Version Compatibility

Reference examples tested with: Bowtie2 2.5.3+, HISAT2 2.2.1+, Trim Galore 0.6.10+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• CLI:

  <tool> --version

  then

  <tool> --help

  to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Bismark Alignment

"Align my bisulfite sequencing reads" → Map WGBS/RRBS reads to an in-silico bisulfite-converted reference genome, producing BAM files with methylation context tags.

• CLI:

  bismark_genome_preparation genome/

  then

  bismark --genome genome/ reads.fq.gz

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
• differential-cpg-testing - Per-CpG testing from coverage data
• sequence-io/read-sequences - FASTQ handling
• alignment-files/sam-bam-basics - BAM manipulation