BOWTIE2-ALIGN-S(1)

💖 BOWTIE2-ALIGN-S 💖

okay bestie, welcome to the bowtie2-align-s page from the User Commands corner of the manual — yes this is THE backend queen, the one doing the actual work behind your bowtie2 command, and she is 💅 ultrafast and 💅 memory-efficient. We're aligning sequencing reads to long reference sequences and we're gonna have a good time.

---

✨ NAME ✨

bowtie2-align-s — ultrafast and memory-efficient</ backend tool for aligning sequencing reads to long reference sequences. the girl the legend the moment 👑

---

🌸 DESCRIPTION 🌸

Bowtie 2 version 2.5.5 by Ben Langmead (langmea@cs.jhu.edu, www.cs.jhu.edu/~langmea) 🫶

---

🎀 USAGE 🎀

bowtie2-align [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r> |
--interleaved <i> | -b <bam>} [-S <sam>]

• <bt2-idx> — the index filename prefix, minus that trailing .X.bt2. ⚠️ NOTE — Bowtie 1 and Bowtie 2 indexes are not compatible, they do not mix, they are not friends, do not try it bestie.
• <m1> — files with #1 mates, paired with the files in <m2>.
• <m2> — files with #2 mates, paired with the files in <m1>.
• <r> — files with unpaired reads.
• <i> — files with interleaved paired-end FASTQ/FASTA reads.
• <bam> — files are unaligned BAM sorted by read name.
• <sam> — file for SAM output (default: stdout).

and here's the lil secret: <m1>, <m2> and <r> can be comma-separated lists (no whitespace pls) and can be specified many times. like, for example:

-U file1.fq,file2.fq -U file3.fq

---

💜 OPTIONS 💜

(defaults are chilling in parentheses, iykyk)

🍓 Input:

• -q — query input files are FASTQ .fq/.fastq (default)
• --tab5 — query input files are TAB5 .tab5
• --tab6 — query input files are TAB6 .tab6
• --qseq — query input files are in Illumina's qseq format
• -f — query input files are (multi-)FASTA .fa/.mfa
• -r — query input files are raw one-sequence-per-line
• -F k:<int>,i:<int> — query input files are continuous FASTA where reads are substrings (k-mers) extracted from the FASTA file and aligned at offsets 1, 1+i, 1+2i ... end of reference
• -c — <m1>, <m2>, <r> are sequences themselves, not files
• -s/--skip <int> — skip the first <int> reads/pairs in the input (none)
• -u/--upto <int> — stop after first <int> reads/pairs (no limit)
• -5/--trim5 <int> — trim <int> bases from 5'/left end of reads (0)
• -3/--trim3 <int> — trim <int> bases from 3'/right end of reads (0)
• --trim-to [3:|5:]<int> — trim reads exceeding <int> bases from either 3' or 5' end. If the read end is not specified then it defaults to 3 (0) 🙈
• --phred33 — qualities are Phred+33 (default)
• --phred64 — qualities are Phred+64
• --int-quals — qualities encoded as space-delimited integers

🌈 Presets:

okay so these are all shortcuts where the preset = these exact flags. same as:

For --end-to-end:

• --very-fast — -D 5 -R 1 -N 0 -L 22 -i S,0,2.50
• --fast — -D 10 -R 2 -N 0 -L 22 -i S,0,2.50
• --sensitive — -D 15 -R 2 -N 0 -L 22 -i S,1,1.15 (default) ⭐
• --very-sensitive — -D 20 -R 3 -N 0 -L 20 -i S,1,0.50

For --local:

• --very-fast-local — -D 5 -R 1 -N 0 -L 25 -i S,1,2.00
• --fast-local — -D 10 -R 2 -N 0 -L 22 -i S,1,1.75
• --sensitive-local — -D 15 -R 2 -N 0 -L 20 -i S,1,0.75 (default) ⭐
• --very-sensitive-local — -D 20 -R 3 -N 0 -L 20 -i S,1,0.50

🦋 Alignment:

• -N <int> — max # mismatches in seed alignment; can be 0 or 1 (0)
• -L <int> — length of seed substrings; must be >3, <32 (22)
• -i <func> — interval between seed substrings w/r/t read len (S,1,1.15)
• --n-ceil <func> — func for max # non-A/C/G/Ts permitted in aln (L,0,0.15)
• --dpad <int> — include <int> extra ref chars on sides of DP table (15)
• --gbar <int> — disallow gaps within <int> nucs of read extremes (4)
• --ignore-quals — treat all quality values as 30 on Phred scale (off)
• --nofw — do not align forward (original) version of read (off)
• --norc — do not align reverse-complement version of read (off)
• --no-1mm-upfront — do not allow 1 mismatch alignments before attempting to scan for the optimal seeded alignments
• --end-to-end — entire read must align; no clipping (on)

OR ✨

• --local — local alignment; ends might be soft clipped (off)

💅 Scoring:

• --ma <int> — match bonus (0 for --end-to-end, 2 for --local)
• --mp <int> — max penalty for mismatch; lower qual = lower penalty (6)
• --np <int> — penalty for non-A/C/G/Ts in read/ref (1)
• --rdg <int>,<int> — read gap open, extend penalties (5,3)
• --rfg <int>,<int> — reference gap open, extend penalties (5,3)
• --score-min <func> — min acceptable alignment score w/r/t read length (G,20,8 for local, L,-0.6,-0.6 for end-to-end)

👑 Reporting:

• (default) — look for multiple alignments, report best, with MAPQ

OR

• -k <int> — report up to <int> alns per read; MAPQ not meaningful
• -a/--all — report all alignments; very slow without -l, MAPQ not meaningful

🔥 Effort:

• -l/--lowseeds <n> — ignore any low quality seeds with ranges over threshold (0=no cut, if percentage, mili or nano, relative to idx size)
• -D <int> — give up extending after <int> failed extends in a row (15)
• -R <int> — for reads w/ repetitive seeds, try <int> sets of seeds (2)
• -d/--deterministic-seeds — Consider all seeds in order (no subsampling, best with -a)

💞 Paired-end:

• -I/--minins <int> — minimum fragment length (0)
• -X/--maxins <int> — maximum fragment length (500)
• --fr/--rf/--ff — -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr)
• --no-mixed — suppress unpaired alignments for paired reads
• --no-discordant — suppress discordant alignments for paired reads
• --dovetail — concordant when mates extend past each other
• --no-contain — not concordant when one mate alignment contains other
• --no-overlap — not concordant when mates overlap at all

🧬 BAM:

• --align-paired-reads — Bowtie2 will, by default, attempt to align unpaired BAM reads. Use this option to align paired-end reads instead.
• --preserve-tags — Preserve tags from the original BAM record by appending them to the end of the corresponding SAM output.

🌟 Output:

• -t/--time — print wall-clock time taken by search phases
• --quiet — print nothing to stderr except serious errors 🤫
• --met-file <path> — send metrics to file at <path> (off)
• --met-stderr — send metrics to stderr (off)
• --met <int> — report internal counters & metrics every <int> secs (1)
• --no-unal — suppress SAM records for unaligned reads
• --no-head — suppress header lines, i.e. lines starting with @
• --no-sq — suppress @SQ header lines
• --rg-id <text> — set read group id, reflected in @RG line and RG:Z: opt field
• --rg <text> — add <text> ("lab:value") to @RG line of SAM header. Note: @RG line only printed when --rg-id is set.
• --omit-sec-seq — put '*' in SEQ and QUAL fields for secondary alignments.
• --sam-no-qname-trunc — Suppress standard behavior of truncating readname at first whitespace at the expense of generating non-standard SAM.
• --xeq — Use '='/'X', instead of 'M,' to specify matches/mismatches in SAM record.
• --soft-clipped-unmapped-tlen — Exclude soft-clipped bases when reporting TLEN.
• --sam-append-comment — Append FASTA/FASTQ comment to SAM record.
• --sam-opt-config <config> — Use <config>, example '-MD,YP,-AS', to toggle SAM Optional fields.

⚡ Performance:

• -p/--threads <int> — number of alignment threads to launch (1)
• --reorder — force SAM output order to match order of input reads
• --mm — use memory-mapped I/O for index; many 'bowtie's can share

🫶 Other:

• --qc-filter — filter out reads that are bad according to QSEQ filter
• --seed <int> — seed for random number generator (0)
• --non-deterministic — seed rand. gen. arbitrarily instead of using read attributes
• --version — print version information and quit
• -h/--help — print this usage message

---

💌 and that's the whole page bestie!! every flag, every default, nothing skipped, no cap. go align something iconic. 🧚✨

bowtie2-align-s 2.5.5 · February 2026 · mwah, kissing this manpage goodnight 💋🌙