Bowtie2 Alignment

Build Index

# Build index from reference FASTA
bowtie2-build reference.fa reference_index

# With threads (faster)
bowtie2-build --threads 8 reference.fa reference_index

# Creates: reference_index.1.bt2, .2.bt2, .3.bt2, .4.bt2, .rev.1.bt2, .rev.2.bt2

Basic Alignment

# Paired-end reads
bowtie2 -p 8 -x reference_index -1 reads_1.fq.gz -2 reads_2.fq.gz -S aligned.sam

# Single-end reads
bowtie2 -p 8 -x reference_index -U reads.fq.gz -S aligned.sam

# Direct to sorted BAM
bowtie2 -p 8 -x reference_index -1 r1.fq.gz -2 r2.fq.gz | \
    samtools sort -@ 4 -o aligned.sorted.bam -

Alignment Modes

# End-to-end mode (default) - align entire read
bowtie2 --end-to-end -x index -1 r1.fq -2 r2.fq -S aligned.sam

# Local mode - soft-clip ends for better alignment
bowtie2 --local -x index -1 r1.fq -2 r2.fq -S aligned.sam

Sensitivity Presets

# Very fast (less sensitive)
bowtie2 --very-fast -x index -1 r1.fq -2 r2.fq -S aligned.sam

# Fast
bowtie2 --fast -x index -1 r1.fq -2 r2.fq -S aligned.sam

# Sensitive (default)
bowtie2 --sensitive -x index -1 r1.fq -2 r2.fq -S aligned.sam

# Very sensitive (slower but more accurate)
bowtie2 --very-sensitive -x index -1 r1.fq -2 r2.fq -S aligned.sam

# Local mode equivalents
bowtie2 --very-sensitive-local -x index -1 r1.fq -2 r2.fq -S aligned.sam

ChIP-seq Alignment

# Typical ChIP-seq settings
bowtie2 -p 8 \
    --very-sensitive \
    --no-mixed \
    --no-discordant \
    -x index -1 chip_1.fq.gz -2 chip_2.fq.gz | \
    samtools view -bS -q 30 -F 4 - | \
    samtools sort -o chip.sorted.bam -

ATAC-seq Alignment

# ATAC-seq with size selection
bowtie2 -p 8 \
    --very-sensitive \
    -X 2000 \                    # Max fragment length
    --no-mixed \
    --no-discordant \
    -x index -1 atac_1.fq.gz -2 atac_2.fq.gz | \
    samtools view -bS -q 30 - | \
    samtools sort -o atac.sorted.bam -

Fragment Size Options

# Set expected insert size range
bowtie2 -p 8 \
    -I 100 \     # Minimum fragment length
    -X 500 \     # Maximum fragment length
    -x index -1 r1.fq -2 r2.fq -S aligned.sam

Read Group and Output Options

# Add read group
bowtie2 -p 8 \
    --rg-id sample1 \
    --rg SM:sample1 \
    --rg PL:ILLUMINA \
    --rg LB:lib1 \
    -x index -1 r1.fq -2 r2.fq -S aligned.sam

Multi-mapping Reads

# Report up to k alignments per read
bowtie2 -k 5 -x index -1 r1.fq -2 r2.fq -S aligned.sam

# Report all alignments
bowtie2 -a -x index -1 r1.fq -2 r2.fq -S aligned.sam

Output Unmapped Reads

# Write unmapped reads to separate files
bowtie2 -p 8 \
    --un-conc-gz unmapped_%.fq.gz \
    -x index -1 r1.fq.gz -2 r2.fq.gz -S aligned.sam

Key Parameters

Parameter	Default	Description
-p	1	Number of threads
-x	-	Index basename
-1/-2	-	Paired-end reads
-U	-	Single-end reads
-I	0	Min fragment length
-X	500	Max fragment length
-k	1	Report up to k alignments
--no-mixed	off	Suppress unpaired alignments
--no-discordant	off	Suppress discordant alignments

Alignment Statistics

# Bowtie2 prints alignment summary to stderr
bowtie2 -p 8 -x index -1 r1.fq -2 r2.fq -S aligned.sam 2> alignment_stats.txt

Example output:

1000000 reads; of these:
  1000000 (100.00%) were paired; of these:
    50000 (5.00%) aligned concordantly 0 times
    900000 (90.00%) aligned concordantly exactly 1 time
    50000 (5.00%) aligned concordantly >1 times
95.00% overall alignment rate

Related Skills

• read-qc/fastp-workflow - Preprocess reads before alignment
• alignment-files/alignment-sorting - Post-alignment processing
• chip-seq/peak-calling - ChIP-seq analysis
• atac-seq/atac-peak-calling - ATAC-seq analysis