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