Bowtie.aligner (v4)

Bowtie2 (v. 2.1.0) is an ultrafast and memory-efficient short read aligner.

Author: Ben Langmead and Cole Trapnell, Johns Hopkins Bloomberg School of Public Health and the University of Maryland CBCB; Marc-Danie Nazaire,, Broad Institute


Algorithm Version: 2.1.0


Bowtie.aligner is an ultrafast, memory-efficient short read aligner geared toward quickly aligning large sets of short DNA sequences (reads) to large genomes. Bowtie.aligner takes sequence files in FASTA or FASTQ format, as well as a genome index. For more information on the FASTA format, see the NIH description at For more information on the FASTQ format, see the specification at Bowtie.aligner outputs alignments in SAM format. For more details on the SAM format, see the specification at SAM v1.4.

Bowtie.aligner takes a genome index and a set of reads as input and outputs a list of alignments. Bowtie.aligner works best when aligning short reads to large genomes (e.g., human or mouse), though it supports arbitrarily small reference sequences and reads as long as 1024 bases. Bowtie.aligner is designed to be very fast for sets of short reads where:

  • many reads have at least one good, valid alignment
  • many reads are relatively high-quality
  • the number of alignments reported per read is small (close to 1)

Bowtie.aligner was created at the Johns Hopkins Bloomberg School of Public Health and the University of Maryland Center for Bioinformatics and Computational Biology. This document is adapted from the Bowtie documentation for release 2.1.0. For more information about Bowtie, see the Bowtie Web site.

Important Notes

When you use a prebuilt index for the first time (see the prebuilt.bowtie.index parameter below), your GenePattern server must be connected to the Internet.

Bowtie.aligner may cause other processes and user interactions to run considerably slower when the alignment is performed on a laptop or desktop-class machine.

If you receive the following error message, "Can't login to (<server name>):Sorry, max 5 users -- try again later," then wait until one of your Bowtie.aligner processes has completed and then re-run your job.


Langmead B, Salzberg S. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012, 9:357-359.
Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25. (

Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754-1760.


Bowtie aligner documentation: - the-bowtie-aligner


Name Description Command Line Flag
prebuilt.bowtie.index An indexed genome. Many pre-built indexes are available, including:
  • E. coli
  • H. sapiens, UCSC hg19
  • H. sapiens, UCSC hg18
  • M. musculus, UCSC mm10
  • M. musculus, UCSC mm9
If this list does not include the genome the user requires, an indexed genome can be generated using Bowtie.indexer. Either a prebuilt or a custom Bowtie index must be specified.
custom.bowtie.index A ZIP archive containing Bowtie index files. Either a prebuilt or a custom Bowtie index must be specified.  
input.format* The format of the reads file. The available formats are:
  • QSEQ
  • One sequence per line
  • FASTQ: -q
  • FASTA: -f
  • QSEQ: -qseq
  • One sequence per line: -r
reads.pair.1* Unpaired reads file or first mate for paired reads. A file or zip of files containing reads in FASTA or FASTQ format (can be compressed - ie .gz).  
reads.pair.2 Second mate for paired reads. A file or zip of files in FASTA or FASTQ format (can be compressed - ie .gz).  
quality.value.scale Scale to use for quality values. The available scales are:
  • Phred
  • Phred plus 33
  • Phred plus 64
  • Convert Solexa to Phred
Phred quality scores are integers from 0-50 that are stored as single characters after adding 33 (Phred33) or 64 (Phred64) to the character’s ASCII value. Solexa scores are similar, using an offset of 64. See for more information.
Default: Phred
  • Phred plus 33: --pred33
  • Phred plus 64: --pred64
  • Convert Solexa to Phred: --solexa-quals

Whether the quality values are represented as space-separated ASCII integers (i.e 40 40 30 40 ..).
Default: no

--int-quals The maximum number of reads to align. -u

The alignment mode to use. The available modes are:

  • End to end
  • Local

Default: End to end

--end-to-end, --local

A predefined set of options to use. Bowtie 2 comes with some useful combinations of parameters packaged into shorter "preset" parameters. The preset options that come with Bowtie 2 are designed to cover a wide area of the speed/sensitivity/accuracy tradeoff space. The presets ending in "fast" generally being faster but less sensitive and less accurate, and the presets ending in "sensitive" generally being slower but more sensitive and more accurate.

Preset options in end-to-end alignment mode:

  • 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 -L 22 -i S,1,1.15
  • very-sensitive:    -D 20 -R 3 -N 0 -L 20 -i S,1,0.50
Preset options in local alignment mode:
  • very-fast-local:    -D5-R1-N0-L 25 -i S,1,2.00
  • fast-local:    -D 10 -R 2 -N 0 - L 22 -i S,1,1.75
  • sensitive-local:    -D15-R2-N0- L 20 -i S,1,0.75
  • very-sensitive-local:    -D 20 -R 3 -N 0 - L 20 -i S,1,0.50
Default: sensitive

End-to-end alignment mode:
--very-fast, --fast, --very-sensitive, --sensitive

Local alignment mode:
--very-fast-local, --fast-local, --very-sensitive-local, --sensitive-local

min.fragment.length The minimum fragment length for valid paired-end alignments.
Default: 0
max.fragment.length The maximum fragment length for valid paired-end alignments.
Default: 500

The upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand. The available orientations are:


  • Pair 1 and 2 upstream of reverse
  • Pair 1 reverse and pair 2 forward
  • Pair 1 and 2 forward
Default: Pair 1 and 2 upstream of reverse
  • Pair 1 and 2 upstream of reverse: --fr
  • Pair 1 reverse and pair 2 forward: --rf
  • Pair 1 and 2 forward: --ff
random.seed The seed to use for the pseudo-random number generator.
Default: 12345678
output.prefix The prefix to use for the output file name.
Default: <reads.pair.1_basename>_<reads.pair.2_basename>

* - required

Platform Dependencies

Task Type:

CPU Type:

Operating System:
Macintosh, Linux


Version Comments

Version Release Date Description
4 2013-06-17 Updated to Bowtie2 version 2.1.0
3 2012-01-11 Modified so that errors when deleting temp directories do not go to stderr.txt and fixed command line when more than one file is provided for a file parameter
2 2011-04-12 Modified to output a complete SAM file
1 2011-01-12