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<h1>Table of Contents</h1>
<header id="title-block-header">
<h1 class="title">Bowtie 2 Manual</h1>
</header>
<nav id="TOC" role="doc-toc">
<ul>
<li><a href="#introduction" id="toc-introduction">Introduction</a>
<ul>
<li><a href="#how-is-bowtie-2-different-from-bowtie-1"
id="toc-how-is-bowtie-2-different-from-bowtie-1">How is Bowtie 2
different from Bowtie 1?</a></li>
<li><a href="#what-isnt-bowtie-2" id="toc-what-isnt-bowtie-2">What isn't
Bowtie 2?</a></li>
</ul></li>
<li><a href="#obtaining-bowtie-2" id="toc-obtaining-bowtie-2">Obtaining
Bowtie 2</a>
<ul>
<li><a href="#building-from-source"
id="toc-building-from-source">Building from source</a>
<ul>
<li><a href="#building-with-sra-support"
id="toc-building-with-sra-support">Building with SRA support</a></li>
<li><a href="#building-with-libsais-support"
id="toc-building-with-libsais-support">Building with libsais
support</a></li>
<li><a href="#building-with-cmake" id="toc-building-with-cmake">Building
with CMake</a></li>
</ul></li>
<li><a href="#adding-to-path" id="toc-adding-to-path">Adding to
PATH</a></li>
</ul></li>
<li><a href="#the-bowtie2-aligner" id="toc-the-bowtie2-aligner">The
<code>bowtie2</code> aligner</a>
<ul>
<li><a href="#end-to-end-alignment-versus-local-alignment"
id="toc-end-to-end-alignment-versus-local-alignment">End-to-end
alignment versus local alignment</a>
<ul>
<li><a href="#end-to-end-alignment-example"
id="toc-end-to-end-alignment-example">End-to-end alignment
example</a></li>
<li><a href="#local-alignment-example"
id="toc-local-alignment-example">Local alignment example</a></li>
</ul></li>
<li><a href="#scores-higher-more-similar"
id="toc-scores-higher-more-similar">Scores: higher = more similar</a>
<ul>
<li><a href="#end-to-end-alignment-score-example"
id="toc-end-to-end-alignment-score-example">End-to-end alignment score
example</a></li>
<li><a href="#local-alignment-score-example"
id="toc-local-alignment-score-example">Local alignment score
example</a></li>
<li><a
href="#valid-alignments-meet-or-exceed-the-minimum-score-threshold"
id="toc-valid-alignments-meet-or-exceed-the-minimum-score-threshold">Valid
alignments meet or exceed the minimum score threshold</a></li>
</ul></li>
<li><a href="#mapping-quality-higher-more-unique"
id="toc-mapping-quality-higher-more-unique">Mapping quality: higher =
more unique</a></li>
<li><a href="#aligning-pairs" id="toc-aligning-pairs">Aligning pairs</a>
<ul>
<li><a href="#paired-inputs" id="toc-paired-inputs">Paired
inputs</a></li>
<li><a href="#paired-sam-output" id="toc-paired-sam-output">Paired SAM
output</a></li>
<li><a
href="#concordant-pairs-match-pair-expectations-discordant-pairs-dont"
id="toc-concordant-pairs-match-pair-expectations-discordant-pairs-dont">Concordant
pairs match pair expectations, discordant pairs don't</a></li>
<li><a href="#mixed-mode-paired-where-possible-unpaired-otherwise"
id="toc-mixed-mode-paired-where-possible-unpaired-otherwise">Mixed mode:
paired where possible, unpaired otherwise</a></li>
<li><a href="#some-sam-flags-describe-paired-end-properties"
id="toc-some-sam-flags-describe-paired-end-properties">Some SAM FLAGS
describe paired-end properties</a></li>
<li><a
href="#some-sam-optional-fields-describe-more-paired-end-properties"
id="toc-some-sam-optional-fields-describe-more-paired-end-properties">Some
SAM optional fields describe more paired-end properties</a></li>
<li><a href="#mates-can-overlap-contain-or-dovetail-each-other"
id="toc-mates-can-overlap-contain-or-dovetail-each-other">Mates can
overlap, contain, or dovetail each other</a></li>
</ul></li>
<li><a href="#reporting" id="toc-reporting">Reporting</a>
<ul>
<li><a href="#distinct-alignments-map-a-read-to-different-places"
id="toc-distinct-alignments-map-a-read-to-different-places">Distinct
alignments map a read to different places</a></li>
<li><a
href="#default-mode-search-for-multiple-alignments-report-the-best-one"
id="toc-default-mode-search-for-multiple-alignments-report-the-best-one">Default
mode: search for multiple alignments, report the best one</a></li>
<li><a href="#k-mode-search-for-one-or-more-alignments-report-each"
id="toc-k-mode-search-for-one-or-more-alignments-report-each">-k mode:
search for one or more alignments, report each</a></li>
<li><a href="#a-mode-search-for-and-report-all-alignments"
id="toc-a-mode-search-for-and-report-all-alignments">-a mode: search for
and report all alignments</a></li>
<li><a href="#randomness-in-bowtie-2"
id="toc-randomness-in-bowtie-2">Randomness in Bowtie 2</a></li>
</ul></li>
<li><a href="#multiseed-heuristic"
id="toc-multiseed-heuristic">Multiseed heuristic</a>
<ul>
<li><a href="#fm-index-memory-footprint"
id="toc-fm-index-memory-footprint">FM Index memory footprint</a></li>
</ul></li>
<li><a href="#ambiguous-characters"
id="toc-ambiguous-characters">Ambiguous characters</a></li>
<li><a href="#presets-setting-many-settings-at-once"
id="toc-presets-setting-many-settings-at-once">Presets: setting many
settings at once</a></li>
<li><a href="#filtering" id="toc-filtering">Filtering</a></li>
<li><a href="#alignment-summary" id="toc-alignment-summary">Alignment
summary</a></li>
<li><a href="#wrapper-scripts" id="toc-wrapper-scripts">Wrapper
scripts</a></li>
<li><a href="#small-and-large-indexes"
id="toc-small-and-large-indexes">Small and large indexes</a></li>
<li><a href="#performance-tuning"
id="toc-performance-tuning">Performance tuning</a></li>
<li><a href="#command-line" id="toc-command-line">Command Line</a>
<ul>
<li><a href="#setting-function-options"
id="toc-setting-function-options">Setting function options</a></li>
<li><a href="#usage" id="toc-usage">Usage</a></li>
<li><a href="#main-arguments" id="toc-main-arguments">Main
arguments</a></li>
<li><a href="#options" id="toc-options">Options</a></li>
</ul></li>
<li><a href="#sam-output" id="toc-sam-output">SAM output</a></li>
</ul></li>
<li><a href="#the-bowtie2-build-indexer"
id="toc-the-bowtie2-build-indexer">The <code>bowtie2-build</code>
indexer</a>
<ul>
<li><a href="#command-line-1" id="toc-command-line-1">Command Line</a>
<ul>
<li><a href="#main-arguments-1" id="toc-main-arguments-1">Main
arguments</a></li>
<li><a href="#options-1" id="toc-options-1">Options</a></li>
</ul></li>
</ul></li>
<li><a href="#the-bowtie2-inspect-index-inspector"
id="toc-the-bowtie2-inspect-index-inspector">The
<code>bowtie2-inspect</code> index inspector</a>
<ul>
<li><a href="#command-line-2" id="toc-command-line-2">Command Line</a>
<ul>
<li><a href="#main-arguments-2" id="toc-main-arguments-2">Main
arguments</a></li>
<li><a href="#options-2" id="toc-options-2">Options</a></li>
</ul></li>
</ul></li>
<li><a href="#getting-started-with-bowtie-2-lambda-phage-example"
id="toc-getting-started-with-bowtie-2-lambda-phage-example">Getting
started with Bowtie 2: Lambda phage example</a>
<ul>
<li><a href="#indexing-a-reference-genome"
id="toc-indexing-a-reference-genome">Indexing a reference
genome</a></li>
<li><a href="#aligning-example-reads"
id="toc-aligning-example-reads">Aligning example reads</a></li>
<li><a href="#paired-end-example" id="toc-paired-end-example">Paired-end
example</a></li>
<li><a href="#local-alignment-example-1"
id="toc-local-alignment-example-1">Local alignment example</a></li>
<li><a href="#using-samtoolsbcftools-downstream"
id="toc-using-samtoolsbcftools-downstream">Using SAMtools/BCFtools
downstream</a></li>
</ul></li>
</ul>
</nav>
<!--
! This manual is written in "markdown" format and thus contains some
! distracting formatting clutter. See 'MANUAL' for an easier-to-read version
! of this text document, or see the HTML manual online.
! -->
<h1 id="introduction">Introduction</h1>
<p><a href="http://bowtie-bio.sf.net/bowtie2">Bowtie 2</a> is an
ultrafast and memory-efficient tool for aligning sequencing reads to
long reference sequences. It is particularly good at aligning reads of
about 50 up to 100s of characters to relatively long (e.g. mammalian)
genomes. Bowtie 2 indexes the genome with an <a
href="http://en.wikipedia.org/wiki/FM-index">FM Index</a> (based on the
<a
href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform">Burrows-Wheeler
Transform</a> or <a
href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform">BWT</a>)
to keep its memory footprint small: for the human genome, its memory
footprint is typically around 3.2 gigabytes of RAM. Bowtie 2 supports
gapped, local, and paired-end alignment modes. Multiple processors can
be used simultaneously to achieve greater alignment speed.</p>
<p>Bowtie 2 outputs alignments in <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM</a> format, enabling
interoperation with a large number of other tools (e.g. <a
href="http://samtools.sourceforge.net">SAMtools</a>, <a
href="http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit">GATK</a>)
that use SAM. Bowtie 2 is distributed under the <a
href="http://www.gnu.org/licenses/gpl-3.0.html">GPLv3 license</a>, and
it runs on the command line under Windows, Mac OS X and Linux and
BSD.</p>
<p><a href="http://bowtie-bio.sf.net/bowtie2">Bowtie 2</a> is often the
first step in pipelines for comparative genomics, including for
variation calling, ChIP-seq, RNA-seq, BS-seq. <a
href="http://bowtie-bio.sf.net/bowtie2">Bowtie 2</a> and <a
href="http://bowtie-bio.sf.net">Bowtie</a> (also called "<a
href="http://bowtie-bio.sf.net">Bowtie 1</a>" here) are also tightly
integrated into many other tools, some of which <a
href="http://bowtie-bio.sourceforge.net/bowtie2/other_tools.shtml">are
listed here</a>.</p>
<p>If you use <a href="http://bowtie-bio.sf.net/bowtie2">Bowtie 2</a>
for your published research, please cite our work. Papers describing
Bowtie 2 are:</p>
<ul>
<li><p>Langmead B, Wilks C, Antonescu V, Charles R. <a
href="https://doi.org/10.1093/bioinformatics/bty648">Scaling read
aligners to hundreds of threads on general-purpose processors</a>.
<em>Bioinformatics</em>. 2018 Jul 18. doi:
10.1093/bioinformatics/bty648.</p></li>
<li><p>Langmead B, Salzberg SL. <a
href="https://www.nature.com/articles/nmeth.1923">Fast gapped-read
alignment with Bowtie 2</a>. <em>Nature Methods</em>. 2012 Mar
4;9(4):357-9. doi: 10.1038/nmeth.1923.</p></li>
</ul>
<h2 id="how-is-bowtie-2-different-from-bowtie-1">How is Bowtie 2
different from Bowtie 1?</h2>
<p>Bowtie 1 was released in 2009 and was geared toward aligning the
relatively short sequencing reads (up to 25-50 nucleotides) prevalent at
the time. Since then, technology has improved both sequencing throughput
(more nucleotides produced per sequencer per day) and read length (more
nucleotides per read).</p>
<p>The chief differences between Bowtie 1 and Bowtie 2 are:</p>
<ol type="1">
<li><p>For reads longer than about 50 bp Bowtie 2 is generally faster,
more sensitive, and uses less memory than Bowtie 1. For relatively short
reads (e.g. less than 50 bp) Bowtie 1 is sometimes faster and/or more
sensitive.</p></li>
<li><p>Bowtie 2 supports gapped alignment with affine gap penalties.
Number of gaps and gap lengths are not restricted, except by way of the
configurable scoring scheme. Bowtie 1 finds just ungapped
alignments.</p></li>
<li><p>Bowtie 2 supports <a
href="#end-to-end-alignment-versus-local-alignment">local alignment</a>,
which doesn't require reads to align end-to-end. Local alignments might
be "trimmed" ("soft clipped") at one or both extremes in a way that
optimizes alignment score. Bowtie 2 also supports <a
href="#end-to-end-alignment-versus-local-alignment">end-to-end
alignment</a> which, like Bowtie 1, requires that the read align
entirely.</p></li>
<li><p>There is no upper limit on read length in Bowtie 2. Bowtie 1 had
an upper limit of around 1000 bp.</p></li>
<li><p>Bowtie 2 allows alignments to <a
href="#ambiguous-characters">overlap ambiguous characters</a> (e.g.
<code>N</code>s) in the reference. Bowtie 1 does not.</p></li>
<li><p>Bowtie 2 does away with Bowtie 1's notion of alignment "stratum",
and its distinction between "Maq-like" and "end-to-end" modes. In Bowtie
2 all alignments lie along a continuous spectrum of alignment scores
where the <a href="#scores-higher-more-similar">scoring scheme</a>,
similar to <a
href="http://en.wikipedia.org/wiki/Needleman-Wunsch_algorithm">Needleman-Wunsch</a>
and <a
href="http://en.wikipedia.org/wiki/Smith_waterman">Smith-Waterman</a>.</p></li>
<li><p>Bowtie 2's <a href="#aligning-pairs">paired-end alignment</a> is
more flexible. E.g. for pairs that do not align in a paired fashion,
Bowtie 2 attempts to find unpaired alignments for each mate.</p></li>
<li><p>Bowtie 2 reports a spectrum of mapping qualities, in contrast for
Bowtie 1 which reports either 0 or high.</p></li>
<li><p>Bowtie 2 does not align colorspace reads.</p></li>
</ol>
<p>Bowtie 2 is not a "drop-in" replacement for Bowtie 1. Bowtie 2's
command-line arguments and genome index format are both different from
Bowtie 1's.</p>
<h2 id="what-isnt-bowtie-2">What isn't Bowtie 2?</h2>
<p>Bowtie 2 is geared toward aligning relatively short sequencing reads
to long genomes. That said, it handles arbitrarily small reference
sequences (e.g. amplicons) and very long reads (i.e. upwards of 10s or
100s of kilobases), though it is slower in those settings. It is
optimized for the read lengths and error modes yielded by typical
Illumina sequencers.</p>
<p>Bowtie 2 does not support alignment of colorspace reads. (Bowtie 1
does.)</p>
<h1 id="obtaining-bowtie-2">Obtaining Bowtie 2</h1>
<p>Bowtie 2 is available from various package managers, notably <a
href="https://anaconda.org/bioconda/bowtie2">Bioconda</a>. With Bioconda
installed, you should be able to install Bowtie 2 with
<code>conda install bowtie2</code>.</p>
<p>You can also download Bowtie 2 sources and binaries from the <a
href="https://sourceforge.net/projects/bowtie-bio/files/bowtie2/">Download</a>
section of the Sourceforge site. Binaries are available for the
<code>x86_64</code> architecture running Linux, Mac OS X, and Windows.
FreeBSD users can obtain the latest version of Bowtie 2 from ports using
<code>pkg install bowtie2</code>. If you plan to compile Bowtie 2
yourself, make sure to get the source package, i.e., the filename that
ends in "-source.zip".</p>
<h2 id="building-from-source">Building from source</h2>
<p>Building from source</p>
<p>Building Bowtie 2 from source requires a GNU-like environment with
Clang/GCC, GNU Make and other basics. It should be possible to build
Bowtie 2 on most vanilla *NIX installations or on a Mac installation
with Xcode installed. Bowtie 2 can also be built on Windows using a
64-bit MinGW distribution and MSYS. In order to simplify the MinGW setup
it might be worth investigating popular MinGW personal builds since
these are coming already prepared with most of the toolchains
needed.</p>
<p>First, download the source package from the <a
href="https://sourceforge.net/projects/bowtie-bio/files/bowtie2/">sourceforge
site</a>. Make sure you're getting the source package; the file
downloaded should end in <code>-source.zip</code>. Unzip the file,
change to the unzipped directory, and build the Bowtie 2 tools by
running GNU <code>make</code> (usually with the command
<code>make</code>, but sometimes with <code>gmake</code>) with no
arguments. If building with MinGW, run <code>make</code> from the MSYS
environment.</p>
<p>The Bowtie 2 Makefile also includes recipes for basic automatic
dependency management. Running
<code>make static-libs && make STATIC_BUILD=1</code> will issue
a series of commands that will: 1. download zstd and zlib 2. compile
them as static libraries 3. link the resulting libraries to the compiled
Bowtie 2 binaries</p>
<h3 id="building-with-sra-support">Building with SRA support</h3>
<p>As of version 2.3.5 bowtie2 now supports aligning SRA reads.
Prepackaged builds will include a package that supports SRA. If you're
building bowtie2 from source please make sure that the Java runtime is
available on your system. You can then proceed with the build by running
<code>make sra-deps && make USE_SRA=1</code>.</p>
<h3 id="building-with-libsais-support">Building with libsais
support</h3>
<p>As of version 2.5.3 <code>bowtie2</code> supports building indexes
using the SAIS algorithm provided by <a
href="https://github.com/IlyaGrebnov/libsais">libsais</a>. SAIS is a
state-of-the-art suffix array construction algorithm that will
bring-forth a significant speed-up to the overall index building
process. There is, however, the downside of a significant increase in
memory usage compared to the persistent blockwise algorithm that
<code>bowtie2-build</code> uses by default. When using SAIS small
indexes can be built for inputs up to 2GB. The
<code>bowtie2-build</code> wrapper will help determine the appropriate
index type for uncompressed and gzipped inputs.</p>
<p>To build <code>bowtie2-build</code> with <a
href="https://github.com/IlyaGrebnov/libsais">libsais</a> first make
sure that the libsais submodule is available. This can be done in one of
the following ways:</p>
<ul>
<li>first time cloning bowtie2 --
<code>git clone --recursive https://github.com/BenLangmead/bowtie2.git</code></li>
<li>existing checkout of bowtie2 --
<code>git submodule init && git submodule update</code></li>
</ul>
<p>Issue the following command line to build libsais:</p>
<ul>
<li>with OpenMP support --
<code>[g]make libsais USE_SAIS_OPENMP=1</code></li>
<li>without OpenMP support --
<code>[g]make libsais USE_SAIS=1</code></li>
</ul>
<p>The choice of using OpenMP will determine whether or not the
algorithm runs multithreaded. The [<code>-p/--threads</code>] argument
to <code>bowtie2-build</code> will be ignored when libsais is compiled
without OpenMP support.</p>
<p>Finally, building the bowtie2-build executable:</p>
<ul>
<li>with OpenMP support --
<code>[g]make bowtie2-build-s USE_SAIS_OPENMP=1</code></li>
<li>without OpenMP support --
<code>[g]make bowtie2-build-s USE_SAIS=1</code></li>
</ul>
<h3 id="building-with-cmake">Building with CMake</h3>
<p>To build Bowtie2 with SRA and libsais support issue the following
command:</p>
<ul>
<li><code>cmake . -D USE_SRA=1 -D USE_SAIS=1 && cmake --build .</code></li>
</ul>
<p>CMake will take care of building and linking against the specified
dependencies.</p>
<h2 id="adding-to-path">Adding to PATH</h2>
<p>By adding your new Bowtie 2 directory to your <a
href="http://en.wikipedia.org/wiki/PATH_(variable)">PATH environment
variable</a>, you ensure that whenever you run <code>bowtie2</code>,
<code>bowtie2-build</code> or <code>bowtie2-inspect</code> from the
command line, you will get the version you just installed without having
to specify the entire path. This is recommended for most users. To do
this, follow your operating system's instructions for adding the
directory to your <a
href="http://en.wikipedia.org/wiki/PATH_(variable)">PATH</a>.</p>
<p>If you would like to install Bowtie 2 by copying the Bowtie 2
executable files to an existing directory in your <a
href="http://en.wikipedia.org/wiki/PATH_(variable)">PATH</a>, make sure
that you copy all the executables, including <code>bowtie2</code>,
<code>bowtie2-align-s</code>, <code>bowtie2-align-l</code>,
<code>bowtie2-build</code>, <code>bowtie2-build-s</code>,
<code>bowtie2-build-l</code>, <code>bowtie2-inspect</code>,
<code>bowtie2-inspect-s</code> and <code>bowtie2-inspect-l</code>.</p>
<h1 id="the-bowtie2-aligner">The <code>bowtie2</code> aligner</h1>
<p><code>bowtie2</code> takes a Bowtie 2 index and a set of sequencing
read files and outputs a set of alignments in SAM format.</p>
<p>"Alignment" is the process by which we discover how and where the
read sequences are similar to the reference sequence. An "alignment" is
a result from this process, specifically: an alignment is a way of
"lining up" some or all of the characters in the read with some
characters from the reference in a way that reveals how they're similar.
For example:</p>
<pre><code> Read: GACTGGGCGATCTCGACTTCG
||||| |||||||||| |||
Reference: GACTG--CGATCTCGACATCG</code></pre>
<p>Where dash symbols represent gaps and vertical bars show where
aligned characters match.</p>
<p>We use alignment to make an educated guess as to where a read
originated with respect to the reference genome. It's not always
possible to determine this with certainty. For instance, if the
reference genome contains several long stretches of As
(<code>AAAAAAAAA</code> etc.) and the read sequence is a short stretch
of As (<code>AAAAAAA</code>), we cannot know for certain exactly where
in the sea of <code>A</code>s the read originated.</p>
<h2 id="end-to-end-alignment-versus-local-alignment">End-to-end
alignment versus local alignment</h2>
<p>By default, Bowtie 2 performs end-to-end read alignment. That is, it
searches for alignments involving all of the read characters. This is
also called an "untrimmed" or "unclipped" alignment.</p>
<p>When the --local option is specified, Bowtie 2 performs local read
alignment. In this mode, Bowtie 2 might "trim" or "clip" some read
characters from one or both ends of the alignment if doing so maximizes
the alignment score.</p>
<h3 id="end-to-end-alignment-example">End-to-end alignment example</h3>
<p>The following is an "end-to-end" alignment because it involves all
the characters in the read. Such an alignment can be produced by Bowtie
2 in either end-to-end mode or in local mode.</p>
<pre><code>Read: GACTGGGCGATCTCGACTTCG
Reference: GACTGCGATCTCGACATCG
Alignment:
Read: GACTGGGCGATCTCGACTTCG
||||| |||||||||| |||
Reference: GACTG--CGATCTCGACATCG</code></pre>
<h3 id="local-alignment-example">Local alignment example</h3>
<p>The following is a "local" alignment because some of the characters
at the ends of the read do not participate. In this case, 4 characters
are omitted (or "soft trimmed" or "soft clipped") from the beginning and
3 characters are omitted from the end. This sort of alignment can be
produced by Bowtie 2 only in local mode.</p>
<pre><code>Read: ACGGTTGCGTTAATCCGCCACG
Reference: TAACTTGCGTTAAATCCGCCTGG
Alignment:
Read: ACGGTTGCGTTAA-TCCGCCACG
||||||||| ||||||
Reference: TAACTTGCGTTAAATCCGCCTGG</code></pre>
<h2 id="scores-higher-more-similar">Scores: higher = more similar</h2>
<p>An alignment score quantifies how similar the read sequence is to the
reference sequence aligned to. The higher the score, the more similar
they are. A score is calculated by subtracting penalties for each
difference (mismatch, gap, etc.) and, in local alignment mode, adding
bonuses for each match.</p>
<p>The scores can be configured with the <a
href="#bowtie2-options-ma"><code>--ma</code></a> (match bonus), <a
href="#bowtie2-options-mp"><code>--mp</code></a> (mismatch penalty), <a
href="#bowtie2-options-np"><code>--np</code></a> (penalty for having an
N in either the read or the reference), <a
href="#bowtie2-options-rdg"><code>--rdg</code></a> (affine read gap
penalty) and <a href="#bowtie2-options-rfg"><code>--rfg</code></a>
(affine reference gap penalty) options.</p>
<h3 id="end-to-end-alignment-score-example">End-to-end alignment score
example</h3>
<p>A mismatched base at a high-quality position in the read receives a
penalty of -6 by default. A length-2 read gap receives a penalty of -11
by default (-5 for the gap open, -3 for the first extension, -3 for the
second extension). Thus, in end-to-end alignment mode, if the read is 50
bp long and it matches the reference exactly except for one mismatch at
a high-quality position and one length-2 read gap, then the overall
score is -(6 + 11) = -17.</p>
<p>The best possible alignment score in end-to-end mode is 0, which
happens when there are no differences between the read and the
reference.</p>
<h3 id="local-alignment-score-example">Local alignment score
example</h3>
<p>A mismatched base at a high-quality position in the read receives a
penalty of -6 by default. A length-2 read gap receives a penalty of -11
by default (-5 for the gap open, -3 for the first extension, -3 for the
second extension). A base that matches receives a bonus of +2 be
default. Thus, in local alignment mode, if the read is 50 bp long and it
matches the reference exactly except for one mismatch at a high-quality
position and one length-2 read gap, then the overall score equals the
total bonus, 2 * 49, minus the total penalty, 6 + 11, = 81.</p>
<p>The best possible score in local mode equals the match bonus times
the length of the read. This happens when there are no differences
between the read and the reference.</p>
<h3
id="valid-alignments-meet-or-exceed-the-minimum-score-threshold">Valid
alignments meet or exceed the minimum score threshold</h3>
<p>For an alignment to be considered "valid" (i.e. "good enough") by
Bowtie 2, it must have an alignment score no less than the minimum score
threshold. The threshold is configurable and is expressed as a function
of the read length. In end-to-end alignment mode, the default minimum
score threshold is <code>-0.6 + -0.6 * L</code>, where <code>L</code> is
the read length. In local alignment mode, the default minimum score
threshold is <code>20 + 8.0 * ln(L)</code>, where L is the read length.
This can be configured with the <a
href="#bowtie2-options-score-min"><code>--score-min</code></a> option.
For details on how to set options like <code>--score-min</code> that
correspond to functions, see the section on <a
href="#setting-function-options">setting function options</a>.</p>
<h2 id="mapping-quality-higher-more-unique">Mapping quality: higher =
more unique</h2>
<p>The aligner cannot always assign a read to its point of origin with
high confidence. For instance, a read that originated inside a repeat
element might align equally well to many occurrences of the element
throughout the genome, leaving the aligner with no basis for preferring
one over the others.</p>
<p>Aligners characterize their degree of confidence in the point of
origin by reporting a mapping quality: a non-negative integer Q = -10
log10 p, where p is an estimate of the probability that the alignment
does not correspond to the read's true point of origin. Mapping quality
is sometimes abbreviated MAPQ, and is recorded in the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM</a>
<code>MAPQ</code> field.</p>
<p>Mapping quality is related to "uniqueness." We say an alignment is
unique if it has a much higher alignment score than all the other
possible alignments. The bigger the gap between the best alignment's
score and the second-best alignment's score, the more unique the best
alignment, and the higher its mapping quality should be.</p>
<p>Accurate mapping qualities are useful for downstream tools like
variant callers. For instance, a variant caller might choose to ignore
evidence from alignments with mapping quality less than, say, 10. A
mapping quality of 10 or less indicates that there is at least a 1 in 10
chance that the read truly originated elsewhere.</p>
<h2 id="aligning-pairs">Aligning pairs</h2>
<p>A "paired-end" or "mate-pair" read consists of pair of mates, called
mate 1 and mate 2. Pairs come with a prior expectation about (a) the
relative orientation of the mates, and (b) the distance separating them
on the original DNA molecule. Exactly what expectations hold for a given
dataset depends on the lab procedures used to generate the data. For
example, a common lab procedure for producing pairs is Illumina's
Paired-end Sequencing Assay, which yields pairs with a relative
orientation of FR ("forward, reverse") meaning that if mate 1 came from
the Watson strand, mate 2 very likely came from the Crick strand and
vice versa. Also, this protocol yields pairs where the expected genomic
distance from end to end is about 200-500 base pairs.</p>
<p>For simplicity, this manual uses the term "paired-end" to refer to
any pair of reads with some expected relative orientation and distance.
Depending on the protocol, these might actually be referred to as
"paired-end" or "mate-paired." Also, we always refer to the individual
sequences making up the pair as "mates."</p>
<h3 id="paired-inputs">Paired inputs</h3>
<p>Pairs are often stored in a pair of files, one file containing the
mate 1s and the other containing the mates 2s. The first mate in the
file for mate 1 forms a pair with the first mate in the file for mate 2,
the second with the second, and so on. When aligning pairs with Bowtie
2, specify the file with the mate 1s mates using the <a
href="#bowtie2-options-1"><code>-1</code></a> argument and the file with
the mate 2s using the <a href="#bowtie2-options-2"><code>-2</code></a>
argument. This causes Bowtie 2 to take the paired nature of the reads
into account when aligning them.</p>
<h3 id="paired-sam-output">Paired SAM output</h3>
<p>When Bowtie 2 prints a SAM alignment for a pair, it prints two
records (i.e. two lines of output), one for each mate. The first record
describes the alignment for mate 1 and the second record describes the
alignment for mate 2. In both records, some of the fields of the SAM
record describe various properties of the alignment; for instance, the
7th and 8th fields (<code>RNEXT</code> and <code>PNEXT</code>
respectively) indicate the reference name and position where the other
mate aligned, and the 9th field indicates the inferred length of the DNA
fragment from which the two mates were sequenced. See the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM specification</a>
for more details regarding these fields.</p>
<h3
id="concordant-pairs-match-pair-expectations-discordant-pairs-dont">Concordant
pairs match pair expectations, discordant pairs don't</h3>
<p>A pair that aligns with the expected relative mate orientation and
with the expected range of distances between mates is said to align
"concordantly". If both mates have unique alignments, but the alignments
do not match paired-end expectations (i.e. the mates aren't in the
expected relative orientation, or aren't within the expected distance
range, or both), the pair is said to align "discordantly". Discordant
alignments may be of particular interest, for instance, when seeking <a
href="http://www.ncbi.nlm.nih.gov/dbvar/content/overview/">structural
variants</a>.</p>
<p>The expected relative orientation of the mates is set using the <a
href="#bowtie2-options-fr"><code>--ff</code></a>, <a
href="#bowtie2-options-fr"><code>--fr</code></a>, or <a
href="#bowtie2-options-fr"><code>--rf</code></a> options. The expected
range of inter-mates distances (as measured from the furthest extremes
of the mates; also called "outer distance") is set with the <a
href="#bowtie2-options-I"><code>-I</code></a> and <a
href="#bowtie2-options-X"><code>-X</code></a> options. Note that setting
<a href="#bowtie2-options-I"><code>-I</code></a> and <a
href="#bowtie2-options-X"><code>-X</code></a> far apart makes Bowtie 2
slower. See documentation for <a
href="#bowtie2-options-I"><code>-I</code></a> and <a
href="#bowtie2-options-X"><code>-X</code></a>.</p>
<p>To declare that a pair aligns discordantly, Bowtie 2 requires that
both mates align uniquely. This is a conservative threshold, but this is
often desirable when seeking structural variants.</p>
<p>By default, Bowtie 2 searches for both concordant and discordant
alignments, though searching for discordant alignments can be disabled
with the <a
href="#bowtie2-options-no-discordant"><code>--no-discordant</code></a>
option.</p>
<h3 id="mixed-mode-paired-where-possible-unpaired-otherwise">Mixed mode:
paired where possible, unpaired otherwise</h3>
<p>If Bowtie 2 cannot find a paired-end alignment for a pair, by default
it will go on to look for unpaired alignments for the constituent mates.
This is called "mixed mode." To disable mixed mode, set the <a
href="#bowtie2-options-no-mixed"><code>--no-mixed</code></a> option.</p>
<p>Bowtie 2 runs a little faster in <code>--no-mixed</code> mode, but
will only consider alignment status of pairs per se, not individual
mates.</p>
<h3 id="some-sam-flags-describe-paired-end-properties">Some SAM FLAGS
describe paired-end properties</h3>
<p>The SAM <code>FLAGS</code> field, the second field in a SAM record,
has multiple bits that describe the paired-end nature of the read and
alignment. The first (least significant) bit (1 in decimal, 0x1 in
hexadecimal) is set if the read is part of a pair. The second bit (2 in
decimal, 0x2 in hexadecimal) is set if the read is part of a pair that
aligned in a paired-end fashion. The fourth bit (8 in decimal, 0x8 in
hexadecimal) is set if the read is part of a pair and the other mate in
the pair had at least one valid alignment. The sixth bit (32 in decimal,
0x20 in hexadecimal) is set if the read is part of a pair and the other
mate in the pair aligned to the Crick strand (or, equivalently, if the
reverse complement of the other mate aligned to the Watson strand). The
seventh bit (64 in decimal, 0x40 in hexadecimal) is set if the read is
mate 1 in a pair. The eighth bit (128 in decimal, 0x80 in hexadecimal)
is set if the read is mate 2 in a pair. See the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM specification</a>
for a more detailed description of the <code>FLAGS</code> field.</p>
<h3
id="some-sam-optional-fields-describe-more-paired-end-properties">Some
SAM optional fields describe more paired-end properties</h3>
<p>The last several fields of each SAM record usually contain SAM
optional fields, which are simply tab-separated strings conveying
additional information about the reads and alignments. A SAM optional
field is formatted like this: "XP:i:1" where "XP" is the
<code>TAG</code>, "i" is the <code>TYPE</code> ("integer" in this case),
and "1" is the <code>VALUE</code>. See the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM specification</a>
for details regarding SAM optional fields.</p>
<h3 id="mates-can-overlap-contain-or-dovetail-each-other">Mates can
overlap, contain, or dovetail each other</h3>
<p>The fragment and read lengths might be such that alignments for the
two mates from a pair overlap each other. Consider this example:</p>
<p>(For these examples, assume we expect mate 1 to align to the left of
mate 2.)</p>
<pre><code>Mate 1: GCAGATTATATGAGTCAGCTACGATATTGTT
Mate 2: TGTTTGGGGTGACACATTACGCGTCTTTGAC
Reference: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGCGTCTTTGAC</code></pre>
<p>It's also possible, though unusual, for one mate alignment to contain
the other, as in these examples:</p>
<pre><code>Mate 1: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGC
Mate 2: TGTTTGGGGTGACACATTACGC
Reference: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGCGTCTTTGAC
Mate 1: CAGCTACGATATTGTTTGGGGTGACACATTACGC
Mate 2: CTACGATATTGTTTGGGGTGAC
Reference: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGCGTCTTTGAC</code></pre>
<p>And it's also possible, though unusual, for the mates to "dovetail",
with the mates seemingly extending "past" each other as in this
example:</p>
<pre><code>Mate 1: GTCAGCTACGATATTGTTTGGGGTGACACATTACGC
Mate 2: TATGAGTCAGCTACGATATTGTTTGGGGTGACACAT
Reference: GCAGATTATATGAGTCAGCTACGATATTGTTTGGGGTGACACATTACGCGTCTTTGAC</code></pre>
<p>In some situations, it's desirable for the aligner to consider all
these cases as "concordant" as long as other paired-end constraints are
not violated. Bowtie 2's default behavior is to consider overlapping and
containing as being consistent with concordant alignment. By default,
dovetailing is considered inconsistent with concordant alignment.</p>
<p>These defaults can be overridden. Setting <a
href="#bowtie2-options-no-overlap"><code>--no-overlap</code></a> causes
Bowtie 2 to consider overlapping mates as non-concordant. Setting <a
href="#bowtie2-options-no-contain"><code>--no-contain</code></a> causes
Bowtie 2 to consider cases where one mate alignment contains the other
as non-concordant. Setting <a
href="#bowtie2-options-dovetail"><code>--dovetail</code></a> causes
Bowtie 2 to consider cases where the mate alignments dovetail as
concordant.</p>
<h2 id="reporting">Reporting</h2>
<p>The reporting mode governs how many alignments Bowtie 2 looks for,
and how to report them. Bowtie 2 has three distinct reporting modes. The
default reporting mode is similar to the default reporting mode of many
other read alignment tools, including <a
href="http://bio-bwa.sourceforge.net/">BWA</a>. It is also similar to
Bowtie 1's <code>-M</code> alignment mode.</p>
<p>In general, when we say that a read has an alignment, we mean that it
has a <a
href="#valid-alignments-meet-or-exceed-the-minimum-score-threshold">valid
alignment</a>. When we say that a read has multiple alignments, we mean
that it has multiple alignments that are valid and distinct from one
another.</p>
<h3 id="distinct-alignments-map-a-read-to-different-places">Distinct
alignments map a read to different places</h3>
<p>Two alignments for the same individual read are "distinct" if they
map the same read to different places. Specifically, we say that two
alignments are distinct if there are no alignment positions where a
particular read offset is aligned opposite a particular reference offset
in both alignments with the same orientation. E.g. if the first
alignment is in the forward orientation and aligns the read character at
read offset 10 to the reference character at chromosome 3, offset
3,445,245, and the second alignment is also in the forward orientation
and also aligns the read character at read offset 10 to the reference
character at chromosome 3, offset 3,445,245, they are not distinct
alignments.</p>
<p>Two alignments for the same pair are distinct if either the mate 1s
in the two paired-end alignments are distinct or the mate 2s in the two
alignments are distinct or both.</p>
<h3
id="default-mode-search-for-multiple-alignments-report-the-best-one">Default
mode: search for multiple alignments, report the best one</h3>
<p>By default, Bowtie 2 searches for distinct, valid alignments for each
read. When it finds a valid alignment, it generally will continue to
look for alignments that are nearly as good or better. It will
eventually stop looking, either because it exceeded a limit placed on
search effort (see <a href="#bowtie2-options-D"><code>-D</code></a> and
<a href="#bowtie2-options-R"><code>-R</code></a>) or because it already
knows all it needs to know to report an alignment. Information from the
best alignments are used to estimate mapping quality (the
<code>MAPQ</code> <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM</a> field) and to
set SAM optional fields, such as <a
href="#bowtie2-build-opt-fields-as"><code>AS:i</code></a> and <a
href="#bowtie2-build-opt-fields-xs"><code>XS:i</code></a>. Bowtie 2 does
not guarantee that the alignment reported is the best possible in terms
of alignment score.</p>
<p>See also: <a href="#bowtie2-options-D"><code>-D</code></a>, which
puts an upper limit on the number of dynamic programming problems (i.e.
seed extensions) that can "fail" in a row before Bowtie 2 stops
searching. Increasing <a href="#bowtie2-options-D"><code>-D</code></a>
makes Bowtie 2 slower, but increases the likelihood that it will report
the correct alignment for a read that aligns many places.</p>
<p>See also: <a href="#bowtie2-options-R"><code>-R</code></a>, which
sets the maximum number of times Bowtie 2 will "re-seed" when attempting
to align a read with repetitive seeds. Increasing <a
href="#bowtie2-options-R"><code>-R</code></a> makes Bowtie 2 slower, but
increases the likelihood that it will report the correct alignment for a
read that aligns many places.</p>
<h3 id="k-mode-search-for-one-or-more-alignments-report-each">-k mode:
search for one or more alignments, report each</h3>
<p>In <a href="#bowtie2-options-k"><code>-k</code></a> mode, Bowtie 2
searches for up to N distinct, valid alignments for each read, where N
equals the integer specified with the <code>-k</code> parameter. That
is, if <code>-k 2</code> is specified, Bowtie 2 will search for at most
2 distinct alignments. It reports all alignments found, in descending
order by alignment score. The alignment score for a paired-end alignment
equals the sum of the alignment scores of the individual mates. Each
reported read or pair alignment beyond the first has the SAM 'secondary'
bit (which equals 256) set in its FLAGS field. Supplementary alignments
will also be assigned a MAPQ of 255. See the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM specification</a>
for details.</p>
<p>Bowtie 2 does not "find" alignments in any specific order, so for
reads that have more than N distinct, valid alignments, Bowtie 2 does
not guarantee that the N alignments reported are the best possible in
terms of alignment score. Still, this mode can be effective and fast in
situations where the user cares more about whether a read aligns (or
aligns a certain number of times) than where exactly it originated.</p>
<h3 id="a-mode-search-for-and-report-all-alignments">-a mode: search for
and report all alignments</h3>
<p><a href="#bowtie2-options-a"><code>-a</code></a> mode is similar to
<a href="#bowtie2-options-k"><code>-k</code></a> mode except that there
is no upper limit on the number of alignments Bowtie 2 should report.
Alignments are reported in descending order by alignment score. The
alignment score for a paired-end alignment equals the sum of the
alignment scores of the individual mates. Each reported read or pair
alignment beyond the first has the SAM 'secondary' bit (which equals
256) set in its FLAGS field. Supplementary alignments will be assigned a
MAPQ of 255. See the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM specification</a>
for details.</p>
<p>Some tools are designed with this reporting mode in mind. Bowtie 2 is
not! For very large genomes, this mode is very slow.</p>
<h3 id="randomness-in-bowtie-2">Randomness in Bowtie 2</h3>
<p>Bowtie 2's search for alignments for a given read is "randomized."
That is, when Bowtie 2 encounters a set of equally-good choices, it uses
a pseudo-random number to choose. For example, if Bowtie 2 discovers a
set of 3 equally-good alignments and wants to decide which to report, it
picks a pseudo-random integer 0, 1 or 2 and reports the corresponding
alignment. Arbitrary choices can crop up at various points during
alignment.</p>
<p>The pseudo-random number generator is re-initialized for every read,
and the seed used to initialize it is a function of the read name,
nucleotide string, quality string, and the value specified with <a
href="#bowtie2-options-seed"><code>--seed</code></a>. If you run the
same version of Bowtie 2 on two reads with identical names, nucleotide
strings, and quality strings, and if <a
href="#bowtie2-options-seed"><code>--seed</code></a> is set the same for
both runs, Bowtie 2 will produce the same output; i.e., it will align
the read to the same place, even if there are multiple equally good
alignments. This is intuitive and desirable in most cases. Most users
expect Bowtie to produce the same output when run twice on the same
input.</p>
<p>However, when the user specifies the <a
href="#bowtie2-options-non-deterministic"><code>--non-deterministic</code></a>
option, Bowtie 2 will use the current time to re-initialize the
pseudo-random number generator. When this is specified, Bowtie 2 might
report different alignments for identical reads. This is
counter-intuitive for some users, but might be more appropriate in
situations where the input consists of many identical reads.</p>
<h2 id="multiseed-heuristic">Multiseed heuristic</h2>
<p>To rapidly narrow the number of possible alignments that must be
considered, Bowtie 2 begins by extracting substrings ("seeds") from the
read and its reverse complement and aligning them in an ungapped fashion
with the help of the <a
href="http://portal.acm.org/citation.cfm?id=796543">FM Index</a>. This
is "multiseed alignment" and it is similar to what <a
href="http://genomebiology.com/2009/10/3/R25">Bowtie 1 does</a>, except
Bowtie 1 attempts to align the entire read this way.</p>
<p>This initial step makes Bowtie 2 much faster than it would be without
such a filter, but at the expense of missing some valid alignments. For
instance, it is possible for a read to have a valid overall alignment
but to have no valid seed alignments because each potential seed
alignment is interrupted by too many mismatches or gaps.</p>
<p>The trade-off between speed and sensitivity/accuracy can be adjusted
by setting the seed length (<a
href="#bowtie2-options-L"><code>-L</code></a>), the interval between
extracted seeds (<a href="#bowtie2-options-i"><code>-i</code></a>), and
the number of mismatches permitted per seed (<a
href="#bowtie2-options-N"><code>-N</code></a>). For more sensitive
alignment, set these parameters to (a) make the seeds closer together,
(b) make the seeds shorter, and/or (c) allow more mismatches. You can
adjust these options one-by-one, though Bowtie 2 comes with some useful
combinations of options prepackaged as "<a
href="#presets-setting-many-settings-at-once">preset options</a>."</p>
<p><a href="#bowtie2-options-D"><code>-D</code></a> and <a
href="#bowtie2-options-R"><code>-R</code></a> are also options that
adjust the trade-off between speed and sensitivity/accuracy.</p>
<h3 id="fm-index-memory-footprint">FM Index memory footprint</h3>
<p>Bowtie 2 uses the <a
href="http://portal.acm.org/citation.cfm?id=796543">FM Index</a> to find
ungapped alignments for seeds. This step accounts for the bulk of Bowtie
2's memory footprint, as the <a
href="http://portal.acm.org/citation.cfm?id=796543">FM Index</a> itself
is typically the largest data structure used. For instance, the memory
footprint of the <a
href="http://portal.acm.org/citation.cfm?id=796543">FM Index</a> for the
human genome is about 3.2 gigabytes of RAM.</p>
<h2 id="ambiguous-characters">Ambiguous characters</h2>
<p>Non-whitespace characters besides A, C, G or T are considered
"ambiguous." N is a common ambiguous character that appears in reference
sequences. Bowtie 2 considers all ambiguous characters in the reference
(including <a href="http://www.bioinformatics.org/sms/iupac.html">IUPAC
nucleotide codes</a>) to be Ns.</p>
<p>Bowtie 2 allows alignments to overlap ambiguous characters in the
reference. An alignment position that contains an ambiguous character in
the read, reference, or both, is penalized according to <a
href="#bowtie2-options-np"><code>--np</code></a>. <a
href="#bowtie2-options-n-ceil"><code>--n-ceil</code></a> sets an upper
limit on the number of positions that may contain ambiguous reference
characters in a valid alignment. The optional field <a
href="#bowtie2-build-opt-fields-xn"><code>XN:i</code></a> reports the
number of ambiguous reference characters overlapped by an alignment.</p>
<p>Note that the <a href="#multiseed-heuristic">multiseed heuristic</a>
cannot find <em>seed</em> alignments that overlap ambiguous reference
characters. For an alignment overlapping an ambiguous reference
character to be found, it must have one or more seed alignments that do
not overlap ambiguous reference characters.</p>
<h2 id="presets-setting-many-settings-at-once">Presets: setting many
settings at once</h2>
<p>Bowtie 2 comes with some useful combinations of parameters packaged
into shorter "preset" parameters. For example, running Bowtie 2 with the
<a
href="#bowtie2-options-very-sensitive"><code>--very-sensitive</code></a>
option is the same as running with options:
<code>-D 20 -R 3 -N 0 -L 20 -i S,1,0.50</code>. The preset options that
come with Bowtie 2 are designed to cover a wide area of the
speed/sensitivity/accuracy trade-off space, with the presets ending in
<code>fast</code> generally being faster but less sensitive and less
accurate, and the presets ending in <code>sensitive</code> generally
being slower but more sensitive and more accurate. See the <a
href="#preset-options-in---end-to-end-mode">documentation for the preset
options</a> for details.</p>
<p>As of Bowtie2 v2.4.0, individual preset values can be overridden by
providing the specific options e.g. the configured seed length of 20 in
the [<code>--very-senitive</code>] preset above can be changed to 25 by
also specifying the <code>-L 25</code> parameter anywhere on the command
line.</p>
<h2 id="filtering">Filtering</h2>
<p>Some reads are skipped or "filtered out" by Bowtie 2. For example,
reads may be filtered out because they are extremely short or have a
high proportion of ambiguous nucleotides. Bowtie 2 will still print a
SAM record for such a read, but no alignment will be reported and the
<code>YF:i</code> SAM optional field will be set to indicate the reason
the read was filtered.</p>
<ul>
<li><code>YF:Z:LN</code>: the read was filtered because it had length
less than or equal to the number of seed mismatches set with the <a
href="#bowtie2-options-N"><code>-N</code></a> option.</li>
<li><code>YF:Z:NS</code>: the read was filtered because it contains a
number of ambiguous characters (usually <code>N</code> or
<code>.</code>) greater than the ceiling specified with <a
href="#bowtie2-options-n-ceil"><code>--n-ceil</code></a>.</li>
<li><code>YF:Z:SC</code>: the read was filtered because the read length
and the match bonus (set with <a
href="#bowtie2-options-ma"><code>--ma</code></a>) are such that the read
can't possibly earn an alignment score greater than or equal to the
threshold set with <a
href="#bowtie2-options-score-min"><code>--score-min</code></a></li>
<li><code>YF:Z:QC</code>: the read was filtered because it was marked as
failing quality control and the user specified the <a
href="#bowtie2-options-qc-filter"><code>--qc-filter</code></a> option.
This only happens when the input is in Illumina's QSEQ format (i.e. when
<a href="#bowtie2-options-qseq"><code>--qseq</code></a> is specified)
and the last (11th) field of the read's QSEQ record contains
<code>1</code>.</li>
</ul>
<p>If a read could be filtered for more than one reason, the value
<code>YF:Z</code> flag will reflect only one of those reasons.</p>
<h2 id="alignment-summary">Alignment summary</h2>
<p>When Bowtie 2 finishes running, it prints messages summarizing what
happened. These messages are printed to the "standard error" ("stderr")
filehandle. For datasets consisting of unpaired reads, the summary might
look like this:</p>
<pre><code>20000 reads; of these:
20000 (100.00%) were unpaired; of these:
1247 (6.24%) aligned 0 times
18739 (93.69%) aligned exactly 1 time
14 (0.07%) aligned >1 times
93.77% overall alignment rate</code></pre>
<p>For datasets consisting of pairs, the summary might look like
this:</p>
<pre><code>10000 reads; of these:
10000 (100.00%) were paired; of these:
650 (6.50%) aligned concordantly 0 times
8823 (88.23%) aligned concordantly exactly 1 time
527 (5.27%) aligned concordantly >1 times
----
650 pairs aligned concordantly 0 times; of these:
34 (5.23%) aligned discordantly 1 time
----
616 pairs aligned 0 times concordantly or discordantly; of these:
1232 mates make up the pairs; of these:
660 (53.57%) aligned 0 times
571 (46.35%) aligned exactly 1 time
1 (0.08%) aligned >1 times
96.70% overall alignment rate</code></pre>
<p>The indentation indicates how subtotals relate to totals.</p>
<h2 id="wrapper-scripts">Wrapper scripts</h2>
<p>The <code>bowtie2</code>, <code>bowtie2-build</code> and
<code>bowtie2-inspect</code> 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. Also, the
<code>bowtie2</code> wrapper provides some key functionality, like the
ability to handle compressed inputs, and the functionality for <a
href="#bowtie2-options-un"><code>--un</code></a>, <a
href="#bowtie2-options-al"><code>--al</code></a> and related
options.</p>
<p>It is recommended that you always run the bowtie2 wrappers and not
run the binaries directly.</p>
<h2 id="small-and-large-indexes">Small and large indexes</h2>
<p><code>bowtie2-build</code> can index reference genomes of any size.
For genomes less than about 4 billion nucleotides in length,
<code>bowtie2-build</code> builds a "small" index using 32-bit numbers
in various parts of the index. When the genome is longer,
<code>bowtie2-build</code> builds a "large" index using 64-bit numbers.
Small indexes are stored in files with the <code>.bt2</code> extension,
and large indexes are stored in files with the <code>.bt2l</code>
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.</p>
<h2 id="performance-tuning">Performance tuning</h2>
<ol type="1">
<li><p>If your computer has multiple processors/cores, use
<code>-p</code></p>
<p>The <a href="#bowtie2-options-p"><code>-p</code></a> option causes
Bowtie 2 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).</p></li>
<li><p>If reporting many alignments per read, try reducing
<code>bowtie2-build --offrate</code></p>
<p>If you are using <a href="#bowtie2-options-k"><code>-k</code></a> or
<a href="#bowtie2-options-a"><code>-a</code></a> options and Bowtie 2 is
reporting many alignments per read, using an index with a denser SA
sample can speed things up considerably. To do this, specify a
smaller-than-default <a
href="#bowtie2-options-o"><code>-o</code>/<code>--offrate</code></a>
value when running <code>bowtie2-build</code>. A denser SA sample yields
a larger index, but is also particularly effective at speeding up
alignment when many alignments are reported per read.</p></li>
<li><p>If <code>bowtie2</code> "thrashes", try increasing
<code>bowtie2-build --offrate</code></p>
<p>If <code>bowtie2</code> runs very slowly on a relatively low-memory
computer, try setting <a
href="#bowtie2-options-o"><code>-o</code>/<code>--offrate</code></a> to
a <em>larger</em> value when building the index. This decreases the
memory footprint of the index.</p></li>
</ol>
<h2 id="command-line">Command Line</h2>
<h3 id="setting-function-options">Setting function options</h3>
<p>Some Bowtie 2 options specify a function rather than an individual
number or setting. In these cases the user specifies three parameters:
(a) a function type <code>F</code>, (b) a constant term <code>B</code>,
and (c) a coefficient <code>A</code>. The available function types are
constant (<code>C</code>), linear (<code>L</code>), square-root
(<code>S</code>), and natural log (<code>G</code>). The parameters are
specified as <code>F,B,A</code> - that is, the function type, the
constant term, and the coefficient are separated by commas with no
whitespace. The constant term and coefficient may be negative and/or
floating-point numbers.</p>
<p>For example, if the function specification is
<code>L,-0.4,-0.6</code>, then the function defined is:</p>
<pre><code>f(x) = -0.4 + -0.6 * x</code></pre>
<p>If the function specification is <code>G,1,5.4</code>, then the
function defined is:</p>
<pre><code>f(x) = 1.0 + 5.4 * ln(x)</code></pre>
<p>See the documentation for the option in question to learn what the
parameter <code>x</code> is for. For example, in the case if the <a
href="#bowtie2-options-score-min"><code>--score-min</code></a> option,
the function <code>f(x)</code> sets the minimum alignment score
necessary for an alignment to be considered valid, and <code>x</code> is
the read length.</p>
<h3 id="usage">Usage</h3>
<pre><code>bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r> | --interleaved <i> | --sra-acc <acc> | -b <bam>} -S [<sam>]</code></pre>
<h3 id="main-arguments">Main arguments</h3>
<table>
<tr>
<td id="bowtie2-options-x">
<pre><code>-x <bt2-idx></code></pre>
</td>
<td>
<p>The basename of the index for the reference genome. The basename is
the name of any of the index files up to but not including the final
<code>.1.bt2</code> / <code>.rev.1.bt2</code> / etc.
<code>bowtie2</code> looks for the specified index first in the current
directory, then in the directory specified in the
<code>BOWTIE2_INDEXES</code> environment variable.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-1">
<pre><code>-1 <m1></code></pre>
</td>
<td>
<p>Comma-separated list of files containing mate 1s (filename usually
includes <code>_1</code>), e.g. <code>-1 flyA_1.fq,flyB_1.fq</code>.
Sequences specified with this option must correspond file-for-file and
read-for-read with those specified in <code><m2></code>. Reads may
be a mix of different lengths. If <code>-</code> is specified,
<code>bowtie2</code> will read the mate 1s from the "standard in" or
"stdin" filehandle.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-2">
<pre><code>-2 <m2></code></pre>
</td>
<td>
<p>Comma-separated list of files containing mate 2s (filename usually
includes <code>_2</code>), e.g. <code>-2 flyA_2.fq,flyB_2.fq</code>.
Sequences specified with this option must correspond file-for-file and
read-for-read with those specified in <code><m1></code>. Reads may
be a mix of different lengths. If <code>-</code> is specified,
<code>bowtie2</code> will read the mate 2s from the "standard in" or
"stdin" filehandle.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-U">
<pre><code>-U <r></code></pre>
</td>
<td>
<p>Comma-separated list of files containing unpaired reads to be
aligned, e.g. <code>lane1.fq,lane2.fq,lane3.fq,lane4.fq</code>. Reads
may be a mix of different lengths. If <code>-</code> is specified,
<code>bowtie2</code> gets the reads from the "standard in" or "stdin"
filehandle.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-interleaved">
<pre><code>--interleaved</code></pre>
</td>
<td>
<p>Reads interleaved FASTQ files where the first two records (8 lines)
represent a mate pair.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-sra-acc">
<pre><code>--sra-acc</code></pre>
</td>
<td>
<p>Reads are SRA accessions. If the accession provided cannot be found
in local storage it will be fetched from the NCBI database. If you find
that SRA alignments are long running please rerun your command with the
<a href="#bowtie2-options-p"><code>-p</code>/<code>--threads</code></a>
parameter set to desired number of threads.</p>
<p>NB: this option is only available if bowtie 2 is compiled with the
necessary SRA libraries. See <a href="#obtaining-bowtie-2">Obtaining
Bowtie 2</a> for details.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-b">
<pre><code>-b <bam></code></pre>
</td>
<td>
<p>Reads are unaligned BAM records sorted by read name. The <a
href="#bowtie2-options-align-paired-reads"><code>--align-paired-reads</code></a>
and <a
href="#bowtie2-options-preserve-tags"><code>--preserve-tags</code></a>
options affect the way Bowtie 2 processes records.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-S">
<pre><code>-S <sam></code></pre>
</td>
<td>
<p>File to write SAM alignments to. By default, alignments are written
to the "standard out" or "stdout" filehandle (i.e. the console).</p>
</td>
</tr>
</table>
<h3 id="options">Options</h3>
<h4 id="input-options">Input options</h4>
<table>
<tr>
<td id="bowtie2-options-q">
<pre><code>-q</code></pre>
</td>
<td>
<p>Reads (specified with <code><m1></code>,
<code><m2></code>, <code><s></code>) are FASTQ files. FASTQ
files usually have extension <code>.fq</code> or <code>.fastq</code>.
FASTQ is the default format. See also: <a
href="#bowtie2-options-solexa-quals"><code>--solexa-quals</code></a> and
<a href="#bowtie2-options-int-quals"><code>--int-quals</code></a>.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-tab5">
<pre><code>--tab5</code></pre>
</td>
<td>
<p>Each read or pair is on a single line. An unpaired read line is
<code>[name]\t[seq]\t[qual]\n</code>. A paired-end read line is
<code>[name]\t[seq1]\t[qual1]\t[seq2]\t[qual2]\n</code>. An input file
can be a mix of unpaired and paired-end reads and Bowtie 2 recognizes
each according to the number of fields, handling each as it should.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-tab6">
<pre><code>--tab6</code></pre>
</td>
<td>
<p>Similar to <a href="#bowtie2-options-tab5"><code>--tab5</code></a>
except, for paired-end reads, the second end can have a different name
from the first:
<code>[name1]\t[seq1]\t[qual1]\t[name2]\t[seq2]\t[qual2]\n</code></p>
</td>
</tr>
<tr>
<td id="bowtie2-options-qseq">
<pre><code>--qseq</code></pre>
</td>
<td>
<p>Reads (specified with <code><m1></code>,
<code><m2></code>, <code><s></code>) are QSEQ files. QSEQ
files usually end in <code>_qseq.txt</code>. See also: <a
href="#bowtie2-options-solexa-quals"><code>--solexa-quals</code></a> and
<a href="#bowtie2-options-int-quals"><code>--int-quals</code></a>.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-f">
<pre><code>-f</code></pre>
</td>
<td>
<p>Reads (specified with <code><m1></code>,
<code><m2></code>, <code><s></code>) are <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a> files.
<a href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a>
files usually have extension <code>.fa</code>, <code>.fasta</code>,
<code>.mfa</code>, <code>.fna</code> or similar. <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a> files
do not have a way of specifying quality values, so when <code>-f</code>
is set, the result is as if <a
href="#bowtie2-options-ignore-quals"><code>--ignore-quals</code></a> is
also set.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-r">
<pre><code>-r</code></pre>
</td>
<td>
<p>Reads (specified with <code><m1></code>,
<code><m2></code>, <code><s></code>) are files with one
input sequence per line, without any other information (no read names,
no qualities). When <code>-r</code> is set, the result is as if <a
href="#bowtie2-options-ignore-quals"><code>--ignore-quals</code></a> is
also set.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-F">
<pre><code>-F k:<int>,i:<int></code></pre>
</td>
<td>
Reads are substrings (k-mers) extracted from a FASTA file. Specifically,
for every reference sequence in the FASTA file, Bowtie 2 aligns the
k-mers at offsets 1, 1+i, 1+2i, ... until reaching the end of the
reference. Each k-mer is aligned as a separate read. Quality values are
set to all Is (40 on Phred scale). Each k-mer (read) is given a name
like <code><sequence>_<offset></code>, where
<code><sequence></code> is the name of the FASTA sequence it was
drawn from and <code><offset></code> is its 0-based offset of
origin with respect to the sequence. Only single k-mers, i.e. unpaired
reads, can be aligned in this way.
</td>
</tr>
<tr>
<td id="bowtie2-options-c">
<pre><code>-c</code></pre>
</td>
<td>
<p>The read sequences are given on command line. I.e.
<code><m1></code>, <code><m2></code> and
<code><singles></code> are comma-separated lists of reads rather
than lists of read files. There is no way to specify read names or
qualities, so <code>-c</code> also implies <a
href="#bowtie2-options-ignore-quals"><code>--ignore-quals</code></a>.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-s">
<pre><code>-s/--skip <int></code></pre>
</td>
<td>
<p>Skip (i.e. do not align) the first <code><int></code> reads or
pairs in the input.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-u">
<pre><code>-u/--qupto <int></code></pre>
</td>
<td>
<p>Align the first <code><int></code> reads or read pairs from the
input (after the <a
href="#bowtie2-options-s"><code>-s</code>/<code>--skip</code></a> reads
or pairs have been skipped), then stop. Default: no limit.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-5">
<pre><code>-5/--trim5 <int></code></pre>
</td>
<td>
<p>Trim <code><int></code> bases from 5' (left) end of each read
before alignment (default: 0).</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-3">
<pre><code>-3/--trim3 <int></code></pre>
</td>
<td>
<p>Trim <code><int></code> bases from 3' (right) end of each read
before alignment (default: 0).</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-trim-to">
<pre><code>--trim-to [3:|5:]<int></code></pre>
</td>
<td>
<p>Trim reads exceeding <code><int></code> bases. Bases will be
trimmed from either the 3' (right) or 5' (left) end of the read. If the
read end if not specified, bowtie 2 will default to trimming from the 3'
(right) end of the read. <a
href="#bowtie2-options-trim-to"><code>--trim-to</code></a> and <a
href="#bowtie2-options-3"><code>-3</code></a>/<a
href="#bowtie2-options-5"><code>-5</code></a> are mutually
exclusive.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-phred33-quals">
<pre><code>--phred33</code></pre>
</td>
<td>
<p>Input qualities are ASCII chars equal to the <a
href="http://en.wikipedia.org/wiki/Phred_quality_score">Phred
quality</a> plus 33. This is also called the "Phred+33" encoding, which
is used by the very latest Illumina pipelines.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-phred64-quals">
<pre><code>--phred64</code></pre>
</td>
<td>
<p>Input qualities are ASCII chars equal to the <a
href="http://en.wikipedia.org/wiki/Phred_quality_score">Phred
quality</a> plus 64. This is also called the "Phred+64" encoding.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-solexa-quals">
<pre><code>--solexa-quals</code></pre>
</td>
<td>
<p>Convert input qualities from <a
href="http://en.wikipedia.org/wiki/Phred_quality_score">Solexa</a>
(which can be negative) to <a
href="http://en.wikipedia.org/wiki/Phred_quality_score">Phred</a> (which
can't). This scheme was used in older Illumina GA Pipeline versions
(prior to 1.3). Default: off.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-int-quals">
<pre><code>--int-quals</code></pre>
</td>
<td>
<p>Quality values are represented in the read input file as
space-separated ASCII integers, e.g., <code>40 40 30 40</code>...,
rather than ASCII characters, e.g., <code>II?I</code>.... Integers are
treated as being on the <a
href="http://en.wikipedia.org/wiki/Phred_quality_score">Phred
quality</a> scale unless <a
href="#bowtie2-options-solexa-quals"><code>--solexa-quals</code></a> is
also specified. Default: off.</p>
</td>
</tr>
</table>
<h4 id="preset-options-in---end-to-end-mode">Preset options in
<code>--end-to-end</code> mode</h4>
<table>
<tr>
<td id="bowtie2-options-very-fast">
<pre><code>--very-fast</code></pre>
</td>
<td>
<p>Same as: <code>-D 5 -R 1 -N 0 -L 22 -i S,0,2.50</code></p>
</td>
</tr>
<tr>
<td id="bowtie2-options-fast">
<pre><code>--fast</code></pre>
</td>
<td>
<p>Same as: <code>-D 10 -R 2 -N 0 -L 22 -i S,0,2.50</code></p>
</td>
</tr>
<tr>
<td id="bowtie2-options-sensitive">
<pre><code>--sensitive</code></pre>
</td>
<td>
<p>Same as: <code>-D 15 -R 2 -N 0 -L 22 -i S,1,1.15</code> (default in
<a href="#bowtie2-options-end-to-end"><code>--end-to-end</code></a>
mode)</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-very-sensitive">
<pre><code>--very-sensitive</code></pre>
</td>
<td>
<p>Same as: <code>-D 20 -R 3 -N 0 -L 20 -i S,1,0.50</code></p>
</td>
</tr>
</table>
<h4 id="preset-options-in---local-mode">Preset options in
<code>--local</code> mode</h4>
<table>
<tr>
<td id="bowtie2-options-very-fast-local">
<pre><code>--very-fast-local</code></pre>
</td>
<td>
<p>Same as: <code>-D 5 -R 1 -N 0 -L 25 -i S,1,2.00</code></p>
</td>
</tr>
<tr>
<td id="bowtie2-options-fast-local">
<pre><code>--fast-local</code></pre>
</td>
<td>
<p>Same as: <code>-D 10 -R 2 -N 0 -L 22 -i S,1,1.75</code></p>
</td>
</tr>
<tr>
<td id="bowtie2-options-sensitive-local">
<pre><code>--sensitive-local</code></pre>
</td>
<td>
<p>Same as: <code>-D 15 -R 2 -N 0 -L 20 -i S,1,0.75</code> (default in
<a href="#bowtie2-options-local"><code>--local</code></a> mode)</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-very-sensitive-local">
<pre><code>--very-sensitive-local</code></pre>
</td>
<td>
<p>Same as: <code>-D 20 -R 3 -N 0 -L 20 -i S,1,0.50</code></p>
</td>
</tr>
</table>
<h4 id="alignment-options">Alignment options</h4>
<table>
<tr>
<td id="bowtie2-options-N">
<pre><code>-N <int></code></pre>
</td>
<td>
<p>Sets the number of mismatches to allowed in a seed alignment during
<a href="#multiseed-heuristic">multiseed alignment</a>. Can be set to 0
or 1. Setting this higher makes alignment slower (often much slower) but
increases sensitivity. Default: 0.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-L">
<pre><code>-L <int></code></pre>
</td>
<td>
<p>Sets the length of the seed substrings to align during <a
href="#multiseed-heuristic">multiseed alignment</a>. Smaller values make
alignment slower but more sensitive. Default: the <a
href="#bowtie2-options-sensitive"><code>--sensitive</code></a> preset is
used by default, which sets <code>-L</code> to 22 and 20 in <a
href="#bowtie2-options-end-to-end"><code>--end-to-end</code></a> mode
and in <a href="#bowtie2-options-local"><code>--local</code></a>
mode.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-i">
<pre><code>-i <func></code></pre>
</td>
<td>
<p>Sets a function governing the interval between seed substrings to use
during <a href="#multiseed-heuristic">multiseed alignment</a>. For
instance, if the read has 30 characters, and seed length is 10, and the
seed interval is 6, the seeds extracted will be:</p>
<pre><code>Read: TAGCTACGCTCTACGCTATCATGCATAAAC
Seed 1 fw: TAGCTACGCT
Seed 1 rc: AGCGTAGCTA
Seed 2 fw: CGCTCTACGC
Seed 2 rc: GCGTAGAGCG
Seed 3 fw: ACGCTATCAT
Seed 3 rc: ATGATAGCGT
Seed 4 fw: TCATGCATAA
Seed 4 rc: TTATGCATGA</code></pre>
<p>Since it's best to use longer intervals for longer reads, this
parameter sets the interval as a function of the read length, rather
than a single one-size-fits-all number. For instance, specifying
<code>-i S,1,2.5</code> sets the interval function <code>f</code> to
<code>f(x) = 1 + 2.5 * sqrt(x)</code>, where x is the read length. See
also: <a href="#setting-function-options">setting function options</a>.
If the function returns a result less than 1, it is rounded up to 1.
Default: the <a
href="#bowtie2-options-sensitive"><code>--sensitive</code></a> preset is
used by default, which sets <code>-i</code> to <code>S,1,1.15</code> in
<a href="#bowtie2-options-end-to-end"><code>--end-to-end</code></a> mode
to <code>-i S,1,0.75</code> in <a
href="#bowtie2-options-local"><code>--local</code></a> mode.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-n-ceil">
<pre><code>--n-ceil <func></code></pre>
</td>
<td>
<p>Sets a function governing the maximum number of ambiguous characters
(usually <code>N</code>s and/or <code>.</code>s) allowed in a read as a
function of read length. For instance, specifying <code>-L,0,0.15</code>
sets the N-ceiling function <code>f</code> to
<code>f(x) = 0 + 0.15 * x</code>, where x is the read length. See also:
<a href="#setting-function-options">setting function options</a>. Reads
exceeding this ceiling are <a href="#filtering">filtered out</a>.
Default: <code>L,0,0.15</code>.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-dpad">
<pre><code>--dpad <int></code></pre>
</td>
<td>
<p>"Pads" dynamic programming problems by <code><int></code>
columns on either side to allow gaps. Default: 15.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-gbar">
<pre><code>--gbar <int></code></pre>
</td>
<td>
<p>Disallow gaps within <code><int></code> positions of the
beginning or end of the read. Default: 4.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-ignore-quals">
<pre><code>--ignore-quals</code></pre>
</td>
<td>
<p>When calculating a mismatch penalty, always consider the quality
value at the mismatched position to be the highest possible, regardless
of the actual value. I.e. input is treated as though all quality values
are high. This is also the default behavior when the input doesn't
specify quality values (e.g. in <a
href="#bowtie2-options-f"><code>-f</code></a>, <a
href="#bowtie2-options-r"><code>-r</code></a>, or <a
href="#bowtie2-options-c"><code>-c</code></a> modes).</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-nofw">
<pre><code>--nofw/--norc</code></pre>
</td>
<td>
<p>If <code>--nofw</code> is specified, <code>bowtie2</code> will not
attempt to align unpaired reads to the forward (Watson) reference
strand. If <code>--norc</code> is specified, <code>bowtie2</code> will
not attempt to align unpaired reads against the reverse-complement
(Crick) reference strand. In paired-end mode, <code>--nofw</code> and
<code>--norc</code> pertain to the fragments; i.e. specifying
<code>--nofw</code> causes <code>bowtie2</code> to explore only those
paired-end configurations corresponding to fragments from the
reverse-complement (Crick) strand. Default: both strands enabled.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-no-1mm-upfront">
<pre><code>--no-1mm-upfront</code></pre>
</td>
<td>
<p>By default, Bowtie 2 will attempt to find either an exact or a
1-mismatch end-to-end alignment for the read <em>before</em> trying the
<a href="#multiseed-heuristic">multiseed heuristic</a>. Such alignments
can be found very quickly, and many short read alignments have exact or
near-exact end-to-end alignments. However, this can lead to unexpected
alignments when the user also sets options governing the <a
href="#multiseed-heuristic">multiseed heuristic</a>, like <a
href="#bowtie2-options-L"><code>-L</code></a> and <a
href="#bowtie2-options-N"><code>-N</code></a>. For instance, if the user
specifies <code>-N 0</code> and <code>-L</code> equal to the length of
the read, the user will be surprised to find 1-mismatch alignments
reported. This option prevents Bowtie 2 from searching for 1-mismatch
end-to-end alignments before using the <a
href="#multiseed-heuristic">multiseed heuristic</a>, which leads to the
expected behavior when combined with options such as <a
href="#bowtie2-options-L"><code>-L</code></a> and <a
href="#bowtie2-options-N"><code>-N</code></a>. This comes at the expense
of speed.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-end-to-end">
<pre><code>--end-to-end</code></pre>
</td>
<td>
<p>In this mode, Bowtie 2 requires that the entire read align from one
end to the other, without any trimming (or "soft clipping") of
characters from either end. The match bonus <a
href="#bowtie2-options-ma"><code>--ma</code></a> always equals 0 in this
mode, so all alignment scores are less than or equal to 0, and the
greatest possible alignment score is 0. This is mutually exclusive with
<a href="#bowtie2-options-local"><code>--local</code></a>.
<code>--end-to-end</code> is the default mode.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-local">
<pre><code>--local</code></pre>
</td>
<td>
<p>In this mode, Bowtie 2 does not require that the entire read align
from one end to the other. Rather, some characters may be omitted ("soft
clipped") from the ends in order to achieve the greatest possible
alignment score. The match bonus <a
href="#bowtie2-options-ma"><code>--ma</code></a> is used in this mode,
and the best possible alignment score is equal to the match bonus (<a
href="#bowtie2-options-ma"><code>--ma</code></a>) times the length of
the read. Specifying <code>--local</code> and one of the presets (e.g.
<code>--local --very-fast</code>) is equivalent to specifying the local
version of the preset (<code>--very-fast-local</code>). This is mutually
exclusive with <a
href="#bowtie2-options-end-to-end"><code>--end-to-end</code></a>.
<code>--end-to-end</code> is the default mode.</p>
</td>
</tr>
</table>
<h4 id="scoring-options">Scoring options</h4>
<table>
<tr>
<td id="bowtie2-options-ma">
<pre><code>--ma <int></code></pre>
</td>
<td>
<p>Sets the match bonus. In <a
href="#bowtie2-options-local"><code>--local</code></a> mode
<code><int></code> is added to the alignment score for each
position where a read character aligns to a reference character and the
characters match. Not used in <a
href="#bowtie2-options-end-to-end"><code>--end-to-end</code></a> mode.
Default: 2.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-mp">
<pre><code>--mp MX,MN</code></pre>
</td>
<td>
<p>Sets the maximum (<code>MX</code>) and minimum (<code>MN</code>)
mismatch penalties, both integers. A number less than or equal to
<code>MX</code> and greater than or equal to <code>MN</code> is
subtracted from the alignment score for each position where a read
character aligns to a reference character, the characters do not match,
and neither is an <code>N</code>. If <a
href="#bowtie2-options-ignore-quals"><code>--ignore-quals</code></a> is
specified, the number subtracted quals <code>MX</code>. Otherwise, the
number subtracted is
<code>MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) )</code> where Q is the
Phred quality value. Default: <code>MX</code> = 6, <code>MN</code> =
2.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-np">
<pre><code>--np <int></code></pre>
</td>
<td>
<p>Sets penalty for positions where the read, reference, or both,
contain an ambiguous character such as <code>N</code>. Default: 1.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-rdg">
<pre><code>--rdg <int1>,<int2></code></pre>
</td>
<td>
<p>Sets the read gap open (<code><int1></code>) and extend
(<code><int2></code>) penalties. A read gap of length N gets a
penalty of <code><int1></code> + N * <code><int2></code>.
Default: 5, 3.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-rfg">
<pre><code>--rfg <int1>,<int2></code></pre>
</td>
<td>
<p>Sets the reference gap open (<code><int1></code>) and extend
(<code><int2></code>) penalties. A reference gap of length N gets
a penalty of <code><int1></code> + N * <code><int2></code>.
Default: 5, 3.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-score-min">
<pre><code>--score-min <func></code></pre>
</td>
<td>
<p>Sets a function governing the minimum alignment score needed for an
alignment to be considered "valid" (i.e. good enough to report). This is
a function of read length. For instance, specifying
<code>L,0,-0.6</code> sets the minimum-score function <code>f</code> to
<code>f(x) = 0 + -0.6 * x</code>, where <code>x</code> is the read
length. See also: <a href="#setting-function-options">setting function
options</a>. The default in <a
href="#bowtie2-options-end-to-end"><code>--end-to-end</code></a> mode is
<code>L,-0.6,-0.6</code> and the default in <a
href="#bowtie2-options-local"><code>--local</code></a> mode is
<code>G,20,8</code>.</p>
</td>
</tr>
</table>
<h4 id="reporting-options">Reporting options</h4>
<table>
<tr>
<td id="bowtie2-options-k">
<pre><code>-k <int></code></pre>
</td>
<td>
<p>By default, <code>bowtie2</code> searches for distinct, valid
alignments for each read. When it finds a valid alignment, it continues
looking for alignments that are nearly as good or better. The best
alignment found is reported (randomly selected from among best if tied).
Information about the best alignments is used to estimate mapping
quality and to set SAM optional fields, such as <a
href="#bowtie2-build-opt-fields-as"><code>AS:i</code></a> and <a
href="#bowtie2-build-opt-fields-xs"><code>XS:i</code></a>.</p>
<p>When <code>-k</code> is specified, however, <code>bowtie2</code>
behaves differently. Instead, it searches for at most
<code><int></code> distinct, valid alignments for each read. The
search terminates when it can't find more distinct valid alignments, or
when it finds <code><int></code>, whichever happens first. All
alignments found are reported in descending order by alignment score.
The alignment score for a paired-end alignment equals the sum of the
alignment scores of the individual mates. Each reported read or pair
alignment beyond the first has the SAM 'secondary' bit (which equals
256) set in its FLAGS field. For reads that have more than
<code><int></code> distinct, valid alignments,
<code>bowtie2</code> does not guarantee that the
<code><int></code> alignments reported are the best possible in
terms of alignment score. <code>-k</code> is mutually exclusive with <a
href="#bowtie2-options-a"><code>-a</code></a>.</p>
<p>Note: Bowtie 2 is not designed with large values for <code>-k</code>
in mind, and when aligning reads to long, repetitive genomes large
<code>-k</code> can be very, very slow.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-a">
<pre><code>-a</code></pre>
</td>
<td>
<p>Like <a href="#bowtie2-options-k"><code>-k</code></a> but with no
upper limit on number of alignments to search for. <code>-a</code> is
mutually exclusive with <a
href="#bowtie2-options-k"><code>-k</code></a>.</p>
<p>Note: Bowtie 2 is not designed with <code>-a</code> mode in mind, and
when aligning reads to long, repetitive genomes this mode can be very,
very slow.</p>
</td>
</tr>
</table>
<h4 id="effort-options">Effort options</h4>
<table>
<tr>
<td id="bowtie2-options-D">
<pre><code>-D <int></code></pre>
</td>
<td>
<p>Up to <code><int></code> consecutive seed extension attempts
can "fail" before Bowtie 2 moves on, using the alignments found so far.
A seed extension "fails" if it does not yield a new best or a new
second-best alignment. This limit is automatically adjusted up when -k
or -a are specified. Default: 15.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-R">
<pre><code>-R <int></code></pre>
</td>
<td>
<p><code><int></code> is the maximum number of times Bowtie 2 will
"re-seed" reads with repetitive seeds. When "re-seeding," Bowtie 2
simply chooses a new set of reads (same length, same number of
mismatches allowed) at different offsets and searches for more
alignments. A read is considered to have repetitive seeds if the total
number of seed hits divided by the number of seeds that aligned at least
once is greater than 300. Default: 2.</p>
</td>
</tr>
</table>
<h4 id="paired-end-options">Paired-end options</h4>
<table>
<tr>
<td id="bowtie2-options-I">
<pre><code>-I/--minins <int></code></pre>
</td>
<td>
<p>The minimum fragment length for valid paired-end alignments. E.g. if
<code>-I 60</code> 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 <a
href="#bowtie2-options-X"><code>-X</code></a> is also satisfied). A
19-bp gap would not be valid in that case. If trimming options <a
href="#bowtie2-options-3"><code>-3</code></a> or <a
href="#bowtie2-options-5"><code>-5</code></a> are also used, the <a
href="#bowtie2-options-I"><code>-I</code></a> constraint is applied with
respect to the untrimmed mates.</p>
<p>The larger the difference between <a
href="#bowtie2-options-I"><code>-I</code></a> and <a
href="#bowtie2-options-X"><code>-X</code></a>, the slower Bowtie 2 will
run. This is because larger differences between <a
href="#bowtie2-options-I"><code>-I</code></a> and <a
href="#bowtie2-options-X"><code>-X</code></a> require that Bowtie 2 scan
a larger window to determine if a concordant alignment exists. For
typical fragment length ranges (200 to 400 nucleotides), Bowtie 2 is
very efficient.</p>
<p>Default: 0 (essentially imposing no minimum)</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-X">
<pre><code>-X/--maxins <int></code></pre>
</td>
<td>
<p>The maximum fragment length for valid paired-end alignments. E.g. if
<code>-X 100</code> 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 <a
href="#bowtie2-options-I"><code>-I</code></a> is also satisfied). A
61-bp gap would not be valid in that case. If trimming options <a
href="#bowtie2-options-3"><code>-3</code></a> or <a
href="#bowtie2-options-5"><code>-5</code></a> are also used, the
<code>-X</code> constraint is applied with respect to the untrimmed
mates, not the trimmed mates.</p>
<p>The larger the difference between <a
href="#bowtie2-options-I"><code>-I</code></a> and <a
href="#bowtie2-options-X"><code>-X</code></a>, the slower Bowtie 2 will
run. This is because larger differences between <a
href="#bowtie2-options-I"><code>-I</code></a> and <a
href="#bowtie2-options-X"><code>-X</code></a> require that Bowtie 2 scan
a larger window to determine if a concordant alignment exists. For
typical fragment length ranges (200 to 400 nucleotides), Bowtie 2 is
very efficient.</p>
<p>Default: 500.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-fr">
<pre><code>--fr/--rf/--ff</code></pre>
</td>
<td>
<p>The upstream/downstream mate orientations for a valid paired-end
alignment against the forward reference strand. E.g., if
<code>--fr</code> is specified and there is a candidate paired-end
alignment where mate 1 appears upstream of the reverse complement of
mate 2 and the fragment length constraints (<a
href="#bowtie2-options-I"><code>-I</code></a> and <a
href="#bowtie2-options-X"><code>-X</code></a>) are met, that alignment
is valid. Also, if mate 2 appears upstream of the reverse complement of
mate 1 and all other constraints are met, that too is valid.
<code>--rf</code> likewise requires that an upstream mate1 be
reverse-complemented and a downstream mate2 be forward-oriented.
<code>--ff</code> requires both an upstream mate 1 and a downstream mate
2 to be forward-oriented. Default: <code>--fr</code> (appropriate for
Illumina's Paired-end Sequencing Assay).</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-no-mixed">
<pre><code>--no-mixed</code></pre>
</td>
<td>
<p>By default, when <code>bowtie2</code> cannot find a concordant or
discordant alignment for a pair, it then tries to find alignments for
the individual mates. This option disables that behavior.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-no-discordant">
<pre><code>--no-discordant</code></pre>
</td>
<td>
<p>By default, <code>bowtie2</code> looks for discordant alignments if
it cannot find any concordant alignments. A discordant alignment is an
alignment where both mates align uniquely, but that does not satisfy the
paired-end constraints (<a
href="#bowtie2-options-fr"><code>--fr</code>/<code>--rf</code>/<code>--ff</code></a>,
<a href="#bowtie2-options-I"><code>-I</code></a>, <a
href="#bowtie2-options-X"><code>-X</code></a>). This option disables
that behavior.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-dovetail">
<pre><code>--dovetail</code></pre>
</td>
<td>
<p>If the mates "dovetail", that is if one mate alignment extends past
the beginning of the other such that the wrong mate begins upstream,
consider that to be concordant. See also: <a
href="#mates-can-overlap-contain-or-dovetail-each-other">Mates can
overlap, contain or dovetail each other</a>. Default: mates cannot
dovetail in a concordant alignment.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-no-contain">
<pre><code>--no-contain</code></pre>
</td>
<td>
<p>If one mate alignment contains the other, consider that to be
non-concordant. See also: <a
href="#mates-can-overlap-contain-or-dovetail-each-other">Mates can
overlap, contain or dovetail each other</a>. Default: a mate can contain
the other in a concordant alignment.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-no-overlap">
<pre><code>--no-overlap</code></pre>
</td>
<td>
<p>If one mate alignment overlaps the other at all, consider that to be
non-concordant. See also: <a
href="#mates-can-overlap-contain-or-dovetail-each-other">Mates can
overlap, contain or dovetail each other</a>. Default: mates can overlap
in a concordant alignment.</p>
</td>
</tr>
</table>
<h4 id="bam-options">BAM options</h4>
<table>
<tr>
<td id="bowtie2-options-align-paired-reads">
<pre><code>--align-paired-reads</code></pre>
</td>
<td>
<p>Bowtie 2 will, by default, attempt to align unpaired BAM reads. Use
this option to align paired-end reads instead.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-preserve-tags">
<pre><code>--preserve-tags</code></pre>
</td>
<td>
<p>Preserve tags from the original BAM record by appending them to the
end of the corresponding Bowtie 2 SAM output.</p>
</td>
</tr>
</table>
<h4 id="output-options">Output options</h4>
<table>
<tr>
<td id="bowtie2-options-t">
<pre><code>-t/--time</code></pre>
</td>
<td>
<p>Print the wall-clock time required to load the index files and align
the reads. This is printed to the "standard error" ("stderr")
filehandle. Default: off.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-un">
<pre><code>--un <path>
--un-gz <path>
--un-bz2 <path>
--un-lz4 <path></code></pre>
</td>
<td>
<p>Write unpaired reads that fail to align to file at
<code><path></code>. These reads correspond to the SAM records
with the FLAGS <code>0x4</code> bit set and neither the
<code>0x40</code> nor <code>0x80</code> bits set. If
<code>--un-gz</code> is specified, output will be gzip compressed. If
<code>--un-bz2</code> or <code>--un-lz4</code> is specified, output will
be bzip2 or lz4 compressed. Reads written in this way will appear
exactly as they did in the input file, without any modification (same
sequence, same name, same quality string, same quality encoding). Reads
will not necessarily appear in the same order as they did in the
input.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-al">
<pre><code>--al <path>
--al-gz <path>
--al-bz2 <path>
--al-lz4 <path></code></pre>
</td>
<td>
<p>Write unpaired reads that align at least once to file at
<code><path></code>. These reads correspond to the SAM records
with the FLAGS <code>0x4</code>, <code>0x40</code>, and
<code>0x80</code> bits unset. If <code>--al-gz</code> is specified,
output will be gzip compressed. If <code>--al-bz2</code> is specified,
output will be bzip2 compressed. Similarly if <code>--al-lz4</code> is
specified, output will be lz4 compressed. Reads written in this way will
appear exactly as they did in the input file, without any modification
(same sequence, same name, same quality string, same quality encoding).
Reads will not necessarily appear in the same order as they did in the
input.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-un-conc">
<pre><code>--un-conc <path>
--un-conc-gz <path>
--un-conc-bz2 <path>
--un-conc-lz4 <path></code></pre>
</td>
<td>
<p>Write paired-end reads that fail to align concordantly to file(s) at
<code><path></code>. These reads correspond to the SAM records
with the FLAGS <code>0x4</code> bit set and either the <code>0x40</code>
or <code>0x80</code> bit set (depending on whether it's mate #1 or #2).
<code>.1</code> and <code>.2</code> strings are added to the filename to
distinguish which file contains mate #1 and mate #2. If a percent
symbol, <code>%</code>, is used in <code><path></code>, the
percent symbol is replaced with <code>1</code> or <code>2</code> to make
the per-mate filenames. Otherwise, <code>.1</code> or <code>.2</code>
are added before the final dot in <code><path></code> to make the
per-mate filenames. Reads written in this way will appear exactly as
they did in the input files, without any modification (same sequence,
same name, same quality string, same quality encoding). Reads will not
necessarily appear in the same order as they did in the inputs.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-al-conc">
<pre><code>--al-conc <path>
--al-conc-gz <path>
--al-conc-bz2 <path>
--al-conc-lz4 <path></code></pre>
</td>
<td>
<p>Write paired-end reads that align concordantly at least once to
file(s) at <code><path></code>. These reads correspond to the SAM
records with the FLAGS <code>0x4</code> bit unset and either the
<code>0x40</code> or <code>0x80</code> bit set (depending on whether
it's mate #1 or #2). <code>.1</code> and <code>.2</code> strings are
added to the filename to distinguish which file contains mate #1 and
mate #2. If a percent symbol, <code>%</code>, is used in
<code><path></code>, the percent symbol is replaced with
<code>1</code> or <code>2</code> to make the per-mate filenames.
Otherwise, <code>.1</code> or <code>.2</code> are added before the final
dot in <code><path></code> to make the per-mate filenames. Reads
written in this way will appear exactly as they did in the input files,
without any modification (same sequence, same name, same quality string,
same quality encoding). Reads will not necessarily appear in the same
order as they did in the inputs.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-quiet">
<pre><code>--quiet</code></pre>
</td>
<td>
<p>Print nothing besides alignments and serious errors.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-met-file">
<pre><code>--met-file <path></code></pre>
</td>
<td>
<p>Write <code>bowtie2</code> metrics to file <code><path></code>.
Having alignment metric can be useful for debugging certain problems,
especially performance issues. See also: <a
href="#bowtie2-options-met"><code>--met</code></a>. Default: metrics
disabled.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-met-stderr">
<pre><code>--met-stderr <path></code></pre>
</td>
<td>
<p>Write <code>bowtie2</code> metrics to the "standard error" ("stderr")
filehandle. This is not mutually exclusive with <a
href="#bowtie2-options-met-file"><code>--met-file</code></a>. Having
alignment metric can be useful for debugging certain problems,
especially performance issues. See also: <a
href="#bowtie2-options-met"><code>--met</code></a>. Default: metrics
disabled.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-met">
<pre><code>--met <int></code></pre>
</td>
<td>
<p>Write a new <code>bowtie2</code> metrics record every
<code><int></code> seconds. Only matters if either <a
href="#bowtie2-options-met-stderr"><code>--met-stderr</code></a> or <a
href="#bowtie2-options-met-file"><code>--met-file</code></a> are
specified. Default: 1.</p>
</td>
</tr>
</table>
<h4 id="sam-options">SAM options</h4>
<table>
<tr>
<td id="bowtie2-options-no-unal">
<pre><code>--no-unal</code></pre>
</td>
<td>
<p>Suppress SAM records for reads that failed to align.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-no-hd">
<pre><code>--no-hd</code></pre>
</td>
<td>
<p>Suppress SAM header lines (starting with <code>@</code>).</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-no-sq">
<pre><code>--no-sq</code></pre>
</td>
<td>
<p>Suppress <code>@SQ</code> SAM header lines.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-rg-id">
<pre><code>--rg-id <text></code></pre>
</td>
<td>
<p>Set the read group ID to <code><text></code>. This causes the
SAM <code>@RG</code> header line to be printed, with
<code><text></code> as the value associated with the
<code>ID:</code> tag. It also causes the <code>RG:Z:</code> extra field
to be attached to each SAM output record, with value set to
<code><text></code>.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-rg">
<pre><code>--rg <text></code></pre>
</td>
<td>
<p>Add <code><text></code> (usually of the form
<code>TAG:VAL</code>, e.g. <code>SM:Pool1</code>) as a field on the
<code>@RG</code> header line. Note: in order for the <code>@RG</code>
line to appear, <a
href="#bowtie2-options-rg-id"><code>--rg-id</code></a> must also be
specified. This is because the <code>ID</code> tag is required by the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM Spec</a>. Specify
<code>--rg</code> multiple times to set multiple fields. See the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM Spec</a> for details
about what fields are legal.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-omit-sec-seq">
<pre><code>--omit-sec-seq</code></pre>
</td>
<td>
<p>When printing secondary alignments, Bowtie 2 by default will write
out the <code>SEQ</code> and <code>QUAL</code> strings. Specifying this
option causes Bowtie 2 to print an asterisk in those fields instead.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-soft-clipped-unmapped-tlen">
<pre><code>--soft-clipped-unmapped-tlen</code></pre>
</td>
<td>
<p>Consider soft-clipped bases unmapped when calculating
<code>TLEN</code>. Only available in <a
href="#bowtie2-options-local"><code>--local</code></a> mode.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-sam-no-qname-trunc">
<pre><code>--sam-no-qname-trunc</code></pre>
</td>
<td>
<p>Suppress standard behavior of truncating readname at first whitespace
at the expense of generating non-standard SAM</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-xeq">
<pre><code>--xeq</code></pre>
</td>
<td>
<p>Use <code>'='/'X'</code>, instead of <code>'M'</code>, to specify
matches/mismatches in SAM record</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-sam-append-comment">
<pre><code>--sam-append-comment</code></pre>
</td>
<td>
<p>Append FASTA/FASTQ comment to SAM record, where a comment is
everything after the first space in the read name.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-sam-opt-config">
<pre><code>--sam-opt-config <config></code></pre>
</td>
<td>
<p>Use <code><config></code> to toggle SAM Optional Fields where
<code><config></code> is a string of comma delimited,
case-insensitive, two-letter tags. Tags prefixed with a "-" will be
turned off and hence will not be included in the SAM output. The example
below turns off the "MD" tag and enables the
<code>bowtie2</code>-specific "YP" tag. The config is additive, so, any
default OPT flags that need to be turned off will have to explicitly
specified.</p>
<pre><code>`bowtie2 ... --sam-opt-config "-md,yp"</code></pre>
</td>
</tr>
</table>
<h4 id="performance-options">Performance options</h4>
<table>
<tr>
<td id="bowtie2-options-o">
<pre><code>-o/--offrate <int></code></pre>
</td>
<td>
<p>Override the offrate of the index with <code><int></code>. If
<code><int></code> 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. <code><int></code> must be
greater than the value used to build the index.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-p">
<pre><code>-p/--threads NTHREADS</code></pre>
</td>
<td>
<p>Launch <code>NTHREADS</code> 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 close to linear. Increasing
<code>-p</code> increases Bowtie 2's memory footprint. E.g. when
aligning to a human genome index, increasing <code>-p</code> from 1 to 8
increases the memory footprint by a few hundred megabytes. This option
is only available if <code>bowtie</code> is linked with the
<code>pthreads</code> library (i.e. if <code>BOWTIE_PTHREADS=0</code> is
not specified at build time).</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-reorder">
<pre><code>--reorder</code></pre>
</td>
<td>
<p>Guarantees that output SAM records are printed in an order
corresponding to the order of the reads in the original input file, even
when <a href="#bowtie2-options-p"><code>-p</code></a> is set greater
than 1. Specifying <code>--reorder</code> and setting <a
href="#bowtie2-options-p"><code>-p</code></a> greater than 1 causes
Bowtie 2 to run somewhat slower and use somewhat more memory than if
<code>--reorder</code> were not specified. Has no effect if <a
href="#bowtie2-options-p"><code>-p</code></a> is set to 1, since output
order will naturally correspond to input order in that case.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-mm">
<pre><code>--mm</code></pre>
</td>
<td>
<p>Use memory-mapped I/O to load the index, rather than typical file
I/O. Memory-mapping allows many concurrent <code>bowtie</code> 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 <code>bowtie</code> in situations
where using <a href="#bowtie2-options-p"><code>-p</code></a> is not
possible or not preferable.</p>
</td>
</tr>
</table>
<h4 id="other-options">Other options</h4>
<table>
<tr>
<td id="bowtie2-options-qc-filter">
<pre><code>--qc-filter</code></pre>
</td>
<td>
<p>Filter out reads for which the QSEQ filter field is non-zero. Only
has an effect when read format is <a
href="#bowtie2-options-qseq"><code>--qseq</code></a>. Default: off.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-seed">
<pre><code>--seed <int></code></pre>
</td>
<td>
<p>Use <code><int></code> as the seed for pseudo-random number
generator. Default: 0.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-non-deterministic">
<pre><code>--non-deterministic</code></pre>
</td>
<td>
<p>Normally, Bowtie 2 re-initializes its pseudo-random generator for
each read. It seeds the generator with a number derived from (a) the
read name, (b) the nucleotide sequence, (c) the quality sequence, (d)
the value of the <a href="#bowtie2-options-seed"><code>--seed</code></a>
option. This means that if two reads are identical (same name, same
nucleotides, same qualities) Bowtie 2 will find and report the same
alignment(s) for both, even if there was ambiguity. When
<code>--non-deterministic</code> is specified, Bowtie 2 re-initializes
its pseudo-random generator for each read using the current time. This
means that Bowtie 2 will not necessarily report the same alignment for
two identical reads. This is counter-intuitive for some users, but might
be more appropriate in situations where the input consists of many
identical reads.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-version">
<pre><code>--version</code></pre>
</td>
<td>
<p>Print version information and quit.</p>
</td>
</tr>
<tr>
<td id="bowtie2-options-h">
<pre><code>-h/--help</code></pre>
</td>
<td>
<p>Print usage information and quit.</p>
</td>
</tr>
</table>
<h2 id="sam-output">SAM output</h2>
<p>Following is a brief description of the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM</a> format as output
by <code>bowtie2</code>. For more details, see the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM format
specification</a>.</p>
<p>By default, <code>bowtie2</code> prints a SAM header with
<code>@HD</code>, <code>@SQ</code> and <code>@PG</code> lines. When one
or more <a href="#bowtie2-options-rg"><code>--rg</code></a> arguments
are specified, <code>bowtie2</code> will also print an <code>@RG</code>
line that includes all user-specified <a
href="#bowtie2-options-rg"><code>--rg</code></a> tokens separated by
tabs.</p>
<p>Each subsequent line describes an alignment or, if the read failed to
align, a read. Each line is a collection of at least 12 fields separated
by tabs; from left to right, the fields are:</p>
<ol type="1">
<li><p>Name of read that aligned.</p>
<p>Note that the <a href="http://samtools.sourceforge.net/SAM1.pdf">SAM
specification</a> 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. The standard behavior of truncating at the first whitespace
can be suppressed with <code>--sam-no-qname-trunc</code> at the expense
of generating non-standard SAM.</p></li>
<li><p>Sum of all applicable flags. Flags relevant to Bowtie are:</p>
<table>
<tr>
<td>
<pre><code>1</code></pre>
</td>
<td>
<p>The read is one of a pair</p>
</td>
</tr>
<tr>
<td>
<pre><code>2</code></pre>
</td>
<td>
<p>The alignment is one end of a proper paired-end alignment</p>
</td>
</tr>
<tr>
<td>
<pre><code>4</code></pre>
</td>
<td>
<p>The read has no reported alignments</p>
</td>
</tr>
<tr>
<td>
<pre><code>8</code></pre>
</td>
<td>
<p>The read is one of a pair and has no reported alignments</p>
</td>
</tr>
<tr>
<td>
<pre><code>16</code></pre>
</td>
<td>
<p>The alignment is to the reverse reference strand</p>
</td>
</tr>
<tr>
<td>
<pre><code>32</code></pre>
</td>
<td>
<p>The other mate in the paired-end alignment is aligned to the reverse
reference strand</p>
</td>
</tr>
<tr>
<td>
<pre><code>64</code></pre>
</td>
<td>
<p>The read is mate 1 in a pair</p>
</td>
</tr>
<tr>
<td>
<pre><code>128</code></pre>
</td>
<td>
<p>The read is mate 2 in a pair</p>
</td>
</tr>
</table>
<p>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).</p></li>
<li><p>Name of reference sequence where alignment occurs</p></li>
<li><p>1-based offset into the forward reference strand where leftmost
character of the alignment occurs</p></li>
<li><p>Mapping quality</p></li>
<li><p>CIGAR string representation of alignment</p></li>
<li><p>Name of reference sequence where mate's alignment occurs. Set to
<code>=</code> if the mate's reference sequence is the same as this
alignment's, or <code>*</code> if there is no mate.</p></li>
<li><p>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.</p></li>
<li><p>Inferred fragment length. Size is negative if the mate's
alignment occurs upstream of this alignment. Size is 0 if the mates did
not align concordantly. However, size is non-0 if the mates aligned
discordantly to the same chromosome.</p></li>
<li><p>Read sequence (reverse-complemented if aligned to the reverse
strand)</p></li>
<li><p>ASCII-encoded read qualities (reverse-complemented if the read
aligned to the reverse strand). The encoded quality values are on the <a
href="http://en.wikipedia.org/wiki/Phred_quality_score">Phred
quality</a> scale and the encoding is ASCII-offset by 33 (ASCII char
<code>!</code>), similarly to a <a
href="http://en.wikipedia.org/wiki/FASTQ_format">FASTQ</a>
file.</p></li>
<li><p>Optional fields. Fields are tab-separated. <code>bowtie2</code>
outputs zero or more of these optional fields for each alignment,
depending on the type of the alignment:</p></li>
</ol>
<table>
<tr>
<td id="bowtie2-build-opt-fields-as">
<pre><code>AS:i:<N></code></pre>
</td>
<td>
<p>Alignment score. Can be negative. Can be greater than 0 in <a
href="#bowtie2-options-local"><code>--local</code></a> mode (but not in
<a href="#bowtie2-options-end-to-end"><code>--end-to-end</code></a>
mode). Only present if SAM record is for an aligned read.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-xs">
<pre><code>XS:i:<N></code></pre>
</td>
<td>
<p>Alignment score for the best-scoring alignment found other than the
alignment reported. Can be negative. Can be greater than 0 in <a
href="#bowtie2-options-local"><code>--local</code></a> mode (but not in
<a href="#bowtie2-options-end-to-end"><code>--end-to-end</code></a>
mode). Only present if the SAM record is for an aligned read and more
than one alignment was found for the read. Note that, when the read is
part of a concordantly-aligned pair, this score could be greater than <a
href="#bowtie2-build-opt-fields-as"><code>AS:i</code></a>.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-ys">
<pre><code>YS:i:<N></code></pre>
</td>
<td>
<p>Alignment score for opposite mate in the paired-end alignment. Only
present if the SAM record is for a read that aligned as part of a
paired-end alignment.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-xn">
<pre><code>XN:i:<N></code></pre>
</td>
<td>
<p>The number of ambiguous bases in the reference covering this
alignment. Only present if SAM record is for an aligned read.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-xm">
<pre><code>XM:i:<N></code></pre>
</td>
<td>
<p>The number of mismatches in the alignment. Only present if SAM record
is for an aligned read.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-xo">
<pre><code>XO:i:<N></code></pre>
</td>
<td>
<p>The number of gap opens, for both read and reference gaps, in the
alignment. Only present if SAM record is for an aligned read.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-xg">
<pre><code>XG:i:<N></code></pre>
</td>
<td>
<p>The number of gap extensions, for both read and reference gaps, in
the alignment. Only present if SAM record is for an aligned read.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-nm">
<pre><code>NM:i:<N></code></pre>
</td>
<td>
<p>The edit distance; that is, the minimal number of one-nucleotide
edits (substitutions, insertions and deletions) needed to transform the
read string into the reference string. Only present if SAM record is for
an aligned read.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-yf">
<pre><code>YF:Z:<S></code></pre>
</td>
<td>
<p>String indicating reason why the read was filtered out. See also: <a
href="#filtering">Filtering</a>. Only appears for reads that were
filtered out.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-yt">
<pre><code>YT:Z:<S></code></pre>
</td>
<td>
<p>Value of <code>UU</code> indicates the read was not part of a pair.
Value of <code>CP</code> indicates the read was part of a pair and the
pair aligned concordantly. Value of <code>DP</code> indicates the read
was part of a pair and the pair aligned discordantly. Value of
<code>UP</code> indicates the read was part of a pair but the pair
failed to aligned either concordantly or discordantly.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-opt-fields-md">
<pre><code>MD:Z:<S></code></pre>
</td>
<td>
<p>A string representation of the mismatched reference bases in the
alignment. See <a
href="https://samtools.github.io/hts-specs/SAMtags.pdf">SAM Tags format
specification</a> for details. Only present if SAM record is for an
aligned read.</p>
</td>
</tr>
</table>
<h1 id="the-bowtie2-build-indexer">The <code>bowtie2-build</code>
indexer</h1>
<p><code>bowtie2-build</code> builds a Bowtie index from a set of DNA
sequences. <code>bowtie2-build</code> outputs a set of 6 files with
suffixes <code>.1.bt2</code>, <code>.2.bt2</code>, <code>.3.bt2</code>,
<code>.4.bt2</code>, <code>.rev.1.bt2</code>, and
<code>.rev.2.bt2</code>. In the case of a large index these suffixes
will have a <code>bt2l</code> termination. These files together
constitute the index: they are all that is needed to align reads to that
reference. The original sequence <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a> files
are no longer used by Bowtie 2 once the index is built.</p>
<p>Bowtie 2's <code>.bt2</code> index format is different from Bowtie
1's <code>.ebwt</code> format, and they are not compatible with each
other.</p>
<p>Use of Karkkainen's <a
href="http://portal.acm.org/citation.cfm?id=1314852">blockwise
algorithm</a> allows <code>bowtie2-build</code> to trade off between
running time and memory usage. <code>bowtie2-build</code> has three
options governing how it makes this trade: <a
href="#bowtie2-build-options-p"><code>-p</code>/<code>--packed</code></a>,
<a href="#bowtie2-build-options-bmax"><code>--bmax</code></a>/<a
href="#bowtie2-build-options-bmaxdivn"><code>--bmaxdivn</code></a>, and
<a href="#bowtie2-build-options-dcv"><code>--dcv</code></a>. By default,
<code>bowtie2-build</code> will automatically search for the settings
that yield the best running time without exhausting memory. This
behavior can be disabled using the <a
href="#bowtie2-build-options-a"><code>-a</code>/<code>--noauto</code></a>
option.</p>
<p>The indexer provides options pertaining to the "shape" of the index,
e.g. <a href="#bowtie2-build-options-o"><code>--offrate</code></a>
governs the fraction of <a
href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform">Burrows-Wheeler</a>
rows that are "marked" (i.e., the density of the suffix-array sample;
see the original <a
href="http://portal.acm.org/citation.cfm?id=796543">FM Index</a> 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 <a
href="#performance-tuning">Performance tuning</a> for details.</p>
<p><code>bowtie2-build</code> can generate either <a
href="#small-and-large-indexes">small or large indexes</a>. The wrapper
will decide which based on the length of the input genome. If the
reference does not exceed 4 billion characters but a large index is
preferred, the user can specify <a
href="#bowtie2-build-options-large-index"><code>--large-index</code></a>
to force <code>bowtie2-build</code> to build a large index instead.</p>
<p>The Bowtie 2 index is based on the <a
href="http://portal.acm.org/citation.cfm?id=796543">FM Index</a> of
Ferragina and Manzini, which in turn is based on the <a
href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform">Burrows-Wheeler</a>
transform. The algorithm used to build the index is based on the <a
href="http://portal.acm.org/citation.cfm?id=1314852">blockwise
algorithm</a> of Karkkainen.</p>
<h2 id="command-line-1">Command Line</h2>
<p>Usage:</p>
<pre><code>bowtie2-build [options]* <reference_in> <bt2_base></code></pre>
<h3 id="main-arguments-1">Main arguments</h3>
<table>
<tr>
<td>
<pre><code><reference_in></code></pre>
</td>
<td>
<p>A comma-separated list of <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a> files
containing the reference sequences to be aligned to, or, if <a
href="#bowtie2-build-options-c"><code>-c</code></a> is specified, the
sequences themselves. E.g., <code><reference_in></code> might be
<code>chr1.fa,chr2.fa,chrX.fa,chrY.fa</code>, or, if <a
href="#bowtie2-build-options-c"><code>-c</code></a> is specified, this
might be <code>GGTCATCCT,ACGGGTCGT,CCGTTCTATGCGGCTTA</code>.</p>
</td>
</tr>
<tr>
<td>
<pre><code><bt2_base></code></pre>
</td>
<td>
<p>The basename of the index files to write. By default,
<code>bowtie2-build</code> writes files named <code>NAME.1.bt2</code>,
<code>NAME.2.bt2</code>, <code>NAME.3.bt2</code>,
<code>NAME.4.bt2</code>, <code>NAME.rev.1.bt2</code>, and
<code>NAME.rev.2.bt2</code>, where <code>NAME</code> is
<code><bt2_base></code>.</p>
</td>
</tr>
</table>
<h3 id="options-1">Options</h3>
<table>
<tr>
<td>
<pre><code>-f</code></pre>
</td>
<td>
<p>The reference input files (specified as
<code><reference_in></code>) are <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a> files
(usually having extension <code>.fa</code>, <code>.mfa</code>,
<code>.fna</code> or similar).</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-c">
<pre><code>-c</code></pre>
</td>
<td>
<p>The reference sequences are given on the command line. I.e.
<code><reference_in></code> is a comma-separated list of sequences
rather than a list of <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a>
files.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-large-index">
<pre><code>--large-index</code></pre>
</td>
<td>
<p>Force <code>bowtie2-build</code> to build a <a
href="#small-and-large-indexes">large index</a>, even if the reference
is less than ~ 4 billion nucleotides inlong.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-a">
<pre><code>-a/--noauto</code></pre>
</td>
<td>
<p>Disable the default behavior whereby <code>bowtie2-build</code>
automatically selects values for the <a
href="#bowtie2-build-options-bmax"><code>--bmax</code></a>, <a
href="#bowtie2-build-options-dcv"><code>--dcv</code></a> and <a
href="#bowtie2-build-options-p"><code>--packed</code></a> 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>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-p">
<pre><code>-p/--packed</code></pre>
</td>
<td>
<p>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
href="#bowtie2-build-options-a"><code>-a</code>/<code>--noauto</code></a>
to configure manually.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-bmax">
<pre><code>--bmax <int></code></pre>
</td>
<td>
<p>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 <a
href="#bowtie2-build-options-bmax"><code>--bmax</code></a>, or <a
href="#bowtie2-build-options-bmaxdivn"><code>--bmaxdivn</code></a>.
Default (in terms of the <a
href="#bowtie2-build-options-bmaxdivn"><code>--bmaxdivn</code></a>
parameter) is <a
href="#bowtie2-build-options-bmaxdivn"><code>--bmaxdivn</code></a> 4 *
number of threads. This is configured automatically by default; use <a
href="#bowtie2-build-options-a"><code>-a</code>/<code>--noauto</code></a>
to configure manually.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-bmaxdivn">
<pre><code>--bmaxdivn <int></code></pre>
</td>
<td>
<p>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 <a
href="#bowtie2-build-options-bmax"><code>--bmax</code></a>, or <a
href="#bowtie2-build-options-bmaxdivn"><code>--bmaxdivn</code></a>.
Default: <a
href="#bowtie2-build-options-bmaxdivn"><code>--bmaxdivn</code></a> 4 *
number of threads. This is configured automatically by default; use <a
href="#bowtie2-build-options-a"><code>-a</code>/<code>--noauto</code></a>
to configure manually.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-dcv">
<pre><code>--dcv <int></code></pre>
</td>
<td>
<p>Use <code><int></code> 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
href="#bowtie2-build-options-a"><code>-a</code>/<code>--noauto</code></a>
to configure manually.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-nodc">
<pre><code>--nodc</code></pre>
</td>
<td>
<p>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.</p>
</td>
</tr>
<tr>
<td>
<pre><code>-r/--noref</code></pre>
</td>
<td>
<p>Do not build the <code>NAME.3.bt2</code> and <code>NAME.4.bt2</code>
portions of the index, which contain a bitpacked version of the
reference sequences and are used for paired-end alignment.</p>
</td>
</tr>
<tr>
<td>
<pre><code>-3/--justref</code></pre>
</td>
<td>
<p>Build only the <code>NAME.3.bt2</code> and <code>NAME.4.bt2</code>
portions of the index, which contain a bitpacked version of the
reference sequences and are used for paired-end alignment.</p>
</td>
</tr>
<tr>
<td id="bowtie2-build-options-o">
<pre><code>-o/--offrate <int></code></pre>
</td>
<td>
<p>To map alignments back to positions on the reference sequences, it's
necessary to annotate ("mark") some or all of the <a
href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform">Burrows-Wheeler</a>
rows with their corresponding location on the genome. <a
href="#bowtie2-build-options-o"><code>-o</code>/<code>--offrate</code></a>
governs how many rows get marked: the indexer will mark every
2^<code><int></code> 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).</p>
</td>
</tr>
<tr>
<td>
<pre><code>-t/--ftabchars <int></code></pre>
</td>
<td>
<p>The ftab is the lookup table used to calculate an initial <a
href="http://en.wikipedia.org/wiki/Burrows-Wheeler_transform">Burrows-Wheeler</a>
range with respect to the first <code><int></code> characters of
the query. A larger <code><int></code> yields a larger lookup
table but faster query times. The ftab has size
4^(<code><int></code>+1) bytes. The default setting is 10 (ftab is
4MB).</p>
</td>
</tr>
<tr>
<td>
<pre><code>--seed <int></code></pre>
</td>
<td>
<p>Use <code><int></code> as the seed for pseudo-random number
generator.</p>
</td>
</tr>
<tr>
<td>
<pre><code>--cutoff <int></code></pre>
</td>
<td>
<p>Index only the first <code><int></code> bases of the reference
sequences (cumulative across sequences) and ignore the rest.</p>
</td>
</tr>
<tr>
<td>
<pre><code>-q/--quiet</code></pre>
</td>
<td>
<p><code>bowtie2-build</code> is verbose by default. With this option
<code>bowtie2-build</code> will print only error messages.</p>
</td>
</tr>
<tr>
<td>
<pre><code>--threads <int></code></pre>
</td>
<td>
<p>By default <code>bowtie2-build</code> is using only one thread.
Increasing the number of threads will speed up the index building
considerably in most cases.</p>
</td>
</tr>
<tr>
<td>
<pre><code>-h/--help</code></pre>
</td>
<td>
<p>Print usage information and quit.</p>
</td>
</tr>
<tr>
<td>
<pre><code>--version</code></pre>
</td>
<td>
<p>Print version information and quit.</p>
</td>
</tr>
</table>
<h1 id="the-bowtie2-inspect-index-inspector">The
<code>bowtie2-inspect</code> index inspector</h1>
<p><code>bowtie2-inspect</code> 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 <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a> file
containing the sequences of the original references (with all
non-<code>A</code>/<code>C</code>/<code>G</code>/<code>T</code>
characters converted to <code>N</code>s). It can also be used to extract
just the reference sequence names using the <a
href="#bowtie2-inspect-options-n"><code>-n</code>/<code>--names</code></a>
option or a more verbose summary using the <a
href="#bowtie2-inspect-options-s"><code>-s</code>/<code>--summary</code></a>
option.</p>
<h2 id="command-line-2">Command Line</h2>
<p>Usage:</p>
<pre><code>bowtie2-inspect [options]* <bt2_base></code></pre>
<h3 id="main-arguments-2">Main arguments</h3>
<table>
<tr>
<td>
<pre><code><bt2_base></code></pre>
</td>
<td>
<p>The basename of the index to be inspected. The basename is name of
any of the index files but with the <code>.X.bt2</code> or
<code>.rev.X.bt2</code> suffix omitted. <code>bowtie2-inspect</code>
first looks in the current directory for the index files, then in the
directory specified in the <code>BOWTIE2_INDEXES</code> environment
variable.</p>
</td>
</tr>
</table>
<h3 id="options-2">Options</h3>
<table>
<tr>
<td>
<pre><code>-a/--across <int></code></pre>
</td>
<td>
<p>When printing <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a>
output, output a newline character every <code><int></code> bases
(default: 60).</p>
</td>
</tr>
<tr>
<td id="bowtie2-inspect-options-n">
<pre><code>-n/--names</code></pre>
</td>
<td>
<p>Print reference sequence names, one per line, and quit.</p>
</td>
</tr>
<tr>
<td id="bowtie2-inspect-options-s">
<pre><code>-s/--summary</code></pre>
</td>
<td>
<p>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:</p>
<pre><code>Colorspace <0 or 1>
SA-Sample 1 in <sample>
FTab-Chars <chars>
Sequence-1 <name> <len>
Sequence-2 <name> <len>
...
Sequence-N <name> <len></code></pre>
<p>Fields are separated by tabs. Colorspace is always set to 0 for
Bowtie 2.</p>
</td>
</tr>
<tr>
<td id="bowtie2-inspect-options-o">
<pre><code>-o/--output <filename></code></pre>
</td>
<td>
<p>Save output to user-specified filename (default: stdout)</p>
</td>
</tr>
<tr>
<td>
<pre><code>-v/--verbose</code></pre>
</td>
<td>
<p>Print verbose output (for debugging).</p>
</td>
</tr>
<tr>
<td>
<pre><code>--version</code></pre>
</td>
<td>
<p>Print version information and quit.</p>
</td>
</tr>
<tr>
<td>
<pre><code>-h/--help</code></pre>
</td>
<td>
<p>Print usage information and quit.</p>
</td>
</tr>
</table>
<h1 id="getting-started-with-bowtie-2-lambda-phage-example">Getting
started with Bowtie 2: Lambda phage example</h1>
<p>Bowtie 2 comes with some example files to get you started. The
example files are not scientifically significant; we use the <a
href="http://en.wikipedia.org/wiki/Lambda_phage">Lambda phage</a>
reference genome simply because it's short, and the reads were generated
by a computer program, not a sequencer. However, these files will let
you start running Bowtie 2 and downstream tools right away.</p>
<p>First follow the manual instructions to <a
href="#obtaining-bowtie-2">obtain Bowtie 2</a>. Set the
<code>BT2_HOME</code> environment variable to point to the new Bowtie 2
directory containing the <code>bowtie2</code>,
<code>bowtie2-build</code> and <code>bowtie2-inspect</code> binaries.
This is important, as the <code>BT2_HOME</code> variable is used in the
commands below to refer to that directory.</p>
<h2 id="indexing-a-reference-genome">Indexing a reference genome</h2>
<p>To create an index for the <a
href="http://en.wikipedia.org/wiki/Lambda_phage">Lambda phage</a>
reference genome included with Bowtie 2, create a new temporary
directory (it doesn't matter where), change into that directory, and
run:</p>
<pre><code>$BT2_HOME/bowtie2-build $BT2_HOME/example/reference/lambda_virus.fa lambda_virus</code></pre>
<p>The command should print many lines of output then quit. When the
command completes, the current directory will contain four new files
that all start with <code>lambda_virus</code> and end with
<code>.1.bt2</code>, <code>.2.bt2</code>, <code>.3.bt2</code>,
<code>.4.bt2</code>, <code>.rev.1.bt2</code>, and
<code>.rev.2.bt2</code>. These files constitute the index - you're
done!</p>
<p>You can use <code>bowtie2-build</code> to create an index for a set
of <a href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a>
files obtained from any source, including sites such as <a
href="http://genome.ucsc.edu/cgi-bin/hgGateway">UCSC</a>, <a
href="http://www.ncbi.nlm.nih.gov/sites/genome">NCBI</a>, and <a
href="http://www.ensembl.org/">Ensembl</a>. When indexing multiple <a
href="https://en.wikipedia.org/wiki/FASTA"><code>FASTA</code></a> files,
specify all the files using commas to separate file names. For more
details on how to create an index with <code>bowtie2-build</code>, see
the <a href="#the-bowtie2-build-indexer">manual section on index
building</a>. You may also want to bypass this process by obtaining a
pre-built index. See <a href="#using-a-pre-built-index">using a
pre-built index</a> below for an example.</p>
<h2 id="aligning-example-reads">Aligning example reads</h2>
<p>Stay in the directory created in the previous step, which now
contains the <code>lambda_virus</code> index files. Next, run:</p>
<pre><code>$BT2_HOME/bowtie2 -x lambda_virus -U $BT2_HOME/example/reads/reads_1.fq -S eg1.sam</code></pre>
<p>This runs the Bowtie 2 aligner, which aligns a set of unpaired reads
to the <a href="http://en.wikipedia.org/wiki/Lambda_phage">Lambda
phage</a> reference genome using the index generated in the previous
step. The alignment results in SAM format are written to the file
<code>eg1.sam</code>, and a short alignment summary is written to the
console. (Actually, the summary is written to the "standard error" or
"stderr" filehandle, which is typically printed to the console.)</p>
<p>To see the first few lines of the SAM output, run:</p>
<pre><code>head eg1.sam</code></pre>
<p>You will see something like this:</p>
<pre><code>@HD VN:1.0 SO:unsorted
@SQ SN:gi|9626243|ref|NC_001416.1| LN:48502
@PG ID:bowtie2 PN:bowtie2 VN:2.0.1
r1 0 gi|9626243|ref|NC_001416.1| 18401 42 122M * 0 0 TGAATGCGAACTCCGGGACGCTCAGTAATGTGACGATAGCTGAAAACTGTACGATAAACNGTACGCTGAGGGCAGAAAAAATCGTCGGGGACATTNTAAAGGCGGCGAGCGCGGCTTTTCCG +"@6<:27(F&5)9"B):%B+A-%5A?2$HCB0B+0=D<7E/<.03#!.F77@6B==?C"7>;))%;,3-$.A06+<-1/@@?,26">=?*@'0;$:;??G+:#+(A?9+10!8!?()?7C> AS:i:-5 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:59G13G21G26 YT:Z:UU
r2 0 gi|9626243|ref|NC_001416.1| 8886 42 275M * 0 0 NTTNTGATGCGGGCTTGTGGAGTTCAGCCGATCTGACTTATGTCATTACCTATGAAATGTGAGGACGCTATGCCTGTACCAAATCCTACAATGCCGGTGAAAGGTGCCGGGATCACCCTGTGGGTTTATAAGGGGATCGGTGACCCCTACGCGAATCCGCTTTCAGACGTTGACTGGTCGCGTCTGGCAAAAGTTAAAGACCTGACGCCCGGCGAACTGACCGCTGAGNCCTATGACGACAGCTATCTCGATGATGAAGATGCAGACTGGACTGC (#!!'+!$""%+(+)'%)%!+!(&++)''"#"#&#"!'!("%'""("+&%$%*%%#$%#%#!)*'(#")(($&$'&%+&#%*)*#*%*')(%+!%%*"$%"#+)$&&+)&)*+!"*)!*!("&&"*#+"&"'(%)*("'!$*!!%$&&&$!!&&"(*"$&"#&!$%'%"#)$#+%*+)!&*)+(""#!)!%*#"*)*')&")($+*%%)!*)!('(%""+%"$##"#+(('!*(($*'!"*('"+)&%#&$+('**$$&+*&!#%)')'(+(!%+ AS:i:-14 XN:i:0 XM:i:8 XO:i:0 XG:i:0 NM:i:8 MD:Z:0A0C0G0A108C23G9T81T46 YT:Z:UU
r3 16 gi|9626243|ref|NC_001416.1| 11599 42 338M * 0 0 GGGCGCGTTACTGGGATGATCGTGAAAAGGCCCGTCTTGCGCTTGAAGCCGCCCGAAAGAAGGCTGAGCAGCAGACTCAAGAGGAGAAAAATGCGCAGCAGCGGAGCGATACCGAAGCGTCACGGCTGAAATATACCGAAGAGGCGCAGAAGGCTNACGAACGGCTGCAGACGCCGCTGCAGAAATATACCGCCCGTCAGGAAGAACTGANCAAGGCACNGAAAGACGGGAAAATCCTGCAGGCGGATTACAACACGCTGATGGCGGCGGCGAAAAAGGATTATGAAGCGACGCTGTAAAAGCCGAAACAGTCCAGCGTGAAGGTGTCTGCGGGCGAT 7F$%6=$:9B@/F'>=?!D?@0(:A*)7/>9C>6#1<6:C(.CC;#.;>;2'$4D:?&B!>689?(0(G7+0=@37F)GG=>?958.D2E04C<E,*AD%G0.%$+A:'H;?8<72:88?E6((CF)6DF#.)=>B>D-="C'B080E'5BH"77':"@70#4%A5=6.2/1>;9"&-H6)=$/0;5E:<8G!@::1?2DC7C*;@*#.1C0.D>H/20,!"C-#,6@%<+<D(AG-).?&#0.00'@)/F8?B!&"170,)>:?<A7#1(A@0E#&A.*DC.E")AH"+.,5,2>5"2?:G,F"D0B8D-6$65D<D!A/38860.*4;4B<*31?6 AS:i:-22 XN:i:0 XM:i:8 XO:i:0 XG:i:0 NM:i:8 MD:Z:80C4C16A52T23G30A8T76A41 YT:Z:UU
r4 0 gi|9626243|ref|NC_001416.1| 40075 42 184M * 0 0 GGGCCAATGCGCTTACTGATGCGGAATTACGCCGTAAGGCCGCAGATGAGCTTGTCCATATGACTGCGAGAATTAACNGTGGTGAGGCGATCCCTGAACCAGTAAAACAACTTCCTGTCATGGGCGGTAGACCTCTAAATCGTGCACAGGCTCTGGCGAAGATCGCAGAAATCAAAGCTAAGT(=8B)GD04*G%&4F,1'A>.C&7=F$,+#6!))43C,5/5+)?-/0>/D3=-,2/+.1?@->;)00!'3!7BH$G)HG+ADC'#-9F)7<7"$?&.>0)@5;4,!0-#C!15CF8&HB+B==H>7,/)C5)5*+(F5A%D,EA<(>G9E0>7&/E?4%;#'92)<5+@7:A.(BG@BG86@.G AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:77C106 YT:Z:UU
r5 0 gi|9626243|ref|NC_001416.1| 48010 42 138M * 0 0 GTCAGGAAAGTGGTAAAACTGCAACTCAATTACTGCAATGCCCTCGTAATTAAGTGAATTTACAATATCGTCCTGTTCGGAGGGAAGAACGCGGGATGTTCATTCTTCATCACTTTTAATTGATGTATATGCTCTCTT 9''%<D)A03E1-*7=),:F/0!6,D9:H,<9D%:0B(%'E,(8EFG$E89B$27G8F*2+4,-!,0D5()&=(FGG:5;3*@/.0F-G#5#3->('FDFEG?)5.!)"AGADB3?6(@H(:B<>6!>;>6>G,."?% AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:138 YT:Z:UU
r6 16 gi|9626243|ref|NC_001416.1| 41607 42 72M2D119M * 0 0 TCGATTTGCAAATACCGGAACATCTCGGTAACTGCATATTCTGCATTAAAAAATCAACGCAAAAAATCGGACGCCTGCAAAGATGAGGAGGGATTGCAGCGTGTTTTTAATGAGGTCATCACGGGATNCCATGTGCGTGACGGNCATCGGGAAACGCCAAAGGAGATTATGTACCGAGGAAGAATGTCGCT 1H#G;H"$E*E#&"*)2%66?=9/9'=;4)4/>@%+5#@#$4A*!<D=="8#1*A9BA=:(1+#C&.#(3#H=9E)AC*5,AC#E'536*2?)H14?>9'B=7(3H/B:+A:8%1-+#(E%&$$&14"76D?>7(&20H5%*&CF8!G5B+A4F$7(:"'?0$?G+$)B-?2<0<F=D!38BH,%=8&5@+ AS:i:-13 XN:i:0 XM:i:2 XO:i:1 XG:i:2 NM:i:4 MD:Z:72^TT55C15A47 YT:Z:UU
r7 16 gi|9626243|ref|NC_001416.1| 4692 42 143M * 0 0 TCAGCCGGACGCGGGCGCTGCAGCCGTACTCGGGGATGACCGGTTACAACGGCATTATCGCCCGTCTGCAACAGGCTGCCAGCGATCCGATGGTGGACAGCATTCTGCTCGATATGGACANGCCCGGCGGGATGGTGGCGGGG -"/@*7A0)>2,AAH@&"%B)*5*23B/,)90.B@%=FE,E063C9?,:26$-0:,.,1849'4.;F>FA;76+5&$<C":$!A*,<B,<)@<'85D%C*:)30@85;?.B$05=@95DCDH<53!8G:F:B7/A.E':434> AS:i:-6 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:98G21C22 YT:Z:UU</code></pre>
<p>The first few lines (beginning with <code>@</code>) are SAM header
lines, and the rest of the lines are SAM alignments, one line per read
or mate. See the <a href="#sam-output">Bowtie 2 manual section on SAM
output</a> and the <a
href="http://samtools.sourceforge.net/SAM1.pdf">SAM specification</a>
for details about how to interpret the SAM file format.</p>
<h2 id="paired-end-example">Paired-end example</h2>
<p>To align paired-end reads included with Bowtie 2, stay in the same
directory and run:</p>
<pre><code>$BT2_HOME/bowtie2 -x lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam</code></pre>
<p>This aligns a set of paired-end reads to the reference genome, with
results written to the file <code>eg2.sam</code>.</p>
<h2 id="local-alignment-example-1">Local alignment example</h2>
<p>To use <a href="#end-to-end-alignment-versus-local-alignment">local
alignment</a> to align some longer reads included with Bowtie 2, stay in
the same directory and run:</p>
<pre><code>$BT2_HOME/bowtie2 --local -x lambda_virus -U $BT2_HOME/example/reads/longreads.fq -S eg3.sam</code></pre>
<p>This aligns the long reads to the reference genome using local
alignment, with results written to the file <code>eg3.sam</code>.</p>
<h2 id="using-samtoolsbcftools-downstream">Using SAMtools/BCFtools
downstream</h2>
<p><a href="http://samtools.sourceforge.net">SAMtools</a> is a
collection of tools for manipulating and analyzing SAM and BAM alignment
files. <a
href="http://samtools.sourceforge.net/mpileup.shtml">BCFtools</a> is a
collection of tools for calling variants and manipulating VCF and BCF
files, and it is typically distributed with <a
href="http://samtools.sourceforge.net">SAMtools</a>. Using these tools
together allows you to get from alignments in SAM format to variant
calls in VCF format. This example assumes that <code>samtools</code> and
<code>bcftools</code> are installed and that the directories containing
these binaries are in your <a
href="http://en.wikipedia.org/wiki/PATH_(variable)">PATH environment
variable</a>.</p>
<p>Run the paired-end example:</p>
<pre><code>$BT2_HOME/bowtie2 -x $BT2_HOME/example/index/lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam</code></pre>
<p>Use <code>samtools view</code> to convert the SAM file into a BAM
file. BAM is the binary format corresponding to the SAM text format.
Run:</p>
<pre><code>samtools view -bS eg2.sam > eg2.bam</code></pre>
<p>Use <code>samtools sort</code> to convert the BAM file to a sorted
BAM file.</p>
<pre><code>samtools sort eg2.bam -o eg2.sorted.bam</code></pre>
<p>We now have a sorted BAM file called <code>eg2.sorted.bam</code>.
Sorted BAM is a useful format because the alignments are (a) compressed,
which is convenient for long-term storage, and (b) sorted, which is
conveneint for variant discovery. To generate variant calls in VCF
format, run:</p>
<pre><code>bcftools mpileup -f $BT2_HOME/example/reference/lambda_virus.fa eg2.sorted.bam | bcftools view -Ov - > eg2.raw.bcf</code></pre>
<p>Then to view the variants, run:</p>
<pre><code>bcftools view eg2.raw.bcf</code></pre>
<p>See the official SAMtools guide to <a
href="http://samtools.sourceforge.net/mpileup.shtml">Calling SNPs/INDELs
with SAMtools/BCFtools</a> for more details and variations on this
process.</p>
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