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. Alternatives: strobealign, bwa-mem2. Always pair with samtools. license: MIT metadata: version: "1.0.1" metagenomics.category: "analysis-tools" metagenomics.tags: "mapping, alignment, short-read, coverage" metagenomics.upstream: "https://github.com/BenLangmead/bowtie2" metagenomics.stage: "mapping" metagenomics.maturity: "reviewed" metagenomics.last-reviewed: "2026-10-24" metagenomics.command-status: "Bowtie2/Samtools paired-end coverage template statically reviewed; not executed here"

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

How to run

Build one index per immutable assembly, then map each manifest-defined read pair into a separate output directory.

set -Eeuo pipefail
id="${1:?usage: map-one.sh SAMPLE_ID REF R1 R2 OUTPUT_DIR}"
ref="${2:?usage: map-one.sh SAMPLE_ID REF R1 R2 OUTPUT_DIR}"
r1="${3:?usage: map-one.sh SAMPLE_ID REF R1 R2 OUTPUT_DIR}"
r2="${4:?usage: map-one.sh SAMPLE_ID REF R1 R2 OUTPUT_DIR}"
out="${5:?usage: map-one.sh SAMPLE_ID REF R1 R2 OUTPUT_DIR}"
threads="${THREADS:-16}"
[[ "$id" =~ ^[A-Za-z0-9][A-Za-z0-9._-]*$ ]] || exit 2
for input in "$ref" "$r1" "$r2"; do test -s "$input" || exit 2; done
[[ "$threads" =~ ^[1-9][0-9]*$ ]] || exit 2
test ! -e "$out" || { echo "use a new output directory: $out" >&2; exit 2; }
mkdir -p "$out/index"
 
bowtie2-build --threads "$threads" "$ref" "$out/index/reference" \
  >"$out/bowtie2-build.stdout.log" 2>"$out/bowtie2-build.stderr.log"
bowtie2 -x "$out/index/reference" -1 "$r1" -2 "$r2" \
  -p "$threads" --very-sensitive-local \
  2>"$out/${id}.bowtie2.stderr.log" \
  | samtools sort -@ "$threads" -o "$out/${id}.bam" -
samtools index "$out/${id}.bam"

For multi-sample binning, build the depth table from an explicit, ordered BAM manifest; do not rely on an unreviewed wildcard or mix references/mappers.

Key parameters

Output & QC

samtools quickcheck -v "$out/${id}.bam"
samtools view -H "$out/${id}.bam" | grep -q '^@HD.*SO:coordinate'
test -s "$out/${id}.bam.bai"
samtools flagstat -@ 4 -O tsv "$out/${id}.bam" >"$out/${id}.flagstat.tsv"
samtools idxstats "$out/${id}.bam" >"$out/${id}.idxstats.tsv"

Inspect paired/primary mapping, insert-size behavior, reference names, and depth rather than applying a universal mapping-rate cutoff. Preserve Bowtie2/Samtools versions, reference/read checksums, sensitivity and alignment-policy flags, commands, logs, BAM checksum, and QC tables. Failed streams can leave partial BAMs; only expose downstream after these checks pass.

Decision tree

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

Troubleshooting

Related skills

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