library Types

Type Parameter Description

Directional (default) Standard WGBS/RRBS

Non-directional --non_directional All 4 strands

PBAT --pbat Post-bisulfite adapter tagging

Related Skills

• methylation-calling - Extract methylation from Bismark BAM

• methylkit-analysis - Import Bismark output to R

• sequence-io/read-sequences - FASTQ handling

• alignment-files/sam-bam-basics - BAM manipulation