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Build a reference database
#
One of the crucial steps in the analysis of environmental DNA data is the taxonomic assignment of sequences,
i.e. assigning a species, genus or other taxonomic rank to the sequences present in the collected samples.
Taxonomic assignment requires annotated reference sequences, against which the sequences of interest are compared.
These reference sequences form what is known as a reference database, which is a sequence file in
fasta
format,
for a given marker of metabarcoding.">
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<meta property="og:description" content="Build a reference database # One of the crucial steps in the analysis of environmental DNA data is the taxonomic assignment of sequences, i.e. assigning a species, genus or other taxonomic rank to the sequences present in the collected samples.
Taxonomic assignment requires annotated reference sequences, against which the sequences of interest are compared. These reference sequences form what is known as a reference database, which is a sequence file in fasta format, for a given marker of metabarcoding.">
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<h3>Build a reference database</h3>
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<li><a href="#build-a-reference-database">Build a reference database</a>
<ul>
<li><a href="#download-the-sequences">Download the sequences</a></li>
<li><a href="#perform-a-in-silico-pcr-amplification">Perform a <em>in silico</em> PCR amplification</a></li>
<li><a href="#clean-the-database">Clean the database</a>
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<article class="markdown book-article"><h1 id="build-a-reference-database">
Build a reference database
<a class="anchor" href="#build-a-reference-database">#</a>
</h1>
<p>One of the crucial steps in the analysis of environmental DNA data is the taxonomic assignment of sequences,
<em>i.e.</em> assigning a species, genus or other taxonomic rank to the sequences present in the collected samples.</p>
<p>Taxonomic assignment requires annotated reference sequences, against which the sequences of interest are compared.
These reference sequences form what is known as a <em>reference database</em>, which is a sequence file in
<a href="http://metabar:8888/obidoc/formats/fasta/">fasta</a>
format,
for a given marker of metabarcoding.</p>
<p>Here is a quick step-by-step guide to creating a reference database, here for assigning sequences from wolf fecal
samples to study its diet, a dataset used in the
<a href="https://obitools4.metabarcoding.org/docs/cookbook/wolf-tutorial/">metabarcoding analysis tutorial here</a>.</p>
<p>One way to build a reference database is to use the <a href="http://metabar:8888/obidoc/obitools/obipcr/">
<abbr title="obipcr: the electronic PCR tool"><code>obipcr</code></abbr>
</a> program to simulate a PCR and extract all sequences
from a general purpose DNA database such as
<a href="https://www.ncbi.nlm.nih.gov/nucleotide/">GenBank</a> or
<a href="https://www.ebi.ac.uk/ena/browser/home">EMBL</a>
that can be amplified <em>in silico</em> by the two primers used for PCR amplification.</p>
<p>The steps to create a reference database are:</p>
<ol>
<li>Download sequences from a public database such as GenBank or EMBL</li>
<li>Perform an <em>in silico</em> PCR amplification of these sequences with a given marker with <a href="http://metabar:8888/obidoc/obitools/obipcr/">
<abbr title="obipcr: the electronic PCR tool"><code>obipcr</code></abbr>
</a></li>
<li>Clean up the database by deleting sequences that do not provide sufficient taxonomic information and are redundant</li>
</ol>
<p>Since Genbank and the taxonomy associated with sequences are constantly evolving, you may not get exactly the same results when using the following commands.</p>
<h2 id="download-the-sequences">
Download the sequences
<a class="anchor" href="#download-the-sequences">#</a>
</h2>
<p>In this example, the sequences are downloaded from the
<a href="https://ftp.ncbi.nlm.nih.gov/genbank/">GenBank FTP server</a>.
Please note that the download takes more than a day and currently occupies around 1.5 TB,
so make sure you have the necessary storage capacity before launching it.
To have a local copy of GenBank sequences, please go to the
<a href="https://obitools4.metabarcoding.org/docs/cookbook/local_genbank/">Prepare a local copy of GenBank</a> page.</p>
<h2 id="perform-a-in-silico-pcr-amplification">
Perform a <em>in silico</em> PCR amplification
<a class="anchor" href="#perform-a-in-silico-pcr-amplification">#</a>
</h2>
<p>In this example, we amplify the <em>12S-V5</em> region [@Riaz2011-gn] with the forward primer <strong>TTAGATACCCCACTATGC</strong>
and the reverse primer <strong>TAGAACAGGCTCCTCTAG</strong>, with the following command, to study the wolf diet
(see the
<a href="https://obitools4.metabarcoding.org/docs/cookbook/wolf-tutorial/">tutorial</a>).
Do not forget to update the release number of GenBank in the command line.</p>
<div class="highlight"><pre tabindex="0" style="color:#f8f8f2;background-color:#272822;-moz-tab-size:4;-o-tab-size:4;tab-size:4;"><code class="language-bash" data-lang="bash"><span style="display:flex;"><span>obipcr -e <span style="color:#ae81ff">3</span> -l <span style="color:#ae81ff">50</span> -L <span style="color:#ae81ff">150</span> <span style="color:#ae81ff">\ </span>
</span></span><span style="display:flex;"><span> --forward TTAGATACCCCACTATGC <span style="color:#ae81ff">\
</span></span></span><span style="display:flex;"><span><span style="color:#ae81ff"></span> --reverse TAGAACAGGCTCCTCTAG <span style="color:#ae81ff">\
</span></span></span><span style="display:flex;"><span><span style="color:#ae81ff"></span> --no-order <span style="color:#ae81ff">\
</span></span></span><span style="display:flex;"><span><span style="color:#ae81ff"></span> genbank/Release_264/fasta/*
</span></span><span style="display:flex;"><span> &gt; v05_pcr.fasta
</span></span></code></pre></div><p>The <code>-l</code> and <code>-L</code> options define the minimum and maximum sizes of sequence fragments to be amplified.
Three mismatches with primer sequences are allowed here (-e 3), and we recommend using the <code>--no-order</code> option
to speed up the program (see <a href="http://metabar:8888/obidoc/obitools/obipcr/">
<abbr title="obipcr: the electronic PCR tool"><code>obipcr</code></abbr>
</a> documentation).</p>
<p>This previous command produces a
<a href="http://metabar:8888/obidoc/formats/fasta/">fasta</a>
file, with the computed amplified sequences.</p>
<h2 id="clean-the-database">
Clean the database
<a class="anchor" href="#clean-the-database">#</a>
</h2>
<p>We choose to apply these different steps of filtering to clean up the sequences obtained with <a href="http://metabar:8888/obidoc/obitools/obipcr/">
<abbr title="obipcr: the electronic PCR tool"><code>obipcr</code></abbr>
</a>:</p>
<ol>
<li>Keep the sequences with a taxid and a taxonomic description to family, genus and species ranks (<a href="http://metabar:8888/obidoc/obitools/obigrep/">
<abbr title="obigrep: filter a sequence file"><code>obigrep</code></abbr>
</a>)</li>
<li>Remove redundant sequences (dereplicate)</li>
<li>Ensure that the dereplicated sequences have a taxid (taxon identifier) at the family level</li>
<li>Ensure that sequences each have a unique identification ID with <a href="http://metabar:8888/obidoc/obitools/obiannotate/">
<abbr title="obiannotate: edit sequence annotations"><code>obiannotate</code></abbr>
</a></li>
<li>Index the database</li>
</ol>
<h4 id="keep-annotated-sequences">
Keep annotated sequences
<a class="anchor" href="#keep-annotated-sequences">#</a>
</h4>
<p>To use the <code>-t</code> taxonomy option on all <em>OBITools</em> commands,
you can either enter the path to the taxonomy if you have downloaded
the sequences from the help page
<a href="https://obitools4.metabarcoding.org/docs/cookbook/local_genbank/">here</a>
which looks like <code>Release_264/taxonomy</code>, or download the taxdump file online with <code>curl</code>.</p>
<div class="highlight"><pre tabindex="0" style="color:#f8f8f2;background-color:#272822;-moz-tab-size:4;-o-tab-size:4;tab-size:4;"><code class="language-bash" data-lang="bash"><span style="display:flex;"><span>curl http://ftp.ncbi.nih.gov/pub/taxonomy/taxdump.tar.gz
</span></span></code></pre></div><p>The <a href="http://metabar:8888/obidoc/obitools/obigrep/">
<abbr title="obigrep: filter a sequence file"><code>obigrep</code></abbr>
</a> program allows to filter sequences, to keep only those with a taxid and a sufficient taxonomic description.</p>
<div class="highlight"><pre tabindex="0" style="color:#f8f8f2;background-color:#272822;-moz-tab-size:4;-o-tab-size:4;tab-size:4;"><code class="language-bash" data-lang="bash"><span style="display:flex;"><span>obigrep -t taxdump.tar.gz <span style="color:#ae81ff">\
</span></span></span><span style="display:flex;"><span><span style="color:#ae81ff"></span> -A taxid <span style="color:#ae81ff">\
</span></span></span><span style="display:flex;"><span><span style="color:#ae81ff"></span> --require-rank species <span style="color:#ae81ff">\
</span></span></span><span style="display:flex;"><span><span style="color:#ae81ff"></span> --require-rank genus <span style="color:#ae81ff">\
</span></span></span><span style="display:flex;"><span><span style="color:#ae81ff"></span> --require-rank family <span style="color:#ae81ff">\
</span></span></span><span style="display:flex;"><span><span style="color:#ae81ff"></span> v05_pcr.fasta &gt; v05_clean.fasta
</span></span></code></pre></div><h4 id="dereplicate-sequences">
Dereplicate sequences
<a class="anchor" href="#dereplicate-sequences">#</a>
</h4>
<p>The <a href="http://metabar:8888/obidoc/obitools/obiuniq/">
<abbr title="obiuniq: dereplicate a sequence file"><code>obiuniq</code></abbr>
</a> program is able to dereplicate the sequences.</p>
<div class="highlight"><pre tabindex="0" style="color:#f8f8f2;background-color:#272822;-moz-tab-size:4;-o-tab-size:4;tab-size:4;"><code class="language-bash" data-lang="bash"><span style="display:flex;"><span>obiuniq -c taxid v05_clean.fasta &gt; v05_clean_uniq.fasta
</span></span></code></pre></div><h4 id="ensure-that-the-dereplicated-sequences-have-a-taxid-at-the-family-level">
Ensure that the dereplicated sequences have a taxid at the family level
<a class="anchor" href="#ensure-that-the-dereplicated-sequences-have-a-taxid-at-the-family-level">#</a>
</h4>
<p>Some sequences lose taxonomic information at the dereplication stage if certain versions
of the sequence did not have this information beforehand. So we apply a second filter of this type.</p>
<div class="highlight"><pre tabindex="0" style="color:#f8f8f2;background-color:#272822;-moz-tab-size:4;-o-tab-size:4;tab-size:4;"><code class="language-bash" data-lang="bash"><span style="display:flex;"><span>obigrep -t taxdump.tar.gz --require-rank<span style="color:#f92672">=</span>family v05_clean_uniq.fasta &gt; v05_clean_uniq.fasta
</span></span></code></pre></div><h4 id="ensure-that-sequences-each-have-a-unique-identifier">
Ensure that sequences each have a unique identifier
<a class="anchor" href="#ensure-that-sequences-each-have-a-unique-identifier">#</a>
</h4>
<h4 id="index-the-database">
Index the database
<a class="anchor" href="#index-the-database">#</a>
</h4>
<div class="highlight"><pre tabindex="0" style="color:#f8f8f2;background-color:#272822;-moz-tab-size:4;-o-tab-size:4;tab-size:4;"><code class="language-bash" data-lang="bash"><span style="display:flex;"><span>obirefidx -t taxdump.tar.gz v05_clean_uniq.fasta &gt; v05_clean_uniq_indexed.fasta
</span></span></code></pre></div><p>The database provided in the
<a href="https://obitools4.metabarcoding.org/docs/cookbook/wolf-tutorial/">tutorial</a>
is called <code>wolf_data/db_v05_r117_indexed.fasta</code>.</p>
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