Table of Contents

What is Bowtie?

Bowtie is an ultrafast, memory-efficient short read aligner geared toward quickly aligning large sets of short DNA sequences (reads) to large genomes. It aligns 35-base-pair reads to the human genome at a rate of 25 million reads per hour on a typical workstation. Bowtie indexes the genome with a Burrows-Wheeler index to keep its memory footprint small: for the human genome, the index is typically about 2.2 GB (for unpaired alignment) or 2.9 GB (for paired-end or colorspace alignment). Multiple processors can be used simultaneously to achieve greater alignment speed. Bowtie can also output alignments in the standard SAM format, allowing Bowtie to interoperate with other tools supporting SAM, including the SAMtools consensus, SNP, and indel callers. Bowtie runs on the command line under Windows, Mac OS X, Linux, and Solaris.

Bowtie also forms the basis for other tools, including TopHat: a fast splice junction mapper for RNA-seq reads, Cufflinks: a tool for transcriptome assembly and isoform quantitiation from RNA-seq reads, Crossbow: a cloud-computing software tool for large-scale resequencing data,and Myrna: a cloud computing tool for calculating differential gene expression in large RNA-seq datasets.

If you use Bowtie for your published research, please cite the Bowtie paper.

What isn't Bowtie?

Bowtie is not a general-purpose alignment tool like MUMmer, BLAST or Vmatch. Bowtie works best when aligning short reads to large genomes, though it supports arbitrarily small reference sequences (e.g. amplicons) and reads as long as 1024 bases. Bowtie is designed to be extremely fast for sets of short reads where (a) many of the reads have at least one good, valid alignment, (b) many of the reads are relatively high-quality, and (c) the number of alignments reported per read is small (close to 1).

Bowtie does not yet report gapped alignments; this is future work.

Obtaining Bowtie

You may download either Bowtie sources or binaries for your platform from the Download section of the Sourceforge project site. Binaries are currently available for 64-bit Intel architectures running Linux, Windows, and Mac OS X.

Building from source

Building Bowtie from source requires a GNU-like environment that includes GCC, GNU Make and other basics. It should be possible to build Bowtie on a vanilla Linux or Mac installation. Bowtie can also be built on Windows using MinGW. We recommend TDM's MinGW Build. You also must also have MSYS installed.

To build Bowtie, extract the sources, change to the extracted directory, and run GNU make (usually with the command make, but sometimes with gmake) with no arguments. If building with MinGW, run make from the MSYS command line.

To support the -p (multithreading) option, Bowtie needs the pthreads library on posix platforms like linux or will try to use native threads on Windows. For threading synchronization Bowtie is using by default a spinlocking mechanism. Spinlocking is in general much faster. However if the need arise to not use spinlocking Bowtie can also be compiled using EXTRA_FLAGS=-DNO_SPINLOCK parameter.

The bowtie aligner

bowtie takes an index and a set of reads as input and outputs a list of alignments. Alignments are selected according to a combination of the -v/-n/-e/-l options (plus the -I/-X/--fr/--rf/ --ff options for paired-end alignment), which define which alignments are legal, and the -k/-a/-m/-M/--best/--strata options which define which and how many legal alignments should be reported.

By default, Bowtie enforces an alignment policy similar to Maq's default quality-aware policy (-n 2 -l 28 -e 70). See the -n alignment mode section of the manual for details about this mode. But Bowtie can also enforce a simpler end-to-end k-difference policy (e.g. with -v 2). See the -v alignment mode section of the manual for details about that mode. The -n alignment mode and the -v alignment mode are mutually exclusive.

Bowtie 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 is designed to be very fast for sets of short reads where a) many reads have at least one good, valid alignment, b) many reads are relatively high-quality, c) the number of alignments reported per read is small (close to 1). These criteria are generally satisfied in the context of modern short-read analyses such as RNA-seq, ChIP-seq, other types of -seq, and mammalian resequencing. You may observe longer running times in other research contexts.

If bowtie is too slow for your application, try some of the performance-tuning hints described in the Performance Tuning section below.

Alignments involving one or more ambiguous reference characters (N, -, R, Y, etc.) are considered invalid by Bowtie. This is true only for ambiguous characters in the reference; alignments involving ambiguous characters in the read are legal, subject to the alignment policy. Ambiguous characters in the read mismatch all other characters. Alignments that "fall off" the reference sequence are not considered valid.

The process by which bowtie chooses an alignment to report is randomized in order to avoid "mapping bias" - the phenomenon whereby an aligner systematically fails to report a particular class of good alignments, causing spurious "holes" in the comparative assembly. Whenever bowtie reports a subset of the valid alignments that exist, it makes an effort to sample them randomly. This randomness flows from a simple seeded pseudo-random number generator and is deterministic in the sense that Bowtie will always produce the same results for the same read when run with the same initial "seed" value (see --seed option).

In the default mode, bowtie can exhibit strand bias. Strand bias occurs when input reference and reads are such that (a) some reads align equally well to sites on the forward and reverse strands of the reference, and (b) the number of such sites on one strand is different from the number on the other strand. When this happens for a given read, bowtie effectively chooses one strand or the other with 50% probability, then reports a randomly-selected alignment for that read from among the sites on the selected strand. This tends to overassign alignments to the sites on the strand with fewer sites and underassign to sites on the strand with more sites. The effect is mitigated, though it may not be eliminated, when reads are longer or when paired-end reads are used. Running Bowtie in --best mode eliminates strand bias by forcing Bowtie to select one strand or the other with a probability that is proportional to the number of best sites on the strand.

Gapped alignments are not currently supported in Bowtie, but they are supported in Bowtie 2.

The -n alignment mode

When the -n option is specified (which is the default), bowtie determines which alignments are valid according to the following policy, which is similar to Maq's default policy.

1. Alignments may have no more than N mismatches (where N is a number 0-3, set with -n) in the first L bases (where L is a number 5 or greater, set with -l) on the high-quality (left) end of the read. The first L bases are called the "seed".

2. The sum of the Phred quality values at all mismatched positions (not just in the seed) may not exceed E (set with -e). Where qualities are unavailable (e.g. if the reads are from a FASTA file), the Phred quality defaults to 40.

The -n option is mutually exclusive with the -v option.

If there are many possible alignments satisfying these criteria, Bowtie gives preference to alignments with fewer mismatches and where the sum from criterion 2 is smaller. When the --best option is specified, Bowtie guarantees the reported alignment(s) are "best" in terms of these criteria (criterion 1 has priority), and that the alignments are reported in best-to-worst order. Bowtie is somewhat slower when --best is specified.

Note that Maq internally rounds base qualities to the nearest 10 and rounds qualities greater than 30 to 30. To maintain compatibility, Bowtie does the same. Rounding can be suppressed with the --nomaqround option.

Bowtie is not fully sensitive in -n 2 and -n 3 modes by default. In these modes Bowtie imposes a "backtracking limit" to limit effort spent trying to find valid alignments for low-quality reads unlikely to have any. This may cause bowtie to miss some legal 2- and 3-mismatch alignments. The limit is set to a reasonable default (125 without --best, 800 with --best), but the user may decrease or increase the limit using the --maxbts and/or -y options. -y mode is relatively slow but guarantees full sensitivity.

The -v alignment mode

In -v mode, alignments may have no more than V mismatches, where V may be a number from 0 through 3 set using the -v option. Quality values are ignored. The -v option is mutually exclusive with the -n option.

If there are many legal alignments, Bowtie gives preference to alignments with fewer mismatches. When the --best option is specified, Bowtie guarantees the reported alignment(s) are "best" in terms of the number of mismatches, and that the alignments are reported in best-to-worst order. Bowtie is somewhat slower when --best is specified.

Strata

In the -n alignment mode, an alignment's "stratum" is defined as the number of mismatches in the "seed" region, i.e. the leftmost L bases, where L is set with the -l option. In the -v alignment mode, an alignment's stratum is defined as the total number of mismatches in the entire alignment. Some of Bowtie's options (e.g. --strata and -m use the notion of "stratum" to limit or expand the scope of reportable alignments.

Reporting Modes

With the -k, -a, -m, -M, --best and --strata options, the user can flexibily select which alignments are reported. Below we demonstrate a few ways in which these options can be combined. All examples are using the e_coli index packaged with Bowtie. The --suppress option is used to keep the output concise and some output is elided for clarity.

Example 1: -a

$ ./bowtie -a -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
-   gi|110640213|ref|NC_008253.1|   148810  10:A>G,13:C>G
-   gi|110640213|ref|NC_008253.1|   2852852 8:T>A
-   gi|110640213|ref|NC_008253.1|   4930433 4:G>T,6:C>G
-   gi|110640213|ref|NC_008253.1|   905664  6:A>G,7:G>T
+   gi|110640213|ref|NC_008253.1|   1093035 2:T>G,15:A>T

Specifying -a instructs bowtie to report all valid alignments, subject to the alignment policy: -v 2. In this case, bowtie finds 5 inexact hits in the E. coli genome; 1 hit (the 2nd one listed) has 1 mismatch, and the other 4 hits have 2 mismatches. Four are on the reverse reference strand and one is on the forward strand. Note that they are not listed in best-to-worst order.

Example 2: -k 3

$ ./bowtie -k 3 -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
-   gi|110640213|ref|NC_008253.1|   148810  10:A>G,13:C>G
-   gi|110640213|ref|NC_008253.1|   2852852 8:T>A
-   gi|110640213|ref|NC_008253.1|   4930433 4:G>T,6:C>G

Specifying -k 3 instructs bowtie to report up to 3 valid alignments. In this case, a total of 5 valid alignments exist (see Example 1); bowtie reports 3 out of those 5. -k can be set to any integer greater than 0.

Example 3: -k 6

$ ./bowtie -k 6 -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
-   gi|110640213|ref|NC_008253.1|   148810  10:A>G,13:C>G
-   gi|110640213|ref|NC_008253.1|   2852852 8:T>A
-   gi|110640213|ref|NC_008253.1|   4930433 4:G>T,6:C>G
-   gi|110640213|ref|NC_008253.1|   905664  6:A>G,7:G>T
+   gi|110640213|ref|NC_008253.1|   1093035 2:T>G,15:A>T

Specifying -k 6 instructs bowtie to report up to 6 valid alignments. In this case, a total of 5 valid alignments exist, so bowtie reports all 5.

Example 4: default (-k 1)

$ ./bowtie -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
-   gi|110640213|ref|NC_008253.1|   148810  10:A>G,13:C>G

Leaving the reporting options at their defaults causes bowtie to report the first valid alignment it encounters. Because --best was not specified, we are not guaranteed that bowtie will report the best alignment, and in this case it does not (the 1-mismatch alignment from the previous example would have been better). The default reporting mode is equivalent to -k 1.

Example 5: -a --best

$ ./bowtie -a --best -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
-   gi|110640213|ref|NC_008253.1|   2852852 8:T>A
+   gi|110640213|ref|NC_008253.1|   1093035 2:T>G,15:A>T
-   gi|110640213|ref|NC_008253.1|   905664  6:A>G,7:G>T
-   gi|110640213|ref|NC_008253.1|   148810  10:A>G,13:C>G
-   gi|110640213|ref|NC_008253.1|   4930433 4:G>T,6:C>G

Specifying -a results in the same alignments being printed as if just -a had been specified, but they are guaranteed to be reported in best-to-worst order.

Example 6: -a --best --strata

$ ./bowtie -a --best --strata -v 2 --suppress 1,5,6,7 e_coli -c ATGCATCATGCGCCAT
-   gi|110640213|ref|NC_008253.1|   2852852 8:T>A

Specifying --strata in addition to -a and --best causes bowtie to report only those alignments in the best alignment "stratum". The alignments in the best stratum are those having the least number of mismatches (or mismatches just in the "seed" portion of the alignment in the case of -n mode). Note that if --strata is specified, --best must also be specified.

Example 7: -a -m 3

$ ./bowtie -a -m 3 -v 2 e_coli -c ATGCATCATGCGCCAT
No alignments

Specifying -m 3 instructs bowtie to refrain from reporting any alignments for reads having more than 3 reportable alignments. The -m option is useful when the user would like to guarantee that reported alignments are "unique", for some definition of unique.

Example 1 showed that the read has 5 reportable alignments when -a and -v 2 are specified, so the -m 3 limit causes bowtie to output no alignments.

Example 8: -a -m 5

$ ./bowtie -a -m 5 -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
-   gi|110640213|ref|NC_008253.1|   148810  10:A>G,13:C>G
-   gi|110640213|ref|NC_008253.1|   2852852 8:T>A
-   gi|110640213|ref|NC_008253.1|   4930433 4:G>T,6:C>G
-   gi|110640213|ref|NC_008253.1|   905664  6:A>G,7:G>T
+   gi|110640213|ref|NC_008253.1|   1093035 2:T>G,15:A>T

Specifying -m 5 instructs bowtie to refrain from reporting any alignments for reads having more than 5 reportable alignments. Since the read has exactly 5 reportable alignments, the -m 5 limit allows bowtie to print them as usual.

Example 9: -a -m 3 --best --strata

$ ./bowtie -a -m 3 --best --strata -v 2 e_coli --suppress 1,5,6,7 -c ATGCATCATGCGCCAT
-   gi|110640213|ref|NC_008253.1|   2852852 8:T>A

Specifying -m 3 instructs bowtie to refrain from reporting any alignments for reads having more than 3 reportable alignments. As we saw in Example 6, the read has only 1 reportable alignment when -a, --best and --strata are specified, so the -m 3 limit allows bowtie to print that alignment as usual.

Intuitively, the -m option, when combined with the --best and --strata options, guarantees a principled, though weaker form of "uniqueness." A stronger form of uniqueness is enforced when -m is specified but --best and --strata are not.

Paired-end Alignment

bowtie can align paired-end reads when properly paired read files are specified using the -1 and -2 options (for pairs of raw, FASTA, or FASTQ read files), or using the --12 option (for Tab-delimited read files). A valid paired-end alignment satisfies these criteria:

1. Both mates have a valid alignment according to the alignment policy defined by the -v/-n/-e/-l options.
2. The relative orientation and position of the mates satisfy the constraints defined by the -I/-X/--fr/--rf/--ff options.

Policies governing which paired-end alignments are reported for a given read are specified using the -k, -a and -m options as usual. The --strata and --best options do not apply in paired-end mode.

A paired-end alignment is reported as a pair of mate alignments, both on a separate line, where the alignment for each mate is formatted the same as an unpaired (singleton) alignment. The alignment for the mate that occurs closest to the beginning of the reference sequence (the "upstream" mate) is always printed before the alignment for the downstream mate. Reads files containing paired-end reads will sometimes name the reads according to whether they are the #1 or #2 mates by appending a /1 or /2 suffix to the read name. If no such suffix is present in Bowtie's input, the suffix will be added when Bowtie prints read names in alignments (except in -S "SAM" mode, where mate information is encoded in the FLAGS field instead).

Finding a valid paired-end alignment where both mates align to repetitive regions of the reference can be very time-consuming. By default, Bowtie avoids much of this cost by imposing a limit on the number of "tries" it makes to match an alignment for one mate with a nearby alignment for the other. The default limit is 100. This causes bowtie to miss some valid paired-end alignments where both mates lie in repetitive regions, but the user may use the --pairtries or -y options to increase Bowtie's sensitivity as desired.

Paired-end alignments where one mate's alignment is entirely contained within the other's are considered invalid.

When colospace alignment is enabled via -C, the default setting for paired-end orientation is --ff. This is because most SOLiD datasets have that orientation. When colorspace alignment is not enabled (default), the default setting for orientation is --fr, since most Illumina datasets have this orientation. The default can be overriden in either case.

Because Bowtie uses an in-memory representation of the original reference string when finding paired-end alignments, its memory footprint is larger when aligning paired-end reads. For example, the human index has a memory footprint of about 2.2 GB in single-end mode and 2.9 GB in paired-end mode. Note that paired-end and unpaired alignment incur the same memory footprint in colorspace (e.g. human incurs about 2.9 GB)

Colorspace Alignment

As of version 0.12.0, bowtie can align colorspace reads against a colorspace index when -C is specified. Colorspace is the characteristic output format of Applied Biosystems' SOLiD system. In a colorspace read, each character is a color rather than a nucleotide, where a color encodes a class of dinucleotides. E.g. the color blue encodes any of the dinucleotides: AA, CC, GG, TT. Colorspace has the advantage of (often) being able to distinguish sequencing errors from SNPs once the read has been aligned. See ABI's Principles of Di-Base Sequencing document for details.

Colorspace reads

All input formats (FASTA -f, FASTQ -q, raw -r, tab-delimited --12, command-line -c) are compatible with colorspace (-C). When -C is specified, read sequences are treated as colors. Colors may be encoded either as numbers (0=blue, 1=green, 2=orange, 3=red) or as characters A/C/G/T (A=blue, C=green, G=orange, T=red).

Some reads include a primer base as the first character; e.g.:

>1_53_33_F3
T2213120002010301233221223311331
>1_53_70_F3
T2302111203131231130300111123220
...

Here, T is the primer base. bowtie detects and handles primer bases properly (i.e., the primer base and the adjacent color are both trimmed away prior to alignment) as long as the rest of the read is encoded as numbers.

bowtie also handles input in the form of parallel .csfasta and _QV.qual files. Use -f to specify the .csfasta files and -Q (for unpaired reads) or --Q1/--Q2 (for paired-end reads) to specify the corresponding _QV.qual files. It is not necessary to first convert to FASTQ, though bowtie also handles FASTQ-formatted colorspace reads (with -q, the default).

Building a colorspace index

A colorspace index is built in the same way as a normal index except that -C must be specified when running bowtie-build. If the user attempts to use bowtie without -C to align against an index that was built with -C (or vice versa), bowtie prints an error message and quits.

Decoding colorspace alignments

Once a colorspace read is aligned, Bowtie decodes the alignment into nucleotides and reports the decoded nucleotide sequence. A principled decoding scheme is necessary because many different possible decodings are usually possible. Finding the true decoding with 100% certainty requires knowing all variants (e.g. SNPs) in the subject's genome beforehand, which is usually not possible. Instead, bowtie employs the approximate decoding scheme described in the BWA paper. This scheme attempts to distinguish variants from sequencing errors according to their relative likelihood under a model that considers the quality values of the colors and the (configurable) global likelihood of a SNP.

Quality values are also "decoded" so that each reported quality value is a function of the two color qualities overlapping it. Bowtie again adopts the scheme described in the BWA paper, i.e., the decoded nucleotide quality is either the sum of the overlapping color qualities (when both overlapping colors correspond to bases that match in the alignment), the quality of the matching color minus the quality of the mismatching color, or 0 (when both overlapping colors correspond to mismatches).

For accurate decoding, --snpphred/--snpfrac should be set according to the user's best guess of the SNP frequency in the subject. The --snpphred parameter sets the SNP penalty directly (on the Phred quality scale), whereas --snpfrac allows the user to specify the fraction of sites expected to be SNPs; the fraction is then converted to a Phred quality internally. For the purpose of decoding, the SNP fraction is defined in terms of SNPs per haplotype base. Thus, if the genome is diploid, heterozygous SNPs have half the weight of homozygous SNPs

Note that in -S/--sam mode, the decoded nucleotide sequence is printed for alignments, but the original color sequence (with A=blue, C=green, G=orange, T=red) is printed for unaligned reads without any reported alignments. As always, the --un, --max and --al parameters print reads exactly as they appeared in the input file.

Paired-end colorspace alignment

Like other platforms, SOLiD supports generation of paired-end reads. When colorspace alignment is enabled, the default paired-end orientation setting is --ff. This is because most SOLiD datasets have that orientation.

Note that SOLiD-generated read files can have "orphaned" mates; i.e. mates without a correpsondingly-named mate in the other file. To avoid problems due to orphaned mates, SOLiD paired-end output should first be converted to .csfastq files with unpaired mates omitted. This can be accomplished using, for example, [Galaxy]'s conversion tool (click "NGS: QC and manipulation", then "SOLiD-to-FASTQ" in the left-hand sidebar).

Wrapper scripts

The bowtie, bowtie-build and bowtie-inspect executables are actually wrapper scripts that call binary programs as appropriate. The wrappers shield users from having to distinguish between "small" and "large" index formats, discussed briefly in the following section. The appropiate index type is selected based on the input size.

It is recommended that you always run the bowtie wrappers and not run the binaries directly.

Small and large indexes

bowtie-build can index reference genomes of any size. For genomes less than about 4 billion nucleotides in length, bowtie-build builds a "small" index using 32-bit numbers in various parts of the index. When the genome is longer, bowtie-build builds a "large" index using 64-bit numbers. Small indexes are stored in files with the .ebwt extension, and large indexes are stored in files with the .ebwtl extension. The user need not worry about whether a particular index is small or large; the wrapper scripts will automatically build and use the appropriate index.

Performance Tuning

1. If your computer has multiple processors/cores, use -p

   The -p option causes Bowtie to launch a specified number of parallel search threads. Each thread runs on a different processor/core and all threads find alignments in parallel, increasing alignment throughput by approximately a multiple of the number of threads (though in practice, speedup is somewhat worse than linear).

2. If reporting many alignments per read, try tweaking bowtie-build --offrate

   If you are using the -k, -a or -m options and Bowtie is reporting many alignments per read (an average of more than about 10 per read) and you have some memory to spare, using an index with a denser SA sample can speed things up considerably.

   To do this, specify a smaller-than-default -o/--offrate value when running bowtie-build. A denser SA sample yields a larger index, but is also particularly effective at speeding up alignment when many alignments are reported per read. For example, decreasing the index's -o/--offrate by 1 could as much as double alignment performance, and decreasing by 2 could quadruple alignment performance, etc.

   On the other hand, decreasing -o/--offrate increases the size of the Bowtie index, both on disk and in memory when aligning reads. At the default -o/--offrate of 5, the SA sample for the human genome occupies about 375 MB of memory when aligning reads. Decreasing the -o/--offrate by 1 doubles the memory taken by the SA sample, and decreasing by 2 quadruples the memory taken, etc.

3. If bowtie "thrashes", try increasing bowtie --offrate

   If bowtie runs very slow on a relatively low-memory machine (having less than about 4 GB of memory), then try setting bowtie -o/--offrate to a larger value than the value used to build the index. For example, bowtie-build's default -o/--offrate is 5 and all pre-built indexes available from the Bowtie website are built with -o/--offrate 5; so if bowtie thrashes when querying such an index, try using bowtie --offrate 6. If bowtie still thrashes, try bowtie --offrate 7, etc. A higher -o/--offrate causes bowtie to use a sparser sample of the suffix array than is stored in the index; this saves memory but makes alignment reporting slower (which is especially slow when using -a or large -k or -m).

Command Line

Usage:

bowtie [options]* <ebwt> {-1 <m1> -2 <m2> | --12 <r> | <s>} [<hit>]

Main arguments

<ebwt>

<m1>

<m2>

<r>

<s>

<hit>

Options

Input

-q

-f

-r

-c

-C/--color

-Q/--quals <files>

--Q1 <files>

--Q2 <files>

-s/--skip <int>

-u/--qupto <int>

-5/--trim5 <int>

-3/--trim3 <int>

--phred33-quals

--phred64-quals

--solexa-quals

--solexa1.3-quals

--integer-quals

Alignment

-v <int>

-n/--seedmms <int>

-e/--maqerr <int>

-l/--seedlen <int>

--nomaqround

-I/--minins <int>

-X/--maxins <int>

--fr/--rf/--ff

--nofw/--norc

--maxbts

--pairtries <int>

-y/--tryhard

--chunkmbs <int>

Reporting

-k <int>

-a/--all

-m <int>

-M <int>

--best

--strata

Output

-t/--time

-B/--offbase <int>

--quiet

--refout

--refidx

--al <filename>

--un <filename>

--max <filename>

--suppress <cols>

--fullref

Colorspace

--snpphred <int>

--snpfrac <dec>

--col-cseq

--col-cqual

--col-keepends

SAM

-S/--sam

--mapq <int>

--sam-nohead

--sam-nosq

--sam-RG <text>

Performance

-o/--offrate <int>

-p/--threads <int>

--mm

--shmem

Other

--seed <int>

--verbose

--version

-h/--help

Default bowtie output

bowtie outputs one alignment per line. Each line is a collection of 8 fields separated by tabs; from left to right, the fields are:

1. Name of read that aligned.

   Note that the [SAM specification] disallows whitespace in the read name. If the read name contains any whitespace characters, Bowtie 2 will truncate the name at the first whitespace character. This is similar to the behavior of other tools.

2. Reference strand aligned to, + for forward strand, - for reverse

3. Name of reference sequence where alignment occurs, or numeric ID if no name was provided

4. 0-based offset into the forward reference strand where leftmost character of the alignment occurs

5. Read sequence (reverse-complemented if orientation is -).

   If the read was in colorspace, then the sequence shown in this column is the sequence of decoded nucleotides, not the original colors. See the Colorspace alignment section for details about decoding. To display colors instead, use the --col-cseq option.

6. ASCII-encoded read qualities (reversed if orientation is -). The encoded quality values are on the Phred scale and the encoding is ASCII-offset by 33 (ASCII char !).

   If the read was in colorspace, then the qualities shown in this column are the decoded qualities, not the original qualities. See the Colorspace alignment section for details about decoding. To display colors instead, use the --col-cqual option.

7. If -M was specified and the prescribed ceiling was exceeded for this read, this column contains the value of the ceiling, indicating that at least that many valid alignments were found in addition to the one reported.

   Otherwise, this column contains the number of other instances where the same sequence aligned against the same reference characters as were aligned against in the reported alignment. This is not the number of other places the read aligns with the same number of mismatches. The number in this column is generally not a good proxy for that number (e.g., the number in this column may be '0' while the number of other alignments with the same number of mismatches might be large).

8. Comma-separated list of mismatch descriptors. If there are no mismatches in the alignment, this field is empty. A single descriptor has the format offset:reference-base>read-base. The offset is expressed as a 0-based offset from the high-quality (5') end of the read.

SAM bowtie output

Following is a brief description of the SAM format as output by bowtie when the -S/--sam option is specified. For more details, see the SAM format specification.

When -S/--sam is specified, bowtie prints a SAM header with @HD, @SQ and @PG lines. When one or more --sam-RG arguments are specified, bowtie will also print an @RG line that includes all user-specified --sam-RG tokens separated by tabs.

Each subsequnt line corresponds to a read or an alignment. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are:

1. Name of read that aligned

2. Sum of all applicable flags. Flags relevant to Bowtie are:

   1	The read is one of a pair
   2	The alignment is one end of a proper paired-end alignment
   4	The read has no reported alignments
   8	The read is one of a pair and has no reported alignments
   16	The alignment is to the reverse reference strand
   32	The other mate in the paired-end alignment is aligned to the reverse reference strand
   64	The read is the first (#1) mate in a pair
   128	The read is the second (#2) mate in a pair

   Thus, an unpaired read that aligns to the reverse reference strand will have flag 16. A paired-end read that aligns and is the first mate in the pair will have flag 83 (= 64 + 16 + 2 + 1).

3. Name of reference sequence where alignment occurs, or ordinal ID if no name was provided

4. 1-based offset into the forward reference strand where leftmost character of the alignment occurs

5. Mapping quality

6. CIGAR string representation of alignment

7. Name of reference sequence where mate's alignment occurs. Set to = if the mate's reference sequence is the same as this alignment's, or * if there is no mate.

8. 1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate.

9. Inferred insert size. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if there is no mate.

10. Read sequence (reverse-complemented if aligned to the reverse strand)

11. ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the Phred quality scale and the encoding is ASCII-offset by 33 (ASCII char !), similarly to a FASTQ file.

12. Optional fields. Fields are tab-separated. For descriptions of all possible optional fields, see the SAM format specification. bowtie outputs some of these optional fields for each alignment, depending on the type of the alignment:

    NM:i:<N>	Aligned read has an edit distance of <N>.
    CM:i:<N>	Aligned read has an edit distance of <N> in colorspace. This field is present in addition to the NM field in -C/--color mode, but is omitted otherwise.
    MD:Z:<S>	For aligned reads, <S> is a string representation of the mismatched reference bases in the alignment. See SAM format specification for details. For colorspace alignments, <S> describes the decoded nucleotide alignment, not the colorspace alignment.
    XA:i:<N>	Aligned read belongs to stratum <N>. See Strata for definition.
    XM:i:<N>	For a read with no reported alignments, <N> is 0 if the read had no alignments. If -m was specified and the read's alignments were supressed because the -m ceiling was exceeded, <N> equals the -m ceiling + 1, to indicate that there were at least that many valid alignments (but all were suppressed). In -M mode, if the alignment was randomly selected because the -M ceiling was exceeded, <N> equals the -M ceiling + 1, to indicate that there were at least that many valid alignments (of which one was reported at random).

The bowtie-build indexer

bowtie-build builds a Bowtie index from a set of DNA sequences. bowtie-build outputs a set of 6 files with suffixes .1.ebwt, .2.ebwt, .3.ebwt, .4.ebwt, .rev.1.ebwt, and .rev.2.ebwt. (If the total length of all the input sequences is greater than about 4 billion, then the index files will end in ebwtl instead of ebwt.) These files together constitute the index: they are all that is needed to align reads to that reference. The original sequence files are no longer used by Bowtie once the index is built.

Use of Karkkainen's blockwise algorithm allows bowtie-build to trade off between running time and memory usage. bowtie-build has three options governing how it makes this trade: -p/--packed, --bmax/--bmaxdivn, and --dcv. By default, bowtie-build will automatically search for the settings that yield the best running time without exhausting memory. This behavior can be disabled using the -a/--noauto option.

The indexer provides options pertaining to the "shape" of the index, e.g. --offrate governs the fraction of Burrows-Wheeler rows that are "marked" (i.e., the density of the suffix-array sample; see the original FM Index paper for details). All of these options are potentially profitable trade-offs depending on the application. They have been set to defaults that are reasonable for most cases according to our experiments. See Performance Tuning for details.

The Bowtie index is based on the FM Index of Ferragina and Manzini, which in turn is based on the Burrows-Wheeler transform. The algorithm used to build the index is based on the blockwise algorithm of Karkkainen.

Command Line

Usage:

bowtie-build [options]* <reference_in> <ebwt_base>

Main arguments

<reference_in>

<ebwt_base>

Options

-f

-c

-C/--color

-a/--noauto

-p/--packed

--bmax <int>

--bmaxdivn <int>

--dcv <int>

--nodc

-r/--noref

-3/--justref

-o/--offrate <int>

-t/--ftabchars <int>

--ntoa

--big --little

--seed <int>

-q/--quiet

-h/--help

--version

The bowtie-inspect index inspector

bowtie-inspect extracts information from a Bowtie index about what kind of index it is and what reference sequences were used to build it. When run without any options, the tool will output a FASTA file containing the sequences of the original references (with all non-A/C/G/T characters converted to Ns). It can also be used to extract just the reference sequence names using the -n/--names option or a more verbose summary using the -s/--summary option.

Command Line

Usage:

bowtie-inspect [options]* <ebwt_base>

Main arguments

<ebwt_base>

Options

-a/--across <int>

-n/--names

-s/--summary

Colorspace  <0 or 1>
SA-Sample   1 in <sample>
FTab-Chars  <chars>
Sequence-1  <name>  <len>
Sequence-2  <name>  <len>
...
Sequence-N  <name>  <len>

-e/--ebwt-ref

-v/--verbose

--version

-h/--help