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.
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.
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 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 build Bowtie including support for the bowtie -p and
bowtie-build --threads
multithreading options, we recommend that you first install the
Thread Building Blocks library, also known as TBB, the default
threading library. TBB is installed by default on many operating
systems.
If TBB is not available, then use the NO_TBB=1 option. On Linux or
Mac OS X, this requires the pthreads library, which is installed by
default. On Windows, native Windows threads will be used, which require
no special libraries.
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 over assign
alignments to the sites on the strand with fewer sites and under assign
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.
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.
-
Alignments may have no more than
Nmismatches (whereNis a number 0-3, set with-n) in the firstLbases (whereLis a number 5 or greater, set with-l) on the high-quality (left) end of the read. The firstLbases are called the "seed". -
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.
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.
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.
With the -k, -a, -m, -M, --best and --strata options, the
user can flexibly 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.
$ ./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.
$ ./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.
$ ./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.
$ ./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.
$ ./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 --best 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.
$ ./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.
$ ./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.
$ ./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.
$ ./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.
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), the --12 option (for Tab-delimited read
files), or using the --interleaved (for interleaved FASTQ).
A valid paired-end alignment satisfies these criteria:
- Both mates have a valid alignment according to the alignment policy
defined by the
-v/-n/-e/-loptions. - The relative orientation and position of the mates satisfy the
constraints defined by the
-I/-X/--fr/--rf/--ffoptions.
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)
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.
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).
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.
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.
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).
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.
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.
-
If your computer has multiple processors/cores, use
--threads--threadsoption causes Bowtie to launch a specified number of parallel threads. Each thread runs on a different processor/core. For alignment, this increases alignment throughput by approximately a multiple of the number of threads (though in practice, it is somewhat worse than linear). For index building, using multiple threads decreases building time. -
If reporting many alignments per read, try tweaking
bowtie-build --offrateIf you are using the
-k,-aor-moptions 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/--offratevalue when runningbowtie-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/--offrateby 1 could as much as double alignment performance, and decreasing by 2 could quadruple alignment performance, etc.On the other hand, decreasing
-o/--offrateincreases the size of the Bowtie index, both on disk and in memory when aligning reads. At the default-o/--offrateof 5, the SA sample for the human genome occupies about 375 MB of memory when aligning reads. Decreasing the-o/--offrateby 1 doubles the memory taken by the SA sample, and decreasing by 2 quadruples the memory taken, etc. -
If bowtie "thrashes", try increasing
bowtie --offrateIf
bowtieruns very slow on a low-memory machine (with less than about 4 GB of memory), then try settingbowtie-o/--offrateto a larger value.bowtie-build's default-o/--offrateis 5 and all pre-built indexes available from the Bowtie website are built with-o/--offrate5; so ifbowtiethrashes when querying such an index, try usingbowtie--offrate6. Ifbowtiestill thrashes, trybowtie--offrate7, etc. A higher-o/--offratecausesbowtieto 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-aor large-kor-m).
Usage:
bowtie [options]* <ebwt> {-1 <m1> -2 <m2> | --12 <r> | --interleaved <i> | <s>} [<hit>]
|
The basename of the index to be searched. The basename is the name of
any of the index files up to but not including the final |
|
Comma-separated list of files containing the #1 mates (filename usually
includes |
|
Comma-separated list of files containing the #2 mates (filename usually
includes |
|
Comma-separated list of files containing a mix of unpaired and
paired-end reads in Tab-delimited format. Tab-delimited format is a
1-read-per-line format where unpaired reads consist of a read name,
sequence and quality string each separated by tabs. A paired-end read
consists of a read name, sequnce of the #1 mate, quality values of the
#1 mate, sequence of the #2 mate, and quality values of the #2 mate
separated by tabs. Quality values can be expressed using any of the
scales supported in FASTQ files. Reads may be a mix of different
lengths and paired-end and unpaired reads may be intermingled in the
same file. If |
|
A comma-separated list of interleaved paired-end FASTQ files, where
the records for the mate #1s are interleaved with the records for the
mate #2s. Reads may be a mix of different lengths. If |
|
A comma-separated list of files containing unpaired reads to be
aligned, or, if |
|
File to write alignments to. By default, alignments are written to the "standard out" filehandle (i.e. the console). |
|
The query input files (specified either as |
|
The query input files (specified either as |
|
The query input files (specified either as |
|
The query sequences are given on command line. I.e. |
|
Align in colorspace. Read characters are interpreted as colors. The
index specified must be a colorspace index (i.e. built with
|
|
Comma-separated list of files containing quality values for
corresponding unpaired CSFASTA reads. Use in combination with |
|
Comma-separated list of files containing quality values for
corresponding CSFASTA #1 mates. Use in combination with |
|
Comma-separated list of files containing quality values for
corresponding CSFASTA #2 mates. Use in combination with |
|
Skip (i.e. do not align) the first |
|
Only align the first |
|
Trim |
|
Trim |
|
Input qualities are ASCII chars equal to the Phred quality plus 33. Default: on. |
|
Input qualities are ASCII chars equal to the Phred quality plus 64. Default: off. |
|
Convert input qualities from Solexa (which can be negative) to Phred (which can't). This is usually the right option for use with (unconverted) reads emitted by GA Pipeline versions prior to 1.3. Default: off. |
|
Same as |
|
Quality values are represented in the read input file as
space-separated ASCII integers, e.g., |
|
Force usage of a 'large' index (those ending in '.ebwtl'), even if a small one is present. Default: off. |
|
Report alignments with at most |
|
Maximum number of mismatches permitted in the "seed", i.e. the first
|
|
Maximum permitted total of quality values at all mismatched read
positions throughout the entire alignment, not just in the "seed". The
default is 70. Like Maq, |
|
The "seed length"; i.e., the number of bases on the high-quality end of
the read to which the |
|
Maq accepts quality values in the Phred quality scale, but
internally rounds values to the nearest 10, with a maximum of 30. By
default, |
|
The minimum insert size for valid paired-end alignments. E.g. if |
|
The maximum insert size for valid paired-end alignments. E.g. if |
|
The upstream/downstream mate orientations for a valid paired-end
alignment against the forward reference strand. E.g., if |
|
Normally, Bowtie will not reported a paired-end alignment for a pair when the two ends overlap exactly the same reference interval, or if the alignment interval for one is contained within the other. This option causes Bowtie to report such cases as normal paired-end alignments. |
|
If |
|
The maximum number of backtracks permitted when aligning a read in
|
|
For paired-end alignment, this is the maximum number of attempts
|
|
Try as hard as possible to find valid alignments when they exist,
including paired-end alignments. This is equivalent to specifying very
high values for the |
|
The number of megabytes of memory a given thread is given to store path
descriptors in |
|
Part of bowtie's batch parsing and used to specify the number of reads that bowtie will consume from the input file at once. Default: 16 |
|
Report up to |
|
Report all valid alignments per read or pair (default: off). Validity
of alignments is determined by the alignment policy (combined effects
of |
|
Suppress all alignments for a particular read or pair if more than
|
|
Behaves like |
|
Make Bowtie guarantee that reported singleton alignments are "best" in
terms of stratum (i.e. number of mismatches, or mismatches in the seed
in the case of |
|
If many valid alignments exist and are reportable (e.g. are not
disallowed via the |
|
Print the amount of wall-clock time taken by each phase. |
|
When outputting alignments in Bowtie format, consider the first base of
a reference sequence to have offset |
|
Print nothing besides alignments. |
|
When a reference sequence is referred to in a reported alignment, refer to it by 0-based index (its offset into the list of references that were indexed) rather than by name. |
|
Write all reads for which at least one alignment was reported to a file
with name |
|
Write all reads that could not be aligned to a file with name
|
|
Write all reads with a number of valid alignments exceeding the limit
set with the |
|
Suppress columns of output in the default output mode. E.g. if
|
|
Print the full refernce sequence name, including whitespace, in
alignment output. By default |
|
When decoding colorspace alignments, use |
|
When decoding colorspace alignments, use |
|
If reads are in colorspace and the default output mode is active,
|
|
If reads are in colorspace and the default output mode is active,
|
|
When decoding colorpsace alignments, |
|
Print alignments in SAM format. See the SAM output section of the
manual for details. To suppress all SAM headers, use |
|
If an alignment is non-repetitive (according to |
|
Suppress header lines (starting with |
|
Suppress |
|
Add |
|
Suppress SAM records for reads that failed to align. |
|
Override the offrate of the index with |
|
Launch |
|
Use memory-mapped I/O to load the index, rather than normal C file I/O.
Memory-mapping the index allows many concurrent |
|
Use shared memory to load the index, rather than normal C file I/O.
Using shared memory allows many concurrent bowtie processes on the same
computer to share the same memory image of the index (i.e. you pay the
memory overhead just once). This facilitates memory-efficient
parallelization of |
|
Use |
|
Print verbose output (for debugging). |
|
Print version information and quit. |
|
Print usage information and quit. |
bowtie outputs one alignment per line. Each line is a collection of
8 fields separated by tabs; from left to right, the fields are:
-
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.
-
Reference strand aligned to,
+for forward strand,-for reverse -
Name of reference sequence where alignment occurs, or numeric ID if no name was provided
-
0-based offset into the forward reference strand where leftmost character of the alignment occurs
-
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-cseqoption. -
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-cqualoption. -
If
-Mwas 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).
-
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.
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:
-
Name of read that aligned
-
Sum of all applicable flags. Flags relevant to Bowtie are:
1The read is one of a pair
2The alignment is one end of a proper paired-end alignment
4The read has no reported alignments
8The read is one of a pair and has no reported alignments
16The alignment is to the reverse reference strand
32The other mate in the paired-end alignment is aligned to the reverse reference strand
64The read is the first (#1) mate in a pair
128The 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).
-
Name of reference sequence where alignment occurs, or ordinal ID if no name was provided
-
1-based offset into the forward reference strand where leftmost character of the alignment occurs
-
Mapping quality
-
CIGAR string representation of alignment
-
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. -
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.
-
Inferred insert size. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if there is no mate.
-
Read sequence (reverse-complemented if aligned to the reverse strand)
-
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. -
Optional fields. Fields are tab-separated. For descriptions of all possible optional fields, see the SAM format specification.
bowtieoutputs some of these optional fields for each alignment, depending on the type of the alignment:
|
Aligned read has an edit distance of |
|
Aligned read has an edit distance of |
|
For aligned reads, |
|
Aligned read belongs to stratum |
|
For a read with no reported alignments, |
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.
Usage:
bowtie-build [options]* <reference_in> <ebwt_base>
|
A comma-separated list of FASTA files containing the reference
sequences to be aligned to, or, if |
|
The basename of the index files to write. By default, |
|
The reference input files (specified as |
|
The reference sequences are given on the command line. I.e.
|
|
Build a colorspace index, to be queried using |
|
Disable the default behavior whereby |
|
Use a packed (2-bits-per-nucleotide) representation for DNA strings.
This saves memory but makes indexing 2-3 times slower. Default: off.
This is configured automatically by default; use |
|
The maximum number of suffixes allowed in a block. Allowing more
suffixes per block makes indexing faster, but increases peak memory
usage. Setting this option overrides any previous setting for
|
|
The maximum number of suffixes allowed in a block, expressed as a
fraction of the length of the reference. Setting this option overrides
any previous setting for |
|
Use |
|
Disable use of the difference-cover sample. Suffix sorting becomes quadratic-time in the worst case (where the worst case is an extremely repetitive reference). Default: off. |
|
Do not build the |
|
Build only the |
|
To map alignments back to positions on the reference sequences, it's
necessary to annotate ("mark") some or all of the Burrows-Wheeler
rows with their corresponding location on the genome. |
|
The ftab is the lookup table used to calculate an initial
Burrows-Wheeler range with respect to the first |
|
Launch |
|
Convert Ns in the reference sequence to As before building the index.
By default, Ns are simply excluded from the index and |
|
Endianness to use when serializing integers to the index file. Default: little-endian (recommended for Intel- and AMD-based architectures). |
|
Use |
|
|
|
Print usage information and quit. |
|
Print version information and quit. |
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.
Usage:
bowtie-inspect [options]* <ebwt_base>
|
The basename of the index to be inspected. The basename is name of any
of the index files but with the |
|
When printing FASTA output, output a newline character every |
|
Print reference sequence names, one per line, and quit. |
|
Print a summary that includes information about index settings, as well as the names and lengths of the input sequences. The summary has this format: Fields are separated by tabs. |
|
By default, when |
|
Print verbose output (for debugging). |
|
Print version information and quit. |
|
Print usage information and quit. |