tools-only 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