Imported Upstream version 0.12.7

[bowtie.git] / MANUAL

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].

[Bowtie]:          http://bowtie-bio.sf.net
[Burrows-Wheeler]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform
[SAM]:             http://samtools.sourceforge.net/SAM1.pdf
[SAMtools]:        http://samtools.sourceforge.net/
[TopHat]:          http://tophat.cbcb.umd.edu/
[Cufflinks]:       http://cufflinks.cbcb.umd.edu/
[Crossbow]:        http://bowtie-bio.sf.net/crossbow
[Myrna]:           http://bowtie-bio.sf.net/myrna
[Bowtie paper]:    http://genomebiology.com/2009/10/3/R25

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.

[MUMmer]: http://mummer.sourceforge.net/
[BLAST]:  http://blast.ncbi.nlm.nih.gov/Blast.cgi
[Vmatch]: http://www.vmatch.de/

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 Intel architectures (`i386` and `x86_64`)
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 [Cygwin] or [MinGW].  We recommend
[TDM's MinGW Build].  If using [MinGW], you 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.  To compile Bowtie without `pthreads` (which
disables `-p`), use `make BOWTIE_PTHREADS=0`.

[Cygwin]:   http://www.cygwin.com/
[MinGW]:    http://www.mingw.org/
[TDM's MinGW Build]: http://www.tdragon.net/recentgcc/
[MSYS]:     http://www.mingw.org/wiki/msys
[Download]: https://sourceforge.net/projects/bowtie-bio/files/bowtie/

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, but support is planned
for a future release.

[Maq]: http://maq.sf.net

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.

[Maq]: http://maq.sf.net
[Phred quality]: http://en.wikipedia.org/wiki/FASTQ_format#Variations

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` `--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.

  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).

[Principles of Di-Base Sequencing]: http://tinyurl.com/ygnb2gn
[BWA paper]: http://bioinformatics.oxfordjournals.org/cgi/content/abstract/25/14/1754

Performance Tuning
------------------

1.  Use 64-bit bowtie if possible

    The 64-bit version of Bowtie is substantially (usually more then
    50%) faster than the 32-bit version, owing to its use of 64-bit
    arithmetic.  If possible, download the 64-bit binaries for Bowtie
    and run on a 64-bit computer.  If you are building Bowtie from
    sources, you may need to pass the `-m64` option to `g++` to compile
    the 64-bit version; you can do this by including `BITS=64` in the
    arguments to the `make` command; e.g.: `make BITS=64 bowtie`.  To
    determine whether your version of bowtie is 64-bit or 32-bit, run
    `bowtie --version`.

2.  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).

3.  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.

4.  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>

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 `.1.ebwt` /
`.rev.1.ebwt` / etc.  `bowtie` looks for the specified index first in
the current directory, then in the `indexes` subdirectory under the
directory where the `bowtie` executable is located, then looks in the
directory specified in the `BOWTIE_INDEXES` environment variable.

    <m1>

Comma-separated list of files containing the #1 mates (filename usually
includes `_1`), or, if `-c` is specified, the mate sequences
themselves.  E.g., this might be `flyA_1.fq,flyB_1.fq`, or, if `-c`
is specified, this might be `GGTCATCCT,ACGGGTCGT`.  Sequences specified
with this option must correspond file-for-file and read-for-read with
those specified in `<m2>`.  Reads may be a mix of different lengths.
If `-` is specified, `bowtie` will read the #1 mates from the "standard
in" filehandle.

    <m2>

Comma-separated list of files containing the #2 mates (filename usually
includes `_2`), or, if `-c` is specified, the mate sequences
themselves.  E.g., this might be `flyA_2.fq,flyB_2.fq`, or, if `-c`
is specified, this might be `GGTCATCCT,ACGGGTCGT`.  Sequences specified
with this option must correspond file-for-file and read-for-read with
those specified in `<m1>`.  Reads may be a mix of different lengths.
If `-` is specified, `bowtie` will read the #2 mates from the "standard
in" filehandle.

    <r>

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 `-` is specified, `bowtie` will read the Tab-delimited
reads from the "standard in" filehandle.

    <s>

A comma-separated list of files containing unpaired reads to be
aligned, or, if `-c` is specified, the unpaired read sequences
themselves.  E.g., this might be
`lane1.fq,lane2.fq,lane3.fq,lane4.fq`, or, if `-c` is specified, this
might be `GGTCATCCT,ACGGGTCGT`.  Reads may be a mix of different
lengths.  If `-` is specified, Bowtie gets the reads from the "standard
in" filehandle.

    <hit>

File to write alignments to.  By default, alignments are written to the
"standard out" filehandle (i.e. the console).

  Options

    Input

    -q

The query input files (specified either as `<m1>` and `<m2>`, or as
`<s>`) are FASTQ files (usually having extension `.fq` or `.fastq`).
This is the default.  See also: `--solexa-quals` and
`--integer-quals`.

    -f

The query input files (specified either as `<m1>` and `<m2>`, or as
`<s>`) are FASTA files (usually having extension `.fa`, `.mfa`, `.fna`
or similar).  All quality values are assumed to be 40 on the [Phred
quality] scale.

    -r

The query input files (specified either as `<m1>` and `<m2>`, or as
`<s>`) are Raw files: one sequence per line, without quality values or
names.  All quality values are assumed to be 40 on the [Phred quality]
scale.

    -c

The query sequences are given on command line.  I.e. `<m1>`, `<m2>` and
`<singles>` are comma-separated lists of reads rather than lists of
read files.

    -C/--color

Align in colorspace.  Read characters are interpreted as colors.  The
index specified must be a colorspace index (i.e. built with
`bowtie-build` `-C`, or `bowtie` will print an error message and quit.
See [Colorspace alignment] for more details.

    -Q/--quals <files>

Comma-separated list of files containing quality values for
corresponding unpaired CSFASTA reads.  Use in combination with `-C`
and `-f`.  `--integer-quals` is set automatically when `-Q`/`--quals`
is specified.

    --Q1 <files>

Comma-separated list of files containing quality values for
corresponding CSFASTA #1 mates.  Use in combination with `-C`, `-f`,
and `-1`.  `--integer-quals` is set automatically when `--Q1`
is specified.

    --Q2 <files>

Comma-separated list of files containing quality values for
corresponding CSFASTA #2 mates.  Use in combination with `-C`, `-f`,
and `-2`.  `--integer-quals` is set automatically when `--Q2`
is specified.

    -s/--skip <int>

Skip (i.e. do not align) the first `<int>` reads or pairs in the input.

    -u/--qupto <int>

Only align the first `<int>` reads or read pairs from the input (after
the `-s`/`--skip` reads or pairs have been skipped).  Default: no
limit.

    -5/--trim5 <int>

Trim `<int>` bases from high-quality (left) end of each read before
alignment (default: 0).

    -3/--trim3 <int>

Trim `<int>` bases from low-quality (right) end of each read before
alignment (default: 0).

    --phred33-quals

Input qualities are ASCII chars equal to the [Phred quality] plus 33.
Default: on.

    --phred64-quals

Input qualities are ASCII chars equal to the [Phred quality] plus 64.
Default: off.

    --solexa-quals

Convert input qualities from [Solexa][Phred quality] (which can be
negative) to [Phred][Phred quality] (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.

    --solexa1.3-quals

Same as `--phred64-quals`.  This is usually the right option for use
with (unconverted) reads emitted by GA Pipeline version 1.3 or later. 
Default: off.

    --integer-quals

Quality values are represented in the read input file as
space-separated ASCII integers, e.g., `40 40 30 40`..., rather than
ASCII characters, e.g., `II?I`....  Integers are treated as being on
the [Phred quality] scale unless `--solexa-quals` is also specified.
Default: off.

    Alignment

    -v <int>

Report alignments with at most `<int>` mismatches.  `-e` and `-l`
options are ignored and quality values have no effect on what
alignments are valid.  `-v` is mutually exclusive with `-n`.

    -n/--seedmms <int>

Maximum number of mismatches permitted in the "seed", i.e. the first
`L` base pairs of the read (where `L` is set with `-l`/`--seedlen`).
This may be 0, 1, 2 or 3 and the default is 2.  This option is mutually
exclusive with the `-v` option.

    -e/--maqerr <int>

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], `bowtie` rounds quality values to the
nearest 10 and saturates at 30; rounding can be disabled with
`--nomaqround`.

    -l/--seedlen <int>

The "seed length"; i.e., the number of bases on the high-quality end of
the read to which the `-n` ceiling applies.  The lowest permitted
setting is 5 and the default is 28.  `bowtie` is faster for larger
values of `-l`.

    --nomaqround

[Maq] accepts quality values in the [Phred quality] scale, but
internally rounds values to the nearest 10, with a maximum of 30.  By
default, `bowtie` also rounds this way.  `--nomaqround` prevents this
rounding in `bowtie`.

    -I/--minins <int>

The minimum insert size for valid paired-end alignments.  E.g. if `-I
60` is specified and a paired-end alignment consists of two 20-bp
alignments in the appropriate orientation with a 20-bp gap between
them, that alignment is considered valid (as long as `-X` is also
satisfied).  A 19-bp gap would not be valid in that case.  If trimming
options `-3` or `-5` are also used, the `-I` constraint is
applied with respect to the untrimmed mates.  Default: 0.

    -X/--maxins <int>

The maximum insert size for valid paired-end alignments.  E.g. if `-X
100` is specified and a paired-end alignment consists of two 20-bp
alignments in the proper orientation with a 60-bp gap between them,
that alignment is considered valid (as long as `-I` is also
satisfied).  A 61-bp gap would not be valid in that case.  If trimming
options `-3` or `-5` are also used, the `-X` constraint is applied
with respect to the untrimmed mates, not the trimmed mates.  Default:
250.

    --fr/--rf/--ff

The upstream/downstream mate orientations for a valid paired-end
alignment against the forward reference strand.  E.g., if `--fr` is
specified and there is a candidate paired-end alignment where mate1
appears upstream of the reverse complement of mate2 and the insert
length constraints are met, that alignment is valid.  Also, if mate2
appears upstream of the reverse complement of mate1 and all other
constraints are met, that too is valid.  `--rf` likewise requires that
an upstream mate1 be reverse-complemented and a downstream mate2 be
forward-oriented. ` --ff` requires both an upstream mate1 and a
downstream mate2 to be forward-oriented.  Default: `--fr` when `-C`
(colorspace alignment) is not specified, `--ff` when `-C` is specified.

    --nofw/--norc

If `--nofw` is specified, `bowtie` will not attempt to align against
the forward reference strand.  If `--norc` is specified, `bowtie` will
not attempt to align against the reverse-complement reference strand.
For paired-end reads using `--fr` or `--rf` modes, `--nofw` and
`--norc` apply to the forward and reverse-complement pair orientations.
I.e. specifying `--nofw` and `--fr` will only find reads in the R/F
orientation where mate 2 occurs upstream of mate 1 with respect to the
forward reference strand.

    --maxbts

The maximum number of backtracks permitted when aligning a read in
`-n` 2 or `-n` 3 mode (default: 125 without `--best`, 800 with
`--best`).  A "backtrack" is the introduction of a speculative
substitution into the alignment.  Without this limit, the default
parameters will sometimes require that `bowtie` try 100s or 1,000s of
backtracks to align a read, especially if the read has many low-quality
bases and/or has no valid alignments, slowing bowtie down
significantly.  However, this limit may cause some valid alignments to
be missed.  Higher limits yield greater sensitivity at the expensive of
longer running times.  See also: `-y`/`--tryhard`.

    --pairtries <int>

For paired-end alignment, this is the maximum number of attempts
`bowtie` will make to match an alignment for one mate up with an
alignment for the opposite mate.  Most paired-end alignments require
only a few such attempts, but pairs where both mates occur in highly
repetitive regions of the reference can require significantly more.
Setting this to a higher number allows `bowtie` to find more paired-
end alignments for repetitive pairs at the expense of speed.  The
default is 100.  See also: `-y`/`--tryhard`.

    -y/--tryhard

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 `--maxbts` and `--pairtries` options.  This
mode is generally much slower than the default settings, but can be
useful for certain problems.  This mode is slower when (a) the
reference is very repetitive, (b) the reads are low quality, or (c) not
many reads have valid alignments.

    --chunkmbs <int>

The number of megabytes of memory a given thread is given to store path
descriptors in `--best` mode.  Best-first search must keep track of
many paths at once to ensure it is always extending the path with the
lowest cumulative cost.  Bowtie tries to minimize the memory impact of
the descriptors, but they can still grow very large in some cases.  If
you receive an error message saying that chunk memory has been
exhausted in `--best` mode, try adjusting this parameter up to
dedicate more memory to the descriptors.  Default: 64.

    Reporting

    -k <int>

Report up to `<int>` valid alignments per read or pair (default: 1).
Validity of alignments is determined by the alignment policy (combined
effects of `-n`, `-v`, `-l`, and `-e`).  If more than one valid
alignment exists and the `--best` and `--strata` options are
specified, then only those alignments belonging to the best alignment
"stratum" will be reported.  Bowtie is designed to be very fast for
small `-k` but bowtie can become significantly slower as `-k`
increases.  If you would like to use Bowtie for larger values of
`-k`, consider building an index with a denser suffix-array sample,
i.e. specify a smaller `-o`/`--offrate` when invoking `bowtie-build`
for the relevant index (see the [Performance tuning] section for
details).

    -a/--all

Report all valid alignments per read or pair (default: off).  Validity
of alignments is determined by the alignment policy (combined effects
of `-n`, `-v`, `-l`, and `-e`).  If more than one valid alignment
exists and the `--best` and `--strata` options are specified, then only
those alignments belonging to the best alignment "stratum" will be
reported.  Bowtie is designed to be very fast for small `-k` but bowtie
can become significantly slower if `-a`/`--all` is specified.  If you
would like to use Bowtie with `-a`, consider building an index with a
denser suffix-array sample, i.e. specify a smaller `-o`/`--offrate`
when invoking `bowtie-build` for the relevant index (see the
[Performance tuning] section for details).

    -m <int>

Suppress all alignments for a particular read or pair if more than
`<int>` reportable alignments exist for it.  Reportable alignments are
those that would be reported given the `-n`, `-v`, `-l`, `-e`, `-k`,
`-a`, `--best`, and `--strata` options.  Default: no limit.  Bowtie is
designed to be very fast for small `-m` but bowtie can become
significantly slower for larger values of `-m`.  If you would like to
use Bowtie for larger values of `-k`, consider building an index with a
denser suffix-array sample, i.e. specify a smaller `-o`/`--offrate` when
invoking `bowtie-build` for the relevant index (see the [Performance
tuning] section for details).

    -M <int>

Behaves like `-m` except that if a read has more than `<int>`
reportable alignments, one is reported at random.  In [default
output mode], the selected alignment's 7th column is set to `<int>`+1 to
indicate the read has at least `<int>`+1 valid alignments.  In
`-S`/`--sam` mode, the selected alignment is given a `MAPQ` (mapping
quality) of 0 and the `XM:I` field is set to `<int>`+1.  This option
requires `--best`; if specified without `--best`, `--best` is enabled
automatically.

    --best

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 `-n` mode) and in terms of the quality values at the
mismatched position(s).  Stratum always trumps quality; e.g. a
1-mismatch alignment where the mismatched position has [Phred quality]
40 is preferred over a 2-mismatch alignment where the mismatched
positions both have [Phred quality] 10.  When `--best` is not
specified, Bowtie may report alignments that are sub-optimal in terms
of stratum and/or quality (though an effort is made to report the best
alignment).  `--best` mode also removes all strand bias.  Note that
`--best` does not affect which alignments are considered "valid" by
`bowtie`, only which valid alignments are reported by `bowtie`.  When
`--best` is specified and multiple hits are allowed (via `-k` or
`-a`), the alignments for a given read are guaranteed to appear in
best-to-worst order in `bowtie`'s output.  `bowtie` is somewhat slower
when `--best` is specified.

    --strata

If many valid alignments exist and are reportable (e.g. are not
disallowed via the `-k` option) and they fall into more than one
alignment "stratum", report only those alignments that fall into the
best stratum.  By default, Bowtie reports all reportable alignments
regardless of whether they fall into multiple strata.  When
`--strata` is specified, `--best` must also be specified. 

    Output

    -t/--time

Print the amount of wall-clock time taken by each phase.

    -B/--offbase <int>

When outputting alignments, number the first base of a reference
sequence as `<int>`.  Default: 0.

    --quiet

Print nothing besides alignments.

    --refout

Write alignments to a set of files named `refXXXXX.map`, where `XXXXX`
is the 0-padded index of the reference sequence aligned to.  This can
be a useful way to break up work for downstream analyses when dealing
with, for example, large numbers of reads aligned to the assembled
human genome.  If `<hits>` is also specified, it will be ignored.

    --refidx

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.

    --al <filename>

Write all reads for which at least one alignment was reported to a file
with name `<filename>`.  Written reads will appear as they did in the
input, without any of the trimming or translation of quality values
that may have taken place within `bowtie`.  Paired-end reads will be
written to two parallel files with `_1` and `_2` inserted in the
filename, e.g., if `<filename>` is `aligned.fq`, the #1 and #2 mates
that fail to align will be written to `aligned_1.fq` and `aligned_2.fq`
respectively.

    --un <filename>

Write all reads that could not be aligned to a file with name
`<filename>`.  Written reads will appear as they did in the input,
without any of the trimming or translation of quality values that may
have taken place within Bowtie.  Paired-end reads will be written to
two parallel files with `_1` and `_2` inserted in the filename, e.g.,
if `<filename>` is `unaligned.fq`, the #1 and #2 mates that fail to
align will be written to `unaligned_1.fq` and `unaligned_2.fq`
respectively.  Unless `--max` is also specified, reads with a number
of valid alignments exceeding the limit set with the `-m` option are
also written to `<filename>`.

    --max <filename>

Write all reads with a number of valid alignments exceeding the limit
set with the `-m` option to a file with name `<filename>`.  Written
reads will appear as they did in the input, without any of the trimming
or translation of quality values that may have taken place within
`bowtie`.  Paired-end reads will be written to two parallel files with
`_1` and `_2` inserted in the filename, e.g., if `<filename>` is
`max.fq`, the #1 and #2 mates that exceed the `-m` limit will be
written to `max_1.fq` and `max_2.fq` respectively.  These reads are not
written to the file specified with `--un`.

    --suppress <cols>

Suppress columns of output in the [default output mode].  E.g. if
`--suppress 1,5,6` is specified, the read name, read sequence, and read
quality fields will be omitted.  See [Default Bowtie output] for field
descriptions.  This option is ignored if the output mode is
`-S`/`--sam`.

    --fullref

Print the full refernce sequence name, including whitespace, in
alignment output.  By default `bowtie` prints everything up to but not
including the first whitespace.

    Colorspace

    --snpphred <int>

When decoding colorspace alignments, use `<int>` as the SNP penalty.
This should be set to the user's best guess of the true ratio of SNPs
per base in the subject genome, converted to the [Phred quality] scale.
E.g., if the user expects about 1 SNP every 1,000 positions,
`--snpphred` should be set to 30 (which is also the default).  To
specify the fraction directly, use `--snpfrac`.

    --snpfrac <dec>

When decoding colorspace alignments, use `<dec>` as the estimated ratio
of SNPs per base.  For best decoding results, this should be set to the
user's best guess of the true ratio.  `bowtie` internally converts the
ratio to a [Phred quality], and behaves as if that quality had been set
via the `--snpphred` option.  Default: 0.001.

    --col-cseq

If reads are in colorspace and the [default output mode] is active,
`--col-cseq` causes the reads' color sequence to appear in the
read-sequence column (column 5) instead of the decoded nucleotide
sequence.  See the [Decoding colorspace alignments] section for details
about decoding.  This option is ignored in `-S`/`--sam` mode.

    --col-cqual

If reads are in colorspace and the [default output mode] is active,
`--col-cqual` causes the reads' original (color) quality sequence to
appear in the quality column (column 6) instead of the decoded
qualities.  See the [Colorspace alignment] section for details about
decoding.  This option is ignored in `-S`/`--sam` mode.

    --col-keepends

When decoding colorpsace alignments, `bowtie` trims off a nucleotide
and quality from the left and right edges of the alignment.  This is
because those nucleotides are supported by only one color, in contrast
to the middle nucleotides which are supported by two.  Specify
`--col-keepends` to keep the extreme-end nucleotides and qualities.

    SAM

    -S/--sam

Print alignments in [SAM] format.  See the [SAM output] section of the
manual for details.  To suppress all SAM headers, use `--sam-nohead`
in addition to `-S/--sam`.  To suppress just the `@SQ` headers (e.g. if
the alignment is against a very large number of reference sequences),
use `--sam-nosq` in addition to `-S/--sam`.  `bowtie` does not write
BAM files directly, but SAM output can be converted to BAM on the fly
by piping `bowtie`'s output to `samtools view`.  `-S`/`--sam` is not
compatible with `--refout`.

    --mapq <int>

If an alignment is non-repetitive (according to `-m`, `--strata` and
other options) set the `MAPQ` (mapping quality) field to this value.
See the [SAM Spec][SAM] for details about the `MAPQ` field  Default: 255.

    --sam-nohead

Suppress header lines (starting with `@`) when output is `-S`/`--sam`.
This must be specified *in addition to* `-S`/`--sam`.  `--sam-nohead`
is ignored unless `-S`/`--sam` is also specified.

    --sam-nosq

Suppress `@SQ` header lines when output is `-S`/`--sam`.  This must be
specified *in addition to* `-S`/`--sam`.  `--sam-nosq` is ignored
unless `-S`/`--sam` is also specified.

    --sam-RG <text>

Add `<text>` (usually of the form `TAG:VAL`, e.g. `ID:IL7LANE2`) as a
field on the `@RG` header line.  Specify `--sam-RG` multiple times to
set multiple fields.  See the [SAM Spec][SAM] for details about what fields
are legal.  Note that, if any `@RG` fields are set using this option,
the `ID` and `SM` fields must both be among them to make the `@RG` line
legal according to the [SAM Spec][SAM].  `--sam-RG` is ignored unless
`-S`/`--sam` is also specified.

    Performance

    -o/--offrate <int>

Override the offrate of the index with `<int>`.  If `<int>` is greater
than the offrate used to build the index, then some row markings are
discarded when the index is read into memory.  This reduces the memory
footprint of the aligner but requires more time to calculate text
offsets.  `<int>` must be greater than the value used to build the
index.

    -p/--threads <int>

Launch `<int>` parallel search threads (default: 1).  Threads will run
on separate processors/cores and synchronize when parsing reads and
outputting alignments.  Searching for alignments is highly parallel,
and speedup is fairly close to linear.  This option is only available
if `bowtie` is linked with the `pthreads` library (i.e. if
`BOWTIE_PTHREADS=0` is not specified at build time).

    --mm

Use memory-mapped I/O to load the index, rather than normal C file I/O.
Memory-mapping the index 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 `bowtie` in situations where using `-p` is not
possible.

    --shmem

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 `bowtie` in situations where using `-p` is not
desirable.  Unlike `--mm`, `--shmem` installs the index into shared
memory permanently, or until the user deletes the shared memory chunks
manually.  See your operating system documentation for details on how
to manually list and remove shared memory chunks (on Linux and Mac OS
X, these commands are `ipcs` and `ipcrm`).  You may also need to
increase your OS's maximum shared-memory chunk size to accomodate
larger indexes; see your OS documentation.

    Other

    --seed <int>

Use `<int>` as the seed for pseudo-random number generator.

    --verbose

Print verbose output (for debugging).

    --version

Print version information and quit.

    -h/--help

Print usage information and quit.

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

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][SAM].

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  mate in a pair

        128

    The read is the second  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).

[SAM format specification]: http://samtools.sf.net/SAM1.pdf
[FASTQ]: http://en.wikipedia.org/wiki/FASTQ_format

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`.  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.

Because `bowtie-build` uses 32-bit pointers internally, it can handle
up to a theoretical maximum of 2^32-1 (somewhat more than 4 billion)
characters in an index, though, with other constraints, the actual
ceiling is somewhat less than that.  If your reference exceeds 2^32-1
characters, `bowtie-build` will print an error message and abort.  To
resolve this, divide your reference sequences into smaller batches
and/or chunks and build a separate index for each.

If your computer has more than 3-4 GB of memory and you would like to
exploit that fact to make index building faster, use a 64-bit version
of the `bowtie-build` binary.  The 32-bit version of the binary is
restricted to using less than 4 GB of memory.  If a 64-bit pre-built
binary does not yet exist for your platform on the sourceforge download
site, you will need to build one from source.

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.

[Blockwise algorithm]: http://portal.acm.org/citation.cfm?id=1314852
[FM Index]: http://portal.acm.org/citation.cfm?id=796543
[Burrows-Wheeler]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform

Command Line
------------

Usage:

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

  Main arguments

    <reference_in>

A comma-separated list of FASTA files containing the reference
sequences to be aligned to, or, if `-c` is specified, the sequences
themselves. E.g., `<reference_in>` might be
`chr1.fa,chr2.fa,chrX.fa,chrY.fa`, or, if `-c` is specified, this might
be `GGTCATCCT,ACGGGTCGT,CCGTTCTATGCGGCTTA`.

    <ebwt_base>

The basename of the index files to write.  By default, `bowtie-build`
writes files named `NAME.1.ebwt`, `NAME.2.ebwt`, `NAME.3.ebwt`,
`NAME.4.ebwt`, `NAME.rev.1.ebwt`, and `NAME.rev.2.ebwt`, where `NAME`
is `<ebwt_base>`.

  Options

    -f

The reference input files (specified as `<reference_in>`) are FASTA
files (usually having extension `.fa`, `.mfa`, `.fna` or similar).

    -c

The reference sequences are given on the command line.  I.e.
`<reference_in>` is a comma-separated list of sequences rather than a
list of FASTA files.

    -C/--color

Build a colorspace index, to be queried using `bowtie` `-C`.

    -a/--noauto

Disable the default behavior whereby `bowtie-build` automatically
selects values for the `--bmax`, `--dcv` and `--packed` parameters
according to available memory.  Instead, user may specify values for
those parameters.  If memory is exhausted during indexing, an error
message will be printed; it is up to the user to try new parameters.

    -p/--packed

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 `-a`/`--noauto` to
configure manually.

    --bmax <int>

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
`--bmax`, or `--bmaxdivn`.  Default (in terms of the `--bmaxdivn`
parameter) is `--bmaxdivn` 4.  This is configured automatically by
default; use `-a`/`--noauto` to configure manually.

    --bmaxdivn <int>

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 `--bmax`, or `--bmaxdivn`.  Default:
`--bmaxdivn` 4.  This is configured automatically by default; use
`-a`/`--noauto` to configure manually.

    --dcv <int>

Use `<int>` as the period for the difference-cover sample.  A larger
period yields less memory overhead, but may make suffix sorting slower,
especially if repeats are present.  Must be a power of 2 no greater
than 4096.  Default: 1024.  This is configured automatically by
default; use `-a`/`--noauto` to configure manually.

    --nodc

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.

    -r/--noref

Do not build the `NAME.3.ebwt` and `NAME.4.ebwt` portions of the index,
which contain a bitpacked version of the reference sequences and are
used for paired-end alignment.

    -3/--justref

Build *only* the `NAME.3.ebwt` and `NAME.4.ebwt` portions of the index,
which contain a bitpacked version of the reference sequences and are
used for paired-end alignment.

    -o/--offrate <int>

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.  `-o`/`--offrate`
governs how many rows get marked: the indexer will mark every 2^`<int>`
rows.  Marking more rows makes reference-position lookups faster, but
requires more memory to hold the annotations at runtime.  The default
is 5 (every 32nd row is marked; for human genome, annotations occupy
about 340 megabytes).  

    -t/--ftabchars <int>

The ftab is the lookup table used to calculate an initial
[Burrows-Wheeler] range with respect to the first `<int>` characters
of the query.  A larger `<int>` yields a larger lookup table but faster
query times.  The ftab has size 4^(`<int>`+1) bytes.  The default
setting is 10 (ftab is 4MB).

    --ntoa

Convert Ns in the reference sequence to As before building the index.
By default, Ns are simply excluded from the index and `bowtie` will not
report alignments that overlap them.

    --big --little

Endianness to use when serializing integers to the index file.
Default: little-endian (recommended for Intel- and AMD-based
architectures).

    --seed <int>

Use `<int>` as the seed for pseudo-random number generator.

    --cutoff <int>

Index only the first `<int>` bases of the reference sequences
(cumulative across sequences) and ignore the rest.

    -q/--quiet

`bowtie-build` is verbose by default.  With this option `bowtie-build`
will print only error messages.

    -h/--help

Print usage information and quit.

    --version

Print version information and quit.

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 `N`s).  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>

The basename of the index to be inspected.  The basename is name of any
of the index files but with the `.X.ebwt` or `.rev.X.ebwt` suffix
omitted.  `bowtie-inspect` first looks in the current directory for the
index files, then looks in the `indexes` subdirectory under the
directory where the currently-running `bowtie` executable is located,
then looks in the directory specified in the `BOWTIE_INDEXES`
environment variable.

  Options

    -a/--across <int>

When printing FASTA output, output a newline character every `<int>`
bases (default: 60).

    -n/--names

Print reference sequence names, one per line, and quit.

    -s/--summary

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:

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

Fields are separated by tabs.

    -e/--ebwt-ref

By default, when `bowtie-inspect` is run without `-s` or `-n`, it
recreates the reference nucleotide sequences using the bit-encoded
reference nucleotides kept in the `.3.ebwt` and `.4.ebwt` index files.
When `-e/--ebwt-ref` is specified, `bowtie-inspect` recreates the
reference sequences from the Burrows-Wheeler-transformed reference
sequence in the `.1.ebwt` file instead.  The reference recreation
process is much slower when `-e/--ebwt-ref` is specified.  Also, when
`-e/--ebwt-ref` is specified and the index is in colorspace, the
reference is printed in colors (A=blue, C=green, G=orange, T=red).

    -v/--verbose

Print verbose output (for debugging).

    --version

Print version information and quit.

    -h/--help

Print usage information and quit.