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