bowtie2

name	bowtie2
description	Gapped short-read alignment to contigs/genomes for coverage used in binning and abundance. Use when indexing assemblies, mapping cleaned PE reads, choosing sensitivity presets, QC of BAM rates, or preparing depths for MetaBAT2/CoverM/BASALT. Upstream: https://github.com/BenLangmead/bowtie2. Alternatives: strobealign, bwa-mem2. Always pair with samtools.
license	MIT
category	analysis-tools
tags	["mapping","alignment","short-read","coverage"]
upstream	https://github.com/BenLangmead/bowtie2
stage	mapping

Bowtie 2

Upstream: BenLangmead/bowtie2

Citation

Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012). https://doi.org/10.1038/nmeth.1923

Analytical thinking

Mapping is the bridge from assembly → binning. Coverage profiles (not raw reads) drive MetaBAT2/MaxBin2/CONCOCT/SemiBin2 and inform BASALT. Index once per assembly; map every sample that should contribute coverage.

Use the same cleaned PE reads that fed assembly (or the declared coverage set from study-design). Mixing host-contaminated and dehosted libraries silently biases bins.

Presets trade sensitivity vs speed: --sensitive / --very-sensitive for catalogue work; --very-sensitive-local allows soft-clipping useful on fragmented metagenome contigs. Do not treat higher sensitivity as “more true species”.

Prerequisites

Requirement	Minimum	Recommended
Contigs	FASTA from assembly	Filtered min-length if huge
Reads	Clean PE FASTQ	From kneaddata
Tools	bowtie2 + samtools	Same env
Threads	8	16–32
Disk	BAM ≈ 1–2× read set	Keep sorted BAM + .bai

How to run

conda install -c bioconda bowtie2 samtools

mkdir -p temp/map
bowtie2-build --threads 8 contigs.fa temp/map/contigs

bowtie2 -x temp/map/contigs \
  -1 temp/hr/${id}_1.fastq -2 temp/hr/${id}_2.fastq \
  -p 16 --very-sensitive-local \
  2> temp/map/${id}.bowtie2.log \
  | samtools sort -@ 8 -o temp/map/${id}.bam
samtools index temp/map/${id}.bam

# Multi-sample depth for MetaBAT2
jgi_summarize_bam_contig_depths --outputDepth temp/map/depth.txt temp/map/*.bam

Key parameters

Option	Guidance
-x	Index prefix from bowtie2-build
-1/-2	PE mates; keep order from KneadData --reorder
-U	SE only when truly single-end
-p	Threads (aligner); samtools sort uses -@
--very-sensitive-local	Soft-clip friendly on short contigs
--no-unal	Smaller SAM/BAM; lose unmapped diagnostics

Output & QC

Artifact	Use
${id}.bam + .bai	CoverM, binners, inStrain
depth.txt	MetaBAT2 -a
stderr overall alignment rate	Library/assembly sanity

samtools flagstat temp/map/${id}.bam
# Typical clean metagenomes: often 50–90% mapped; near-0% ⇒ wrong contigs/reads

Decision tree

Illumina → contig coverage?
├─ Large cohort / speed critical → strobealign (or bwa-mem2)
├─ Lab standard / KneadData ecosystem → bowtie2
└─ Long reads → minimap2 (not bowtie2)

Troubleshooting

Symptom	Action
~0% mapped	Contigs/reads mismatch; wrong sample IDs
PE sync errors	Re-KneadData with --reorder
bowtie2-build RAM blowup	Split huge contig sets or raise node RAM
Unsorted BAM breaks MetaBAT	Always samtools sort + index

Related skills

samtools · strobealign · bwa-mem2 · coverm · metabat2 · basalt · study-design