Bowtie2

Overview

Bowtie2 is a fast and memory-efficient short-read aligner that uses an FM-index based on the Burrows-Wheeler Transform. It supports local and end-to-end alignment modes and is widely used for ChIP-seq, ATAC-seq, and other applications where gapped alignment of Illumina reads is needed. Bowtie2 handles reads of varying lengths and can report multiple alignments per read when required.

Installation

mamba install -c bioconda bowtie2

Basic Usage

Build a genome index, then align paired-end reads. The example below uses settings typical for ATAC-seq, with a maximum fragment length of 2000 bp and the --very-sensitive preset.

# Build index
bowtie2-build reference.fa reference_index

# Align paired-end reads (ATAC-seq example with -X 2000)
bowtie2 -x reference_index \
  -1 sample_R1.fastq.gz -2 sample_R2.fastq.gz \
  -X 2000 --very-sensitive -p 8 \
  | samtools sort -@ 4 -o sample.sorted.bam -

samtools index sample.sorted.bam

Key Parameters

Flag / option	Description
`-x`	Basename of the Bowtie2 index (built with `bowtie2-build`).
`-1` / `-2`	Paired-end input FASTQ files (read 1 and read 2).
`-U`	Unpaired (single-end) input FASTQ file.
`-X`	Maximum fragment length for valid paired-end alignments (default 500). Increase for ATAC-seq.
`--very-sensitive`	Preset that maximises alignment sensitivity (slower). Equivalent to `-D 20 -R 3 -N 0 -L 20 -i S,1,0.50`.
`--local`	Use local alignment mode (soft-clips read ends rather than penalising mismatches at the ends).
`-p`	Number of alignment threads.
`--no-mixed`	Suppress unpaired alignments for reads that cannot be aligned concordantly as a pair.
`--no-discordant`	Suppress discordant alignments for paired-end reads.
`--rg-id`	Set the read group ID. Combine with `--rg` to set additional read group fields.
`-k`	Report up to this many alignments per read (default: best alignment only).

Expected Output

The pipeline above produces:

sample.sorted.bam – a coordinate-sorted BAM file containing all aligned and unaligned reads.
sample.sorted.bam.bai – the BAM index file.

Bowtie2 prints alignment summary statistics to standard error, including the total number of reads, the overall alignment rate, the number of reads that aligned concordantly, and the number that aligned discordantly.

Verify the alignment with:

samtools flagstat sample.sorted.bam