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

bash
# 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

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

Paired-End Alignment

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

Common Options

bash
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

bash
# 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

bash
# Post-Bisulfite Adapter Tagging (e.g., scBS-seq)
bismark --genome /path/to/genome_folder/ --pbat reads.fastq.gz

Non-Directional Libraries

bash
# 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)

bash
# 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

bash
# --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

bash
# 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

bash
# Bismark output is unsorted
samtools sort output.bam -o output.sorted.bam
samtools index output.sorted.bam

Deduplicate (Optional)

bash
# 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

bash
# 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)

bash
# 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