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<span class="sidebar-item-text sidebar-link text-start">The GO *OBITools* library</span>
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<h2 id="toc-title">Table of contents</h2>
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<ul>
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<li><a href="#sequence-attributes" id="toc-sequence-attributes" class="nav-link active" data-scroll-target="#sequence-attributes"><span class="toc-section-number">A.0.1</span> Sequence attributes</a></li>
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<h1 class="title d-none d-lg-block">Appendix A — Annexes</h1>
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<section id="sequence-attributes" class="level3" data-number="A.0.1">
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<h3 data-number="A.0.1" class="anchored" data-anchor-id="sequence-attributes"><span class="header-section-number">A.0.1</span> Sequence attributes</h3>
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<section id="reserved-sequence-attributes" class="level4" data-number="A.0.1.1">
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<h4 data-number="A.0.1.1" class="anchored" data-anchor-id="reserved-sequence-attributes"><span class="header-section-number">A.0.1.1</span> Reserved sequence attributes</h4>
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<h5 data-number="A.0.1.1.1" class="anchored" data-anchor-id="ali_dir"><span class="header-section-number">A.0.1.1.1</span> <code>ali_dir</code></h5>
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<section id="type-string" class="level6" data-number="A.0.1.1.1.1">
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<h6 data-number="A.0.1.1.1.1" class="anchored" data-anchor-id="type-string"><span class="header-section-number">A.0.1.1.1.1</span> Type : <code>string</code></h6>
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<p>The attribute can contain 2 string values <code>"left"</code> or <code>"right".</code></p>
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</section>
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<section id="set-by-the-obipairing-tool" class="level6" data-number="A.0.1.1.1.2">
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<h6 data-number="A.0.1.1.1.2" class="anchored" data-anchor-id="set-by-the-obipairing-tool"><span class="header-section-number">A.0.1.1.1.2</span> Set by the <em>obipairing</em> tool</h6>
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<p>The alignment generated by <em>obipairing</em> is a 3’-end gap free algorithm. Two cases can occur when aligning the forward and reverse reads. If the barcode is long enough, both the reads overlap only on their 3’ ends. In such case, the alignment direction <code>ali_dir</code> is set to <em>left</em>. If the barcode is shorter than the read length, the paired reads overlap by their 5’ ends, and the complete barcode is sequenced by both the reads. In that later case, <code>ali_dir</code> is set to <em>right</em>.</p>
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</section>
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</section>
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<section id="ali_length" class="level5" data-number="A.0.1.1.2">
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<h5 data-number="A.0.1.1.2" class="anchored" data-anchor-id="ali_length"><span class="header-section-number">A.0.1.1.2</span> <code>ali_length</code></h5>
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<section id="set-by-the-obipairing-tool-1" class="level6" data-number="A.0.1.1.2.1">
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<h6 data-number="A.0.1.1.2.1" class="anchored" data-anchor-id="set-by-the-obipairing-tool-1"><span class="header-section-number">A.0.1.1.2.1</span> Set by the <em>obipairing</em> tool</h6>
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<p>Length of the aligned parts when merging forward and reverse reads</p>
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</section>
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</section>
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<section id="count-the-number-of-sequence-occurrences" class="level5" data-number="A.0.1.1.3">
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<h5 data-number="A.0.1.1.3" class="anchored" data-anchor-id="count-the-number-of-sequence-occurrences"><span class="header-section-number">A.0.1.1.3</span> <code>count</code> : the number of sequence occurrences</h5>
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<section id="set-by-the-obiuniq-tool" class="level6" data-number="A.0.1.1.3.1">
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<h6 data-number="A.0.1.1.3.1" class="anchored" data-anchor-id="set-by-the-obiuniq-tool"><span class="header-section-number">A.0.1.1.3.1</span> Set by the <em>obiuniq</em> tool</h6>
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<p>The <code>count</code> attribute indicates how-many strictly identical sequences have been merged in a single record. It contains an integer value. If it is absent this means that the sequence record represents a single occurrence of the sequence.</p>
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</section>
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<section id="getter-method-count" class="level6" data-number="A.0.1.1.3.2">
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<h6 data-number="A.0.1.1.3.2" class="anchored" data-anchor-id="getter-method-count"><span class="header-section-number">A.0.1.1.3.2</span> Getter : method <code>Count()</code></h6>
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<p>The <code>Count()</code> method allows to access to the count attribute as an integer value. If the <code>count</code> attribute is not defined for the given sequence, the value <em>1</em> is returned</p>
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</section>
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</section>
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<section id="merged_" class="level5" data-number="A.0.1.1.4">
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<h5 data-number="A.0.1.1.4" class="anchored" data-anchor-id="merged_"><span class="header-section-number">A.0.1.1.4</span> <code>merged_*</code></h5>
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<section id="type-mapstringint" class="level6" data-number="A.0.1.1.4.1">
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<h6 data-number="A.0.1.1.4.1" class="anchored" data-anchor-id="type-mapstringint"><span class="header-section-number">A.0.1.1.4.1</span> Type : <code>map[string]int</code></h6>
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</section>
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<section id="set-by-the-obiuniq-tool-1" class="level6" data-number="A.0.1.1.4.2">
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<h6 data-number="A.0.1.1.4.2" class="anchored" data-anchor-id="set-by-the-obiuniq-tool-1"><span class="header-section-number">A.0.1.1.4.2</span> Set by the <em>obiuniq</em> tool</h6>
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<p>The <code>-m</code> option of the <em>obiuniq</em> tools allows for keeping track of the distribution of the values stored in given attribute of interest. Often this option is used to summarise distribution of a sequence variant accross samples when <em>obiuniq</em> is run after running <em>obimultiplex</em>. The actual name of the attribute depends on the name of the monitored attribute. If <code>-m</code> option is used with the attribute <em>sample</em>, then this attribute names <em>merged_sample</em>.</p>
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</section>
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</section>
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<section id="mode" class="level5" data-number="A.0.1.1.5">
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<h5 data-number="A.0.1.1.5" class="anchored" data-anchor-id="mode"><span class="header-section-number">A.0.1.1.5</span> <code>mode</code></h5>
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||||
<section id="set-by-the-obipairing-tool-2" class="level6" data-number="A.0.1.1.5.1">
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||||
<h6 data-number="A.0.1.1.5.1" class="anchored" data-anchor-id="set-by-the-obipairing-tool-2"><span class="header-section-number">A.0.1.1.5.1</span> Set by the <em>obipairing</em> tool</h6>
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<p><strong><code>obitag_ref_index</code></strong></p>
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</section>
|
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<section id="set-by-the-obirefidx-tool." class="level6" data-number="A.0.1.1.5.2">
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||||
<h6 data-number="A.0.1.1.5.2" class="anchored" data-anchor-id="set-by-the-obirefidx-tool."><span class="header-section-number">A.0.1.1.5.2</span> Set by the <em>obirefidx</em> tool.</h6>
|
||||
<p>It resumes to which taxonomic annotation a match to that sequence must lead according to the number of differences existing between the query sequence and the reference sequence having that tag.</p>
|
||||
</section>
|
||||
<section id="getter-method-count-1" class="level6" data-number="A.0.1.1.5.3">
|
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<h6 data-number="A.0.1.1.5.3" class="anchored" data-anchor-id="getter-method-count-1"><span class="header-section-number">A.0.1.1.5.3</span> Getter : method <code>Count()</code></h6>
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<a href="./comm_computation.html" class="sidebar-item-text sidebar-link active"><span class="chapter-number">11</span> <span class="chapter-title">Computations on sequences</span></a>
|
||||
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|
||||
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|
||||
<li class="sidebar-item">
|
||||
<div class="sidebar-item-container">
|
||||
<a href="./comm_sampling.html" class="sidebar-item-text sidebar-link"><span class="chapter-number">12</span> <span class="chapter-title">Sequence sampling and filtering</span></a>
|
||||
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<a href="./comm_utilities.html" class="sidebar-item-text sidebar-link"><span class="chapter-number">13</span> <span class="chapter-title">Utilities</span></a>
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<span class="sidebar-item-text sidebar-link text-start">The GO *OBITools* library</span>
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<a href="./annexes.html" class="sidebar-item-text sidebar-link"><span class="chapter-number">A</span> <span class="chapter-title">Annexes</span></a>
|
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|
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<h2 id="toc-title">Table of contents</h2>
|
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<ul>
|
||||
<li><a href="#obipairing" id="toc-obipairing" class="nav-link active" data-scroll-target="#obipairing"><span class="toc-section-number">11.1</span> <code>obipairing</code></a>
|
||||
<ul class="collapse">
|
||||
<li><a href="#alignment-procedure" id="toc-alignment-procedure" class="nav-link" data-scroll-target="#alignment-procedure">Alignment procedure</a></li>
|
||||
<li><a href="#the-scoring-system" id="toc-the-scoring-system" class="nav-link" data-scroll-target="#the-scoring-system">The scoring system</a></li>
|
||||
</ul></li>
|
||||
<li><a href="#obimultiplex" id="toc-obimultiplex" class="nav-link" data-scroll-target="#obimultiplex"><span class="toc-section-number">11.2</span> <code>obimultiplex</code></a></li>
|
||||
<li><a href="#obicomplement" id="toc-obicomplement" class="nav-link" data-scroll-target="#obicomplement"><span class="toc-section-number">11.3</span> <code>obicomplement</code></a></li>
|
||||
<li><a href="#obiclean" id="toc-obiclean" class="nav-link" data-scroll-target="#obiclean"><span class="toc-section-number">11.4</span> <code>obiclean</code></a></li>
|
||||
<li><a href="#obiuniq" id="toc-obiuniq" class="nav-link" data-scroll-target="#obiuniq"><span class="toc-section-number">11.5</span> <code>obiuniq</code></a></li>
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<div class="quarto-title">
|
||||
<h1 class="title d-none d-lg-block"><span class="chapter-number">11</span> <span class="chapter-title">Computations on sequences</span></h1>
|
||||
</div>
|
||||
|
||||
|
||||
|
||||
<div class="quarto-title-meta">
|
||||
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||||
|
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|
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|
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|
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|
||||
</header>
|
||||
|
||||
<section id="obipairing" class="level2" data-number="11.1">
|
||||
<h2 data-number="11.1" class="anchored" data-anchor-id="obipairing"><span class="header-section-number">11.1</span> <code>obipairing</code></h2>
|
||||
<blockquote class="blockquote">
|
||||
<p>Replace the <code>illuminapairedends</code> original <em>OBITools</em></p>
|
||||
</blockquote>
|
||||
<section id="alignment-procedure" class="level3 unnumbered">
|
||||
<h3 class="unnumbered anchored" data-anchor-id="alignment-procedure">Alignment procedure</h3>
|
||||
<p><code>obipairing</code> is introducing a new alignment algorithm compared to the <code>illuminapairedend</code> command of the <code>OBITools V2</code>. Nethertheless this new algorithm has been design to produce the same results than the previous, except in very few cases.</p>
|
||||
<p>The new algorithm is a two-step procedure. First, a FASTN-type algorithm <span class="citation" data-cites="Lipman1985-hw">(<a href="references.html#ref-Lipman1985-hw" role="doc-biblioref">Lipman and Pearson 1985</a>)</span> identifies the best offset between the two matched readings. This identifies the region of overlap.</p>
|
||||
<p>In the second step, the matching regions of the two reads are extracted along with a flanking sequence of <span class="math inline">\(\Delta\)</span> base pairs. The two subsequences are then aligned using a “one side free end-gap” dynamic programming algorithm. This latter step is only called if at least one mismatch is detected by the FASTP step.</p>
|
||||
<p>Unless the similarity between the two reads at their overlap region is very low, the addition of the flanking regions in the second step of the alignment ensures the same alignment as if the dynamic programming alignment was performed on the full reads.</p>
|
||||
</section>
|
||||
<section id="the-scoring-system" class="level3 unnumbered">
|
||||
<h3 class="unnumbered anchored" data-anchor-id="the-scoring-system">The scoring system</h3>
|
||||
<p>In the dynamic programming step, the match and mismatch scores take into account the quality scores of the two aligned nucleotides. By taking these into account, the probability of a true match can be calculated for each aligned base pair.</p>
|
||||
<p>If we consider a nucleotide read with a quality score <span class="math inline">\(Q\)</span>, the probability of misreading this base (<span class="math inline">\(P_E\)</span>) is : <span class="math display">\[
|
||||
P_E = 10^{-\frac{Q}{10}}
|
||||
\]</span></p>
|
||||
<p>Thus, when a given nucleotide <span class="math inline">\(X\)</span> is observed with the quality score <span class="math inline">\(Q\)</span>. The probability that <span class="math inline">\(X\)</span> is really an <span class="math inline">\(X\)</span> is :</p>
|
||||
<p><span class="math display">\[
|
||||
P(X=X) = 1 - P_E
|
||||
\]</span></p>
|
||||
<p>Otherwise, <span class="math inline">\(X\)</span> is actually one of the three other possible nucleotides (<span class="math inline">\(X_{E1}\)</span>, <span class="math inline">\(X_{E2}\)</span> or <span class="math inline">\(X_{E3}\)</span>). If we suppose that the three reading error have the same probability :</p>
|
||||
<p><span class="math display">\[
|
||||
P(X=X_{E1}) = P(X=X_{E3}) = P(X=X_{E3}) = \frac{P_E}{3}
|
||||
\]</span></p>
|
||||
<p>At each position in an alignment where the two nucleotides <span class="math inline">\(X_1\)</span> and <span class="math inline">\(X_2\)</span> face each other (not a gapped position), the probability of a true match varies depending on whether <span class="math inline">\(X_1=X_2\)</span>, an observed match, or <span class="math inline">\(X_1 \neq X_2\)</span>, an observed mismatch.</p>
|
||||
<p><strong>Probability of a true match when <span class="math inline">\(X_1=X_2\)</span></strong></p>
|
||||
<p>That probability can be divided in two parts. First <span class="math inline">\(X_1\)</span> and <span class="math inline">\(X_2\)</span> have been correctly read. The corresponding probability is :</p>
|
||||
<p><span class="math display">\[
|
||||
\begin{aligned}
|
||||
P_{TM} &= (1- PE_1)(1-PE_2)\\
|
||||
&=(1 - 10^{-\frac{Q_1}{10} } )(1 - 10^{-\frac{Q_2}{10}} )
|
||||
\end{aligned}
|
||||
\]</span></p>
|
||||
<p>Secondly, a match can occure if the true nucleotides read as <span class="math inline">\(X_1\)</span> and <span class="math inline">\(X_2\)</span> are not <span class="math inline">\(X_1\)</span> and <span class="math inline">\(X_2\)</span> but identical.</p>
|
||||
<p><span class="math display">\[
|
||||
\begin{aligned}
|
||||
P(X_1==X_{E1}) \cap P(X_2==X_{E1}) &= \frac{P_{E1} P_{E2}}{9} \\
|
||||
P(X_1==X_{Ex}) \cap P(X_2==X_{Ex}) & = \frac{P_{E1} P_{E2}}{3}
|
||||
\end{aligned}
|
||||
\]</span></p>
|
||||
<p>The probability of a true match between <span class="math inline">\(X_1\)</span> and <span class="math inline">\(X_2\)</span> when <span class="math inline">\(X_1 = X_2\)</span> an observed match :</p>
|
||||
<p><span class="math display">\[
|
||||
\begin{aligned}
|
||||
P(MATCH | X_1 = X_2) = (1- PE_1)(1-PE_2) + \frac{P_{E1} P_{E2}}{3}
|
||||
\end{aligned}
|
||||
\]</span></p>
|
||||
<p><strong>Probability of a true match when <span class="math inline">\(X_1 \neq X_2\)</span></strong></p>
|
||||
<p>That probability can be divided in three parts.</p>
|
||||
<ol type="a">
|
||||
<li><span class="math inline">\(X_1\)</span> has been correctly read and <span class="math inline">\(X_2\)</span> is a sequencing error and is actually equal to <span class="math inline">\(X_1\)</span>. <span class="math display">\[
|
||||
P_a = (1-P_{E1})\frac{P_{E2}}{3}
|
||||
\]</span></li>
|
||||
<li><span class="math inline">\(X_2\)</span> has been correctly read and <span class="math inline">\(X_1\)</span> is a sequencing error and is actually equal to <span class="math inline">\(X_2\)</span>. <span class="math display">\[
|
||||
P_b = (1-P_{E2})\frac{P_{E1}}{3}
|
||||
\]</span></li>
|
||||
<li><span class="math inline">\(X_1\)</span> and <span class="math inline">\(X_2\)</span> corresponds to sequencing error but are actually the same base <span class="math inline">\(X_{Ex}\)</span> <span class="math display">\[
|
||||
P_c = 2\frac{P_{E1} P_{E2}}{9}
|
||||
\]</span></li>
|
||||
</ol>
|
||||
<p>Consequently : <span class="math display">\[
|
||||
\begin{aligned}
|
||||
P(MATCH | X_1 \neq X_2) = (1-P_{E1})\frac{P_{E2}}{3} + (1-P_{E2})\frac{P_{E1}}{3} + 2\frac{P_{E1} P_{E2}}{9}
|
||||
\end{aligned}
|
||||
\]</span></p>
|
||||
<p><strong>Probability of a match under the random model</strong></p>
|
||||
<p>The second considered model is a pure random model where every base is equiprobable, hence having a probability of occurrence of a nucleotide equals <span class="math inline">\(0.25\)</span>. Under that hypothesis</p>
|
||||
<p><span class="math display">\[
|
||||
P(MATCH | \text{Random model}) = 0.25
|
||||
\]</span></p>
|
||||
<p><strong>The score is a log ration of likelyhood</strong></p>
|
||||
<p>Score is define as the logarithm of the ratio between the likelyhood of the observations considering the sequencer error model over tha likelyhood u</p>
|
||||
<div class="cell">
|
||||
<div class="cell-output-display">
|
||||
<div class="quarto-figure quarto-figure-center">
|
||||
<figure class="figure">
|
||||
<p><img src="comm_computation_files/figure-html/unnamed-chunk-1-1.png" class="img-fluid figure-img" width="672"></p>
|
||||
<p></p><figcaption class="figure-caption">Evolution of the match and mismatch scores when the quality of base is 20 while the second range from 10 to 40.</figcaption><p></p>
|
||||
</figure>
|
||||
</div>
|
||||
</div>
|
||||
</div>
|
||||
</section>
|
||||
</section>
|
||||
<section id="obimultiplex" class="level2" data-number="11.2">
|
||||
<h2 data-number="11.2" class="anchored" data-anchor-id="obimultiplex"><span class="header-section-number">11.2</span> <code>obimultiplex</code></h2>
|
||||
<blockquote class="blockquote">
|
||||
<p>Replace the <code>ngsfilter</code> original <em>OBITools</em></p>
|
||||
</blockquote>
|
||||
</section>
|
||||
<section id="obicomplement" class="level2" data-number="11.3">
|
||||
<h2 data-number="11.3" class="anchored" data-anchor-id="obicomplement"><span class="header-section-number">11.3</span> <code>obicomplement</code></h2>
|
||||
</section>
|
||||
<section id="obiclean" class="level2" data-number="11.4">
|
||||
<h2 data-number="11.4" class="anchored" data-anchor-id="obiclean"><span class="header-section-number">11.4</span> <code>obiclean</code></h2>
|
||||
</section>
|
||||
<section id="obiuniq" class="level2" data-number="11.5">
|
||||
<h2 data-number="11.5" class="anchored" data-anchor-id="obiuniq"><span class="header-section-number">11.5</span> <code>obiuniq</code></h2>
|
||||
|
||||
|
||||
<div id="refs" class="references csl-bib-body hanging-indent" role="doc-bibliography" style="display: none">
|
||||
<div id="ref-Lipman1985-hw" class="csl-entry" role="doc-biblioentry">
|
||||
Lipman, D J, and W R Pearson. 1985. <span>“<span class="nocase">Rapid and sensitive protein similarity searches</span>.”</span> <em>Science</em> 227 (4693): 1435–41. <a href="http://www.ncbi.nlm.nih.gov/pubmed/2983426">http://www.ncbi.nlm.nih.gov/pubmed/2983426</a>.
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<li><a href="#obigrep-filters-sequence-files-according-to-numerous-conditions" id="toc-obigrep-filters-sequence-files-according-to-numerous-conditions" class="nav-link active" data-scroll-target="#obigrep-filters-sequence-files-according-to-numerous-conditions"><span class="toc-section-number">12.1</span> <code>obigrep</code> – filters sequence files according to numerous conditions</a>
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<h1 class="title d-none d-lg-block"><span class="chapter-number">12</span> <span class="chapter-title">Sequence sampling and filtering</span></h1>
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<h2 data-number="12.1" class="anchored" data-anchor-id="obigrep-filters-sequence-files-according-to-numerous-conditions"><span class="header-section-number">12.1</span> <code>obigrep</code> – filters sequence files according to numerous conditions</h2>
|
||||
<p>The <code>obigrep</code> command is somewhat analogous to the standard Unix <code>grep</code> command. It selects a subset of sequence records from a sequence file. A sequence record is a complex object consisting of an identifier, a set of attributes (a key, defined by its name, associated with a value), a definition, and the sequence itself. Instead of working text line by text line like the standard Unix tool, <code>obigrep</code> selection is done sequence record by sequence record. A large number of options allow you to refine the selection on any element of the sequence. <code>obigrep</code> allows you to specify multiple conditions simultaneously (which take on the value <code>TRUE</code> or <code>FALSE</code>) and only those sequence records which meet all conditions (all conditions are <code>TRUE</code>) are selected. <code>obigrep</code> is able to work on two paired read files. The selection criteria apply to one or the other of the readings in each pair depending on the mode chosen (<strong>--paired-mode</strong> option). In all cases the selection is applied in the same way to both files, thus maintaining their consistency.</p>
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<h4 data-number="12.1.1.1" class="anchored" data-anchor-id="selecting-sequences-based-on-their-caracteristics"><span class="header-section-number">12.1.1.1</span> Selecting sequences based on their caracteristics</h4>
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<p>Sequences can be selected on several of their caracteristics, their length, their id, their sequence. Options allow for specifying the condition if selection.</p>
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<dt><strong>--min-count</strong> | <strong>-c</strong> <em>COUNT</em></dt>
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|
||||
<p>only sequences reprensenting at least <em>COUNT</em> reads will be selected. That option rely on the <code>count</code> attribute. If the <code>count</code> attribute is not defined for a sequence record, it is assumed equal to <span class="math inline">\(1\)</span>.</p>
|
||||
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|
||||
<dt><strong>--max-count</strong> | <strong>-C</strong> <em>COUNT</em></dt>
|
||||
<dd>
|
||||
<p>only sequences reprensenting no more than <em>COUNT</em> reads will be selected. That option rely on the <code>count</code> attribute. If the <code>count</code> attribute is not defined for a sequence record, it is assumed equal to <span class="math inline">\(1\)</span>.</p>
|
||||
</dd>
|
||||
<dt>Example</dt>
|
||||
<dd>
|
||||
<p>Selecting sequence records representing at least five reads in the dataset.</p>
|
||||
</dd>
|
||||
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|
||||
<div class="sourceCode" id="cb1"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obigrep</span> <span class="at">-c</span> 5 data_SPER01.fasta <span class="op">></span> data_norare_SPER01.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
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482
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||||
<li><a href="#obicount" id="toc-obicount" class="nav-link active" data-scroll-target="#obicount"><span class="toc-section-number">13.1</span> <code>obicount</code></a></li>
|
||||
<li><a href="#obidistribute" id="toc-obidistribute" class="nav-link" data-scroll-target="#obidistribute"><span class="toc-section-number">13.2</span> <code>obidistribute</code></a></li>
|
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<h2 data-number="13.1" class="anchored" data-anchor-id="obicount"><span class="header-section-number">13.1</span> <code>obicount</code></h2>
|
||||
<p><code>obicount</code> counts the number of sequence records, the sum of the <code>count</code> attributes, and the sum of the length of all the sequences.</p>
|
||||
<p><em>Example:</em></p>
|
||||
<div class="sourceCode" id="cb1"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obicount</span> seq.fasta </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<p>Prints the number of sequence records contained in the <code>seq.fasta</code> file and the sum of their <code>count</code> attributes.</p>
|
||||
<p><em>Options specific to the command</em></p>
|
||||
<ul>
|
||||
<li><code>--reads|-r</code> Prints read counts.</li>
|
||||
<li><code>--symbols|-s</code> Prints symbol counts.</li>
|
||||
<li><code>--variants|-v</code> Prints variant counts.</li>
|
||||
</ul>
|
||||
</section>
|
||||
<section id="obidistribute" class="level2" data-number="13.2">
|
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|
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|
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<span class="sidebar-item-text sidebar-link text-start">The GO *OBITools* library</span>
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<li><a href="#helpful-options" id="toc-helpful-options" class="nav-link active" data-scroll-target="#helpful-options"><span class="toc-section-number">6.1</span> Helpful options</a></li>
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<h1 class="title d-none d-lg-block"><span class="chapter-number">6</span> <span class="chapter-title">Options common to most of the <em>OBITools</em> commands</span></h1>
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<h2 data-number="6.1" class="anchored" data-anchor-id="helpful-options"><span class="header-section-number">6.1</span> Helpful options</h2>
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<dl>
|
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<dt><strong>--help</strong>, <strong>-h</strong></dt>
|
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<dd>
|
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<p>Display a friendly help message.</p>
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<p><strong>--no-progressbar</strong></p>
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<h2 data-number="6.2" class="anchored" data-anchor-id="system-related-options"><span class="header-section-number">6.2</span> System related options</h2>
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<p><strong>Managing parallel execution of tasks</strong></p>
|
||||
<p>A new feature of <em>OBITools</em> V4 is the ability to run multiple tasks in parallel, reading files, calculating on the data, formatting and writing the results. Each of these tasks can itself be parallelized by dividing the data into batches and running the calculation on several batches in parallel. This allows the overall calculation time of an <em>OBITools</em> command to be reduced considerably. The parameters organizing the parallel calculation are determined automatically to use the maximum capacity of your computer. But in some circumstances, it is necessary to override these default settings either to try to optimize the computation on a given machine, or to limit the OBITools to using only a part of the computational capacity. There are two options for doing this.</p>
|
||||
<dl>
|
||||
<dt><strong>--max-cpu</strong></dt>
|
||||
<dd>
|
||||
<p>OBITools V4 are able to run in parallel on all the CPU cores available on the computer. It is sometime required to limit the computation to a smaller number of cores. That option specify the maximum number of cores that the OBITools command can use. This behaviour can also be set up using the <code>OBIMAXCPU</code> environment variable.</p>
|
||||
</dd>
|
||||
</dl>
|
||||
<p><strong>--workers</strong>, <strong>-w</strong></p>
|
||||
<p>If your computer has 8 cores, but you want to limit <em>OBITools</em> to use only two of them you have several solution:</p>
|
||||
<ul>
|
||||
<li><p>If you want to set the limit for a single execution you can use the <strong>–max-cpu</strong> option</p>
|
||||
<div class="sourceCode" id="cb1"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obiconvert</span> <span class="at">--max-cpu</span> 2 <span class="at">--fasta-output</span> data.fastq <span class="op">></span> data.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<p>or you can precede the command by setting the environment variable <code>OBIMAXCPU</code></p>
|
||||
<div class="sourceCode" id="cb2"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb2-1"><a href="#cb2-1" aria-hidden="true" tabindex="-1"></a><span class="va">OBIMAXCPU</span><span class="op">=</span>2 <span class="ex">obiconvert</span> <span class="at">--fasta-output</span> data.fastq <span class="op">></span> data.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div></li>
|
||||
<li><p>If you want to set the limit to your complete session, you have to export <code>OBIMAXCPU</code></p>
|
||||
<div class="sourceCode" id="cb3"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb3-1"><a href="#cb3-1" aria-hidden="true" tabindex="-1"></a><span class="bu">export</span> <span class="va">OBIMAXCPU</span><span class="op">=</span>2 </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<p>all the following OBITools commands will be limited to use at max 2 CPU cores.</p></li>
|
||||
<li><p>If all the time you want to impose this limit, you must include the above <code>export</code> command in your <code>.bashrc</code> file.</p></li>
|
||||
</ul>
|
||||
<p><strong>OBITools debuging related options</strong></p>
|
||||
<p><strong>--debug</strong></p>
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<li><a href="#variables-usable-in-the-expression" id="toc-variables-usable-in-the-expression" class="nav-link active" data-scroll-target="#variables-usable-in-the-expression"><span class="toc-section-number">7.1</span> Variables usable in the expression</a></li>
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<li><a href="#accessing-to-the-sequence-annotations" id="toc-accessing-to-the-sequence-annotations" class="nav-link" data-scroll-target="#accessing-to-the-sequence-annotations"><span class="toc-section-number">7.3</span> Accessing to the sequence annotations</a></li>
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<h1 class="title d-none d-lg-block"><span class="chapter-number">7</span> <span class="chapter-title">OBITools expression language</span></h1>
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<p>Several OBITools (<em>e.g.</em> obigrep, obiannotate) allow the user to specify some simple expressions to compute values or define predicates. This expressions are parsed and evaluated using the <a href="https://pkg.go.dev/github.com/PaesslerAG/gval" title="Gval (Go eVALuate) for evaluating arbitrary expressions Go-like expressions.">gval</a> go package, which allows for evaluating go-Like expression.</p>
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<h2 data-number="7.1" class="anchored" data-anchor-id="variables-usable-in-the-expression"><span class="header-section-number">7.1</span> Variables usable in the expression</h2>
|
||||
<ul>
|
||||
<li><code>sequence</code> is the sequence object on which the expression is evaluated.</li>
|
||||
<li><code>annotations</code>is a map object containing every annotations associated to the currently processed sequence.</li>
|
||||
</ul>
|
||||
</section>
|
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<section id="function-defined-in-the-language" class="level2" data-number="7.2">
|
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<h2 data-number="7.2" class="anchored" data-anchor-id="function-defined-in-the-language"><span class="header-section-number">7.2</span> Function defined in the language</h2>
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|
||||
<h3 class="unnumbered anchored" data-anchor-id="instrospection-functions">Instrospection functions</h3>
|
||||
<ul>
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||||
<li><code>len(x)</code>is a generic function allowing to retreive the size of a object. It returns the length of a sequences, the number of element in a map like <code>annotations</code>, the number of elements in an array. The reurned value is an <code>int</code>.</li>
|
||||
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|
||||
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|
||||
<section id="cast-functions" class="level3 unnumbered">
|
||||
<h3 class="unnumbered anchored" data-anchor-id="cast-functions">Cast functions</h3>
|
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|
||||
<li><code>int(x)</code> converts if possible the <code>x</code> value to an integer value. The function returns an <code>int</code>.</li>
|
||||
<li><code>numeric(x)</code> converts if possible the <code>x</code> value to a float value. The function returns a <code>float</code>.</li>
|
||||
<li><code>bool(x)</code> converts if possible the <code>x</code> value to a boolean value. The function returns a <code>bool</code>.</li>
|
||||
</ul>
|
||||
</section>
|
||||
<section id="string-related-functions" class="level3 unnumbered">
|
||||
<h3 class="unnumbered anchored" data-anchor-id="string-related-functions">String related functions</h3>
|
||||
<ul>
|
||||
<li><code>printf(format,...)</code> allows to combine several values to build a string. <code>format</code> follows the classical C <code>printf</code> syntax. The function returns a <code>string</code>.</li>
|
||||
<li><code>subspc(x)</code> substitutes every space in the <code>x</code> string by the underscore (<code>_</code>) character. The function returns a <code>string</code>.</li>
|
||||
</ul>
|
||||
</section>
|
||||
</section>
|
||||
<section id="accessing-to-the-sequence-annotations" class="level2" data-number="7.3">
|
||||
<h2 data-number="7.3" class="anchored" data-anchor-id="accessing-to-the-sequence-annotations"><span class="header-section-number">7.3</span> Accessing to the sequence annotations</h2>
|
||||
<p>The <code>annotations</code> variable is a map object containing all the annotations associated to the currently processed sequence. Index of the map are the attribute names. It exists to possibillities to retreive an annotation. It is possible to use the classical <code>[]</code> indexing operator, putting the attribute name quoted by double quotes between them.</p>
|
||||
<div class="sourceCode" id="cb1"><pre class="sourceCode go code-with-copy"><code class="sourceCode go"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a>annotations<span class="op">[</span><span class="st">"direction"</span><span class="op">]</span></span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<p>The above code retreives the <code>direction</code> annotation. A second notation using the dot (<code>.</code>) is often more convenient.</p>
|
||||
<div class="sourceCode" id="cb2"><pre class="sourceCode go code-with-copy"><code class="sourceCode go"><span id="cb2-1"><a href="#cb2-1" aria-hidden="true" tabindex="-1"></a>annotations<span class="op">.</span>direction</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<p>Special attributes of the sequence are accessible only by dedicated methods of the <code>sequence</code> object.</p>
|
||||
<ul>
|
||||
<li>The sequence identifier : <code>Id()</code></li>
|
||||
<li>THe sequence definition : <code>Definition()</code></li>
|
||||
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<div class="sidebar-item-container">
|
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<a href="./comm_utilities.html" class="sidebar-item-text sidebar-link"><span class="chapter-number">13</span> <span class="chapter-title">Utilities</span></a>
|
||||
</div>
|
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|
||||
</ul>
|
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|
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<li class="sidebar-item sidebar-item-section">
|
||||
<span class="sidebar-item-text sidebar-link text-start">The GO *OBITools* library</span>
|
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</li>
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<li class="sidebar-item sidebar-item-section">
|
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<div class="sidebar-item-container">
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<a class="sidebar-item-text sidebar-link text-start" data-bs-toggle="collapse" data-bs-target="#quarto-sidebar-section-4" aria-expanded="true">Appendices</a>
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<a class="sidebar-item-toggle text-start" data-bs-toggle="collapse" data-bs-target="#quarto-sidebar-section-4" aria-expanded="true">
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<i class="bi bi-chevron-right ms-2"></i>
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<li class="sidebar-item">
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<div class="sidebar-item-container">
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<a href="./annexes.html" class="sidebar-item-text sidebar-link"><span class="chapter-number">A</span> <span class="chapter-title">Annexes</span></a>
|
||||
</div>
|
||||
</li>
|
||||
<li class="sidebar-item">
|
||||
<div class="sidebar-item-container">
|
||||
<a href="./references.html" class="sidebar-item-text sidebar-link">References</a>
|
||||
</div>
|
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</li>
|
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|
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<div id="quarto-margin-sidebar" class="sidebar margin-sidebar">
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<nav id="TOC" role="doc-toc" class="toc-active">
|
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<h2 id="toc-title">Table of contents</h2>
|
||||
|
||||
<ul>
|
||||
<li><a href="#the-dna-sequence-data" id="toc-the-dna-sequence-data" class="nav-link active" data-scroll-target="#the-dna-sequence-data"><span class="toc-section-number">3</span> The DNA sequence data</a>
|
||||
<ul class="collapse">
|
||||
<li><a href="#the-iupac-code" id="toc-the-iupac-code" class="nav-link" data-scroll-target="#the-iupac-code"><span class="toc-section-number">3.1</span> The IUPAC Code</a></li>
|
||||
<li><a href="#sec-fasta" id="toc-sec-fasta" class="nav-link" data-scroll-target="#sec-fasta"><span class="toc-section-number">3.2</span> The <em>fasta</em> sequence format</a></li>
|
||||
<li><a href="#sec-fastq" id="toc-sec-fastq" class="nav-link" data-scroll-target="#sec-fastq"><span class="toc-section-number">3.3</span> The <em>fastq</em> sequence format</a>
|
||||
<ul class="collapse">
|
||||
<li><a href="#sequence-quality-scores" id="toc-sequence-quality-scores" class="nav-link" data-scroll-target="#sequence-quality-scores">Sequence quality scores</a></li>
|
||||
</ul></li>
|
||||
<li><a href="#file-extension" id="toc-file-extension" class="nav-link" data-scroll-target="#file-extension"><span class="toc-section-number">3.4</span> File extension</a></li>
|
||||
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|
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|
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|
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<header id="title-block-header" class="quarto-title-block default">
|
||||
<div class="quarto-title">
|
||||
<h1 class="title d-none d-lg-block"><span class="chapter-number">2</span> <span class="chapter-title">File formats usable with <em>OBITools</em></span></h1>
|
||||
</div>
|
||||
|
||||
|
||||
|
||||
<div class="quarto-title-meta">
|
||||
|
||||
|
||||
|
||||
|
||||
</div>
|
||||
|
||||
|
||||
</header>
|
||||
|
||||
<p>OBITools manipulate have to manipulate DNA sequence data and taxonomical data. They can use some supplentary metadata describing the experiment and produce some stats about the processed DNA data. All the manipulated data are stored in text files, following standard data format.</p>
|
||||
<section id="the-dna-sequence-data" class="level1" data-number="3">
|
||||
<h1 data-number="3"><span class="header-section-number">3</span> The DNA sequence data</h1>
|
||||
<p>Sequences can be stored following various format. OBITools knows some of them. The central formats for sequence files manipulated by OBITools scripts are the <a href="#the-fasta-sequence-format"><code>fasta</code></a> and <a href="#the-fastq-sequence-format"><code>fastq</code></a> format. OBITools extends the both these formats by specifying a syntax to include in the definition line data qualifying the sequence. All file formats use the <code>IUPAC</code> code for encoding nucleotides.</p>
|
||||
<p>Moreover these two formats that can be used as input and output formats, <strong>OBITools4</strong> can read the following format :</p>
|
||||
<ul>
|
||||
<li><a href="https://ena-docs.readthedocs.io/en/latest/submit/fileprep/flat-file-example.html">EBML flat file</a> format (use by ENA)</li>
|
||||
<li><a href="https://www.ncbi.nlm.nih.gov/Sitemap/samplerecord.html">Genbank flat file format</a></li>
|
||||
<li><a href="https://pythonhosted.org/OBITools/scripts/ecoPCR.html">ecoPCR output files</a></li>
|
||||
</ul>
|
||||
<section id="the-iupac-code" class="level2" data-number="3.1">
|
||||
<h2 data-number="3.1" class="anchored" data-anchor-id="the-iupac-code"><span class="header-section-number">3.1</span> The IUPAC Code</h2>
|
||||
<p>The International Union of Pure and Applied Chemistry (IUPAC_) defined the standard code for representing protein or DNA sequences.</p>
|
||||
<table class="table">
|
||||
<thead>
|
||||
<tr class="header">
|
||||
<th><strong>Code</strong></th>
|
||||
<th><strong>Nucleotide</strong></th>
|
||||
</tr>
|
||||
</thead>
|
||||
<tbody>
|
||||
<tr class="odd">
|
||||
<td>A</td>
|
||||
<td>Adenine</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td>C</td>
|
||||
<td>Cytosine</td>
|
||||
</tr>
|
||||
<tr class="odd">
|
||||
<td>G</td>
|
||||
<td>Guanine</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td>T</td>
|
||||
<td>Thymine</td>
|
||||
</tr>
|
||||
<tr class="odd">
|
||||
<td>U</td>
|
||||
<td>Uracil</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td>R</td>
|
||||
<td>Purine (A or G)</td>
|
||||
</tr>
|
||||
<tr class="odd">
|
||||
<td>Y</td>
|
||||
<td>Pyrimidine (C, T, or U)</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td>M</td>
|
||||
<td>C or A</td>
|
||||
</tr>
|
||||
<tr class="odd">
|
||||
<td>K</td>
|
||||
<td>T, U, or G</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td>W</td>
|
||||
<td>T, U, or A</td>
|
||||
</tr>
|
||||
<tr class="odd">
|
||||
<td>S</td>
|
||||
<td>C or G</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td>B</td>
|
||||
<td>C, T, U, or G (not A)</td>
|
||||
</tr>
|
||||
<tr class="odd">
|
||||
<td>D</td>
|
||||
<td>A, T, U, or G (not C)</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td>H</td>
|
||||
<td>A, T, U, or C (not G)</td>
|
||||
</tr>
|
||||
<tr class="odd">
|
||||
<td>V</td>
|
||||
<td>A, C, or G (not T, not U)</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td>N</td>
|
||||
<td>Any base (A, C, G, T, or U)</td>
|
||||
</tr>
|
||||
</tbody>
|
||||
</table>
|
||||
</section>
|
||||
<section id="sec-fasta" class="level2" data-number="3.2">
|
||||
<h2 data-number="3.2" class="anchored" data-anchor-id="sec-fasta"><span class="header-section-number">3.2</span> The <em>fasta</em> sequence format</h2>
|
||||
<p>The <strong>fasta format</strong> is certainly the most widely used sequence file format. This is certainly due to its great simplicity. It was originally created for the Lipman and Pearson <a href="http://www.ncbi.nlm.nih.gov/pubmed/3162770?dopt=Citation">FASTA program</a>. OBITools use in more of the classical <code>fasta</code> format an <code>extended version</code> of this format where structured data are included in the title line.</p>
|
||||
<p>In <em>fasta</em> format a sequence is represented by a title line beginning with a <strong><code>></code></strong> character and the sequences by itself following the :doc:<code>iupac</code> code. The sequence is usually split other severals lines of the same length (expect for the last one)</p>
|
||||
<pre><code>>my_sequence this is my pretty sequence
|
||||
ACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGT
|
||||
GTGCTGACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTGTTT
|
||||
AACGACGTTGCAGTACGTTGCAGT</code></pre>
|
||||
<p>This is no special format for the title line excepting that this line should be unique. Usually the first word following the <strong>></strong> character is considered as the sequence identifier. The end of the title line corresponding to a description of the sequence. Several sequences can be concatenated in a same file. The description of the next sequence is just pasted at the end of the record of the previous one</p>
|
||||
<pre><code>>sequence_A this is my first pretty sequence
|
||||
ACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGT
|
||||
GTGCTGACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTGTTT
|
||||
AACGACGTTGCAGTACGTTGCAGT
|
||||
>sequence_B this is my second pretty sequence
|
||||
ACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGT
|
||||
GTGCTGACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTGTTT
|
||||
AACGACGTTGCAGTACGTTGCAGT
|
||||
>sequence_C this is my third pretty sequence
|
||||
ACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGT
|
||||
GTGCTGACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTACGTTGCAGTGTTT
|
||||
AACGACGTTGCAGTACGTTGCAGT</code></pre>
|
||||
</section>
|
||||
<section id="sec-fastq" class="level2" data-number="3.3">
|
||||
<h2 data-number="3.3" class="anchored" data-anchor-id="sec-fastq"><span class="header-section-number">3.3</span> The <em>fastq</em> sequence format<a href="#fn1" class="footnote-ref" id="fnref1" role="doc-noteref"><sup>1</sup></a></h2>
|
||||
<p>The <strong>FASTQ</strong> format is a text file format for storing both biological sequences (only nucleic acid sequences) and the associated quality scores. The sequence and score are each encoded by a single ASCII character. This format was originally developed by the Wellcome Trust Sanger Institute to link a <a href="#the-fasta-sequence-format">FASTA</a> sequence file to the corresponding quality data, but has recently become the de facto standard for storing results from high-throughput sequencers <span class="citation" data-cites="cock2010sanger">(<a href="references.html#ref-cock2010sanger" role="doc-biblioref">Cock et al. 2010</a>)</span>.</p>
|
||||
<p>A fastq file normally uses four lines per sequence.</p>
|
||||
<ul>
|
||||
<li>Line 1 begins with a ‘@’ character and is followed by a sequence identifier and an <em>optional</em> description (like a :ref:<code>fasta</code> title line).</li>
|
||||
<li>Line 2 is the raw sequence letters.</li>
|
||||
<li>Line 3 begins with a ‘+’ character and is <em>optionally</em> followed by the same sequence identifier (and any description) again.</li>
|
||||
<li>Line 4 encodes the quality values for the sequence in Line 2, and must contain the same number of symbols as letters in the sequence.</li>
|
||||
</ul>
|
||||
<p>A fastq file containing a single sequence might look like this:</p>
|
||||
<pre><code>@SEQ_ID
|
||||
GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT
|
||||
+
|
||||
!''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65</code></pre>
|
||||
<p>The character ‘!’ represents the lowest quality while ‘~’ is the highest. Here are the quality value characters in left-to-right increasing order of quality (<code>ASCII</code>):</p>
|
||||
<pre><code>!"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]
|
||||
^_`abcdefghijklmnopqrstuvwxyz{|}~</code></pre>
|
||||
<p>The original Sanger FASTQ files also allowed the sequence and quality strings to be wrapped (split over multiple lines), but this is generally discouraged as it can make parsing complicated due to the unfortunate choice of “@” and “+” as markers (these characters can also occur in the quality string).</p>
|
||||
<section id="sequence-quality-scores" class="level3 unnumbered">
|
||||
<h3 class="unnumbered anchored" data-anchor-id="sequence-quality-scores">Sequence quality scores</h3>
|
||||
<p>The Phred quality value <em>Q</em> is an integer mapping of <em>p</em> (i.e., the probability that the corresponding base call is incorrect). Two different equations have been in use. The first is the standard Sanger variant to assess reliability of a base call, otherwise known as Phred quality score:</p>
|
||||
<p><span class="math display">\[
|
||||
Q_\text{sanger} = -10 \, \log_{10} p
|
||||
\]</span></p>
|
||||
<p>The Solexa pipeline (i.e., the software delivered with the Illumina Genome Analyzer) earlier used a different mapping, encoding the odds <span class="math inline">\(\mathbf{p}/(1-\mathbf{p})\)</span> instead of the probability <span class="math inline">\(\mathbf{p}\)</span>:</p>
|
||||
<p><span class="math display">\[
|
||||
Q_\text{solexa-prior to v.1.3} = -10 \; \log_{10} \frac{p}{1-p}
|
||||
\]</span></p>
|
||||
<p>Although both mappings are asymptotically identical at higher quality values, they differ at lower quality levels (i.e., approximately <span class="math inline">\(\mathbf{p} > 0.05\)</span>, or equivalently, <span class="math inline">\(\mathbf{Q} < 13\)</span>).</p>
|
||||
<div id="fig-Probabilitymetrics" class="quarto-figure quarto-figure-center anchored">
|
||||
<figure class="figure">
|
||||
<p><img src="Probabilitymetrics.png" class="img-fluid figure-img"></p>
|
||||
<p></p><figcaption class="figure-caption">Figure 3.1: Relationship between <em>Q</em> and <em>p</em> using the Sanger (red) and Solexa (black) equations (described above). The vertical dotted line indicates <span class="math inline">\(\mathbf{p}= 0.05\)</span>, or equivalently, <span class="math inline">\(Q = 13\)</span>.</figcaption><p></p>
|
||||
</figure>
|
||||
</div>
|
||||
<section id="encoding" class="level4 unnumbered">
|
||||
<h4 class="unnumbered anchored" data-anchor-id="encoding">Encoding</h4>
|
||||
<p>The <em>fastq</em> format had differente way of encoding the Phred quality score along the time. Here a breif history of these changes is presented.</p>
|
||||
<ul>
|
||||
<li>Sanger format can encode a Phred quality score from 0 to 93 using ASCII 33 to 126 (although in raw read data the Phred quality score rarely exceeds 60, higher scores are possible in assemblies or read maps).</li>
|
||||
<li>Solexa/Illumina 1.0 format can encode a Solexa/Illumina quality score from -5 to 62 using ASCII 59 to 126 (although in raw read data Solexa scores from -5 to 40 only are expected)</li>
|
||||
<li>Starting with Illumina 1.3 and before Illumina 1.8, the format encoded a Phred quality score from 0 to 62 using ASCII 64 to 126 (although in raw read data Phred scores from 0 to 40 only are expected).</li>
|
||||
<li>Starting in Illumina 1.5 and before Illumina 1.8, the Phred scores 0 to 2 have a slightly different meaning. The values 0 and 1 are no longer used and the value 2, encoded by ASCII 66 “B”.</li>
|
||||
</ul>
|
||||
<blockquote class="blockquote">
|
||||
<p>Sequencing Control Software, Version 2.6, (Catalog # SY-960-2601, Part # 15009921 Rev. A, November 2009, page 30) states the following: <em>If a read ends with a segment of mostly low quality (Q15 or below), then all of the quality values in the segment are replaced with a value of 2 (encoded as the letter B in Illumina’s text-based encoding of quality scores)… This Q2 indicator does not predict a specific error rate, but rather indicates that a specific final portion of the read should not be used in further analyses.</em> Also, the quality score encoded as “B” letter may occur internally within reads at least as late as pipeline version 1.6, as shown in the following example:</p>
|
||||
</blockquote>
|
||||
<pre><code>@HWI-EAS209_0006_FC706VJ:5:58:5894:21141#ATCACG/1
|
||||
TTAATTGGTAAATAAATCTCCTAATAGCTTAGATNTTACCTTNNNNNNNNNNTAGTTTCTTGAGA
|
||||
TTTGTTGGGGGAGACATTTTTGTGATTGCCTTGAT
|
||||
+HWI-EAS209_0006_FC706VJ:5:58:5894:21141#ATCACG/1
|
||||
efcfffffcfeefffcffffffddf`feed]`]_Ba_^__[YBBBBBBBBBBRTT\]][ dddd`
|
||||
ddd^dddadd^BBBBBBBBBBBBBBBBBBBBBBBB</code></pre>
|
||||
<p>An alternative interpretation of this ASCII encoding has been proposed. Also, in Illumina runs using PhiX controls, the character ‘B’ was observed to represent an “unknown quality score”. The error rate of ‘B’ reads was roughly 3 phred scores lower the mean observed score of a given run.</p>
|
||||
<ul>
|
||||
<li>Starting in Illumina 1.8, the quality scores have basically returned to the use of the Sanger format (Phred+33).</li>
|
||||
</ul>
|
||||
<p>OBItools follows the Sanger format. Nevertheless, It is possible to read files encoded following the Solexa/Illumina format by applying a shift of 62 (see the option <strong>--solexa</strong> of the OBITools commands).</p>
|
||||
</section>
|
||||
</section>
|
||||
</section>
|
||||
<section id="file-extension" class="level2" data-number="3.4">
|
||||
<h2 data-number="3.4" class="anchored" data-anchor-id="file-extension"><span class="header-section-number">3.4</span> File extension</h2>
|
||||
<p>There is no standard file extension for a FASTQ file, but .fq and .fastq, are commonly used.</p>
|
||||
|
||||
|
||||
<div id="refs" class="references csl-bib-body hanging-indent" role="doc-bibliography" style="display: none">
|
||||
<div id="ref-cock2010sanger" class="csl-entry" role="doc-biblioentry">
|
||||
Cock, Peter JA, Christopher J Fields, Naohisa Goto, Michael L Heuer, and Peter M Rice. 2010. <span>“The Sanger FASTQ File Format for Sequences with Quality Scores, and the Solexa/Illumina FASTQ Variants.”</span> <em>Nucleic Acids Research</em> 38 (6): 1767–71.
|
||||
</div>
|
||||
</div>
|
||||
</section>
|
||||
</section>
|
||||
<section id="footnotes" class="footnotes footnotes-end-of-document" role="doc-endnotes">
|
||||
<hr>
|
||||
<ol>
|
||||
<li id="fn1"><p>This article uses material from the Wikipedia article <a href="http://en.wikipedia.org/wiki/FASTQ_format"><code>FASTQ format</code></a> which is released under the <code>Creative Commons Attribution-Share-Alike License 3.0</code><a href="#fnref1" class="footnote-back" role="doc-backlink">↩︎</a></p></li>
|
||||
</ol>
|
||||
</section>
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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<span class="sidebar-item-text sidebar-link text-start">The GO *OBITools* library</span>
|
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|
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|
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|
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|
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|
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|
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<h1 class="title d-none d-lg-block">OBITools V4</h1>
|
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|
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|
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|
||||
|
||||
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|
||||
|
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|
||||
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|
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|
||||
<p>Eric Coissac </p>
|
||||
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|
||||
</div>
|
||||
|
||||
<div>
|
||||
<div class="quarto-title-meta-heading">Published</div>
|
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<div class="quarto-title-meta-contents">
|
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<p class="date">January 17, 2023</p>
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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<section id="preface" class="level1 unnumbered">
|
||||
<h1 class="unnumbered">Preface</h1>
|
||||
<p>The first version of <em>OBITools</em> started to be developed in 2005. This was at the beginning of the DNA metabarcoding story at the Laboratoire d’Ecologie Alpine (LECA) in Grenoble. At that time, with Pierre Taberlet and François Pompanon, we were thinking about the potential of this new methodology under development. PIerre and François developed more the laboratory methods, while I was thinking more about the tools for analysing the sequences produced. Two ideas were behind this development. I wanted something modular, and something easy to extend. To achieve the first goal, I decided to implement obitools as a suite of unix commands mimicking the classic unix commands but dedicated to sequence files. The basic unix commands are very useful for automatically manipulating, parsing and editing text files. They work in flow, line by line on the input text. The result is a new text file that can be used as input for the next command. Such a design makes it possible to quickly develop a text processing pipeline by chaining simple elementary operations. The <em>OBITools</em> are the exact counterpart of these basic Unix commands, but the basic information they process is a sequence (potentially spanning several lines of text), not a single line of text. Most <em>OBITools</em> consume sequence files and produce sequence files. Thus, the principles of chaining and modularity are respected. In order to be able to easily extend the <em>OBITools</em> to keep up with our evolving ideas about processing DNA metabarcoding data, it was decided to develop them using an interpreted language: Python. Python 2, the version available at the time, allowed us to develop the <em>OBITools</em> efficiently. When parts of the algorithms were computationally demanding, they were implemented in C and linked to the Python code. Even though Python is not the most efficient language available, even though computers were not as powerful as they are today, the size of the data we could produce using 454 sequencers or early solexa machines was small enough to be processed in a reasonable time.</p>
|
||||
<p>The first public version of obitools was <a href="https://metabarcoding.org/obitools"><em>OBITools2</em></a> <span class="citation" data-cites="Boyer2016-gq">(<a href="references.html#ref-Boyer2016-gq" role="doc-biblioref">Boyer et al. 2016</a>)</span>, this was actually a cleaned up and documented version of <em>OBITools</em> that had been running at LECA for years and was not really distributed except to a few collaborators. This is where <em>OBITools</em> started its public life from then on. The DNA metabarcoding spring schools provided and still provide user training every year. But <em>OBITools2</em> soon suffered from two limitations: it was developed in Python2, which was increasingly abandoned in favour of Python3, and the data size kept increasing with the new illumina machines. Python’s intrinsic slowness coupled with the increasing size of the datasets made OBITools computation times increasingly long. The abandonment of all maintenance of Python2 by its developers also imposed the need for a new version of OBITools.</p>
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<p><a href="https://metabarcoding.org/obitools3"><em>OBITools3</em></a> was the first response to this crisis. Developed and maintained by <a href="https://www.celine-mercier.info">Céline Mercier</a>, <em>OBITools3</em> attempted to address several limitations of <em>OBITools2</em>. It is a complete new code, mainly developed in Python3, with most of the lower layer code written in C for efficiency. OBITools3 has also abandoned text files for binary files for the same reason of efficiency. They have been replaced by a database structure that keeps track of every operation performed on the data.</p>
|
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<p>Here we present <em>OBITools4</em> which can be seen as a return to the origins of OBITools. While <em>OBITools3</em> offered traceability of analyses, which is in line with the concept of open science, and faster execution, <em>OBITools2</em> was more versatile and not only usable for the analysis of DNA metabarcoding data. <em>OBITools4</em> is the third full implementation of <em>OBITools</em>. The idea behind this new version is to go back to the original design of <em>OBITools</em> which ran on text files containing sequences, like the classic Unix commands, but running at least as fast as <em>OBITools3</em> and taking advantage of the multicore architecture of all modern laptops. For this, the idea of relying on an interpreted language was abandoned. The <em>OBITools4</em> are now fully implemented in the <a href="https://go.dev">GO</a> language with the exception of a few small pieces of specific code already implemented very efficiently in C. <em>OBITools4</em> also implement a new format for the annotations inserted in the header of every sequences. Rather tha relying on a format specific to <em>OBITools</em>, by default <em>OBITools4</em> use the <a href="https://www.json.org">JSON</a> format. This simplifies the writing of parsers in any languages, and thus allows obitools to easiestly interact with other software.</p>
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Boyer, Frédéric, Céline Mercier, Aurélie Bonin, Yvan Le Bras, Pierre Taberlet, and Eric Coissac. 2016. <span>“<span class="nocase">obitools: a unix-inspired software package for DNA metabarcoding</span>.”</span> <em>Molecular Ecology Resources</em> 16 (1): 176–82. <a href="https://doi.org/10.1111/1755-0998.12428">https://doi.org/10.1111/1755-0998.12428</a>.
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<a href="./comm_sampling.html" class="sidebar-item-text sidebar-link"><span class="chapter-number">12</span> <span class="chapter-title">Sequence sampling and filtering</span></a>
|
||||
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|
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|
||||
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|
||||
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|
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|
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|
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|
||||
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|
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|
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<span class="sidebar-item-text sidebar-link text-start">The GO *OBITools* library</span>
|
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|
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|
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|
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|
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<li><a href="#availability-of-the-obitools" id="toc-availability-of-the-obitools" class="nav-link active" data-scroll-target="#availability-of-the-obitools"><span class="toc-section-number">1.1</span> Availability of the OBITools</a></li>
|
||||
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|
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|
||||
<li><a href="#compilation-from-sources" id="toc-compilation-from-sources" class="nav-link" data-scroll-target="#compilation-from-sources"><span class="toc-section-number">1.4</span> Compilation from sources</a></li>
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<h1 class="title d-none d-lg-block"><span class="chapter-number">1</span> <span class="chapter-title">Installation of the obitools</span></h1>
|
||||
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|
||||
|
||||
|
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|
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||||
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|
||||
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||||
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|
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|
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|
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<p>The <em>OBITools</em> are open source and protected by the <a href="http://www.cecill.info/licences/Licence_CeCILL_V2.1-en.html">CeCILL 2.1 license</a>.</p>
|
||||
<p>All the sources of the <a href="http://metabarcoding.org/obitools4"><em>OBITools4</em></a> can be downloaded from the metabarcoding git server (https://git.metabarcoding.org).</p>
|
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|
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|
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<h2 data-number="1.2" class="anchored" data-anchor-id="prerequisites"><span class="header-section-number">1.2</span> Prerequisites</h2>
|
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<p>The <em>OBITools4</em> are developped using the <a href="https://go.dev/">GO programming language</a>, we stick to the latest version of the language, today the <span class="math inline">\(1.19.5\)</span>. If you want to download and compile the sources yourself, you first need to install the corresponding compiler on your system. Some parts of the soft are also written in C, therefore a recent C compiler is also requested, GCC on Linux or Windows, the Developer Tools on Mac.</p>
|
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|
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|
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<span class="sidebar-item-text sidebar-link text-start">The GO *OBITools* library</span>
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<h2 id="toc-title">Table of contents</h2>
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<li><a href="#aims-of-obitools" id="toc-aims-of-obitools" class="nav-link active" data-scroll-target="#aims-of-obitools">Aims of <em>OBITools</em></a></li>
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<h1 class="title d-none d-lg-block">The OBITools</h1>
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<p>The <em>OBITools4</em> are programs specifically designed for analyzing NGS data in a DNA metabarcoding context, taking into account taxonomic information. It is distributed as an open source software available on the following website: http://metabarcoding.org/obitools4.</p>
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<section id="aims-of-obitools" class="level2">
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<h2 class="anchored" data-anchor-id="aims-of-obitools">Aims of <em>OBITools</em></h2>
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||||
<p>DNA metabarcoding is an efficient approach for biodiversity studies <span class="citation" data-cites="Taberlet2012-pf">(<a href="references.html#ref-Taberlet2012-pf" role="doc-biblioref">Taberlet et al. 2012</a>)</span>. Originally mainly developed by microbiologists <span class="citation" data-cites="Sogin2006-ab">(<em>e.g.</em> <a href="references.html#ref-Sogin2006-ab" role="doc-biblioref">Sogin et al. 2006</a>)</span>, it is now widely used for plants <span class="citation" data-cites="Sonstebo2010-vv Yoccoz2012-ix Parducci2012-rn">(<em>e.g.</em> <a href="references.html#ref-Sonstebo2010-vv" role="doc-biblioref">Sønstebø et al. 2010</a>; <a href="references.html#ref-Yoccoz2012-ix" role="doc-biblioref">Yoccoz et al. 2012</a>; <a href="references.html#ref-Parducci2012-rn" role="doc-biblioref">Parducci et al. 2012</a>)</span> and animals from meiofauna <span class="citation" data-cites="Chariton2010-cz Baldwin2013-yc">(<em>e.g.</em> <a href="references.html#ref-Chariton2010-cz" role="doc-biblioref">Chariton et al. 2010</a>; <a href="references.html#ref-Baldwin2013-yc" role="doc-biblioref">Baldwin et al. 2013</a>)</span> to larger organisms <span class="citation" data-cites="Andersen2012-gj Thomsen2012-au">(<em>e.g.</em> <a href="references.html#ref-Andersen2012-gj" role="doc-biblioref">Andersen et al. 2012</a>; <a href="references.html#ref-Thomsen2012-au" role="doc-biblioref">Thomsen et al. 2012</a>)</span>. Interestingly, this method is not limited to <em>sensu stricto</em> biodiversity surveys, but it can also be implemented in other ecological contexts such as for herbivore <span class="citation" data-cites="Valentini2009-ay Kowalczyk2011-kg">(e.g. <a href="references.html#ref-Valentini2009-ay" role="doc-biblioref">Valentini et al. 2009</a>; <a href="references.html#ref-Kowalczyk2011-kg" role="doc-biblioref">Kowalczyk et al. 2011</a>)</span> or carnivore <span class="citation" data-cites="Deagle2009-yh Shehzad2012-pn">(e.g. <a href="references.html#ref-Deagle2009-yh" role="doc-biblioref">Deagle, Kirkwood, and Jarman 2009</a>; <a href="references.html#ref-Shehzad2012-pn" role="doc-biblioref">Shehzad et al. 2012</a>)</span> diet analyses.</p>
|
||||
<p>Whatever the biological question under consideration, the DNA metabarcoding methodology relies heavily on next-generation sequencing (NGS), and generates considerable numbers of DNA sequence reads (typically million of reads). Manipulation of such large datasets requires dedicated programs usually running on a Unix system. Unix is an operating system, whose first version was created during the sixties. Since its early stages, it is dedicated to scientific computing and includes a large set of simple tools to efficiently process text files. Most of those programs can be viewed as filters extracting information from a text file to create a new text file. These programs process text files as streams, line per line, therefore allowing computation on a huge dataset without requiring a large memory. Unix programs usually print their results to their standard output (<em>stdout</em>), which by default is the terminal, so the results can be examined on screen. The main philosophy of the Unix environment is to allow easy redirection of the <em>stdout</em> either to a file, for saving the results, or to the standard input (<em>stdin</em>) of a second program thus allowing to easily create complex processing from simple base commands. Access to Unix computers is increasingly easier for scientists nowadays. Indeed, the Linux operating system, an open source version of Unix, can be freely installed on every PC machine and the MacOS operating system, running on Apple computers, is also a Unix system. The <em>OBITools</em> programs imitate Unix standard programs because they usually act as filters, reading their data from text files or the <em>stdin</em> and writing their results to the <em>stdout</em>. The main difference with classical Unix programs is that text files are not analyzed line per line but sequence record per sequence record (see below for a detailed description of a sequence record). Compared to packages for similar purposes like mothur <span class="citation" data-cites="Schloss2009-qy">(<a href="references.html#ref-Schloss2009-qy" role="doc-biblioref">Schloss et al. 2009</a>)</span> or QIIME <span class="citation" data-cites="Caporaso2010-ii">(<a href="references.html#ref-Caporaso2010-ii" role="doc-biblioref">Caporaso et al. 2010</a>)</span>, the <em>OBITools</em> mainly rely on filtering and sorting algorithms. This allows users to set up versatile data analysis pipelines (Figure 1), adjustable to the broad range of DNA metabarcoding applications. The innovation of the <em>OBITools</em> is their ability to take into account the taxonomic annotations, ultimately allowing sorting and filtering of sequence records based on the taxonomy.</p>
|
||||
|
||||
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||||
<div id="refs" class="references csl-bib-body hanging-indent" role="doc-bibliography" style="display: none">
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||||
<div id="ref-Andersen2012-gj" class="csl-entry" role="doc-biblioentry">
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||||
Andersen, Kenneth, Karen Lise Bird, Morten Rasmussen, James Haile, Henrik Breuning-Madsen, Kurt H Kjaer, Ludovic Orlando, M Thomas P Gilbert, and Eske Willerslev. 2012. <span>“<span class="nocase">Meta-barcoding of <span class="nocase">ë</span>dirt<span class="nocase">ı́</span>DNA from soil reflects vertebrate biodiversity</span>.”</span> <em>Molecular Ecology</em> 21 (8): 1966–79.
|
||||
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||||
<div id="ref-Baldwin2013-yc" class="csl-entry" role="doc-biblioentry">
|
||||
Baldwin, Darren S, Matthew J Colloff, Gavin N Rees, Anthony A Chariton, Garth O Watson, Leon N Court, Diana M Hartley, et al. 2013. <span>“<span class="nocase">Impacts of inundation and drought on eukaryote biodiversity in semi-arid floodplain soils</span>.”</span> <em>Molecular Ecology</em> 22 (6): 1746–58. <a href="https://doi.org/10.1111/mec.12190">https://doi.org/10.1111/mec.12190</a>.
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||||
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||||
<div id="ref-Caporaso2010-ii" class="csl-entry" role="doc-biblioentry">
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||||
Caporaso, J Gregory, Justin Kuczynski, Jesse Stombaugh, Kyle Bittinger, Frederic D Bushman, Elizabeth K Costello, Noah Fierer, et al. 2010. <span>“<span class="nocase">QIIME allows analysis of high-throughput community sequencing data</span>.”</span> <em>Nature Methods</em> 7 (5): 335–36. <a href="https://doi.org/10.1038/nmeth.f.303">https://doi.org/10.1038/nmeth.f.303</a>.
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||||
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||||
<div id="ref-Chariton2010-cz" class="csl-entry" role="doc-biblioentry">
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||||
Chariton, Anthony A, Anthony C Roach, Stuart L Simpson, and Graeme E Batley. 2010. <span>“<span class="nocase">Influence of the choice of physical and chemistry variables on interpreting patterns of sediment contaminants and their relationships with estuarine macrobenthic communities</span>.”</span> <em>Marine and Freshwater Research</em>. <a href="https://doi.org/10.1071/mf09263">https://doi.org/10.1071/mf09263</a>.
|
||||
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||||
<div id="ref-Deagle2009-yh" class="csl-entry" role="doc-biblioentry">
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||||
Deagle, Bruce E, Roger Kirkwood, and Simon N Jarman. 2009. <span>“<span class="nocase">Analysis of Australian fur seal diet by pyrosequencing prey DNA in faeces</span>.”</span> <em>Molecular Ecology</em> 18 (9): 2022–38. <a href="https://doi.org/10.1111/j.1365-294X.2009.04158.x">https://doi.org/10.1111/j.1365-294X.2009.04158.x</a>.
|
||||
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||||
<div id="ref-Kowalczyk2011-kg" class="csl-entry" role="doc-biblioentry">
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||||
Kowalczyk, Rafał, Pierre Taberlet, Eric Coissac, Alice Valentini, Christian Miquel, Tomasz Kamiński, and Jan M Wójcik. 2011. <span>“<span class="nocase">Influence of management practices on large herbivore diet—Case of European bison in Bia<span class="nocase">ł</span>owie<span class="nocase">ż</span>a Primeval Forest (Poland)</span>.”</span> <em>Forest Ecology and Management</em> 261 (4): 821–28. <a href="https://doi.org/10.1016/j.foreco.2010.11.026">https://doi.org/10.1016/j.foreco.2010.11.026</a>.
|
||||
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||||
<div id="ref-Parducci2012-rn" class="csl-entry" role="doc-biblioentry">
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||||
Parducci, Laura, Tina Jørgensen, Mari Mette Tollefsrud, Ellen Elverland, Torbjørn Alm, Sonia L Fontana, K D Bennett, et al. 2012. <span>“<span class="nocase">Glacial survival of boreal trees in northern Scandinavia</span>.”</span> <em>Science</em> 335 (6072): 1083–86. <a href="https://doi.org/10.1126/science.1216043">https://doi.org/10.1126/science.1216043</a>.
|
||||
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||||
<div id="ref-Schloss2009-qy" class="csl-entry" role="doc-biblioentry">
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||||
Schloss, Patrick D, Sarah L Westcott, Thomas Ryabin, Justine R Hall, Martin Hartmann, Emily B Hollister, Ryan A Lesniewski, et al. 2009. <span>“<span class="nocase">Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities</span>.”</span> <em>Applied and Environmental Microbiology</em> 75 (23): 7537–41. <a href="https://doi.org/10.1128/AEM.01541-09">https://doi.org/10.1128/AEM.01541-09</a>.
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||||
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||||
<div id="ref-Shehzad2012-pn" class="csl-entry" role="doc-biblioentry">
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||||
Shehzad, Wasim, Tiayyba Riaz, Muhammad A Nawaz, Christian Miquel, Carole Poillot, Safdar A Shah, Francois Pompanon, Eric Coissac, and Pierre Taberlet. 2012. <span>“<span class="nocase">Carnivore diet analysis based on next-generation sequencing: Application to the leopard cat (Prionailurus bengalensis) in Pakistan</span>.”</span> <em>Molecular Ecology</em> 21 (8): 1951–65. <a href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-294X.2011.05424.x">https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-294X.2011.05424.x</a>.
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||||
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<div id="ref-Sogin2006-ab" class="csl-entry" role="doc-biblioentry">
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Sogin, Mitchell L, Hilary G Morrison, Julie A Huber, David Mark Welch, Susan M Huse, Phillip R Neal, Jesus M Arrieta, and Gerhard J Herndl. 2006. <span>“<span class="nocase">Microbial diversity in the deep sea and the underexplored "rare biosphere"</span>.”</span> <em>Proceedings of the National Academy of Sciences of the United States of America</em> 103 (32): 12115–20. <a href="https://doi.org/10.1073/pnas.0605127103">https://doi.org/10.1073/pnas.0605127103</a>.
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<div id="ref-Sonstebo2010-vv" class="csl-entry" role="doc-biblioentry">
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Sønstebø, J H, L Gielly, A K Brysting, R Elven, M Edwards, J Haile, E Willerslev, et al. 2010. <span>“<span class="nocase">Using next-generation sequencing for molecular reconstruction of past Arctic vegetation and climate</span>.”</span> <em>Molecular Ecology Resources</em> 10 (6): 1009–18. <a href="https://doi.org/10.1111/j.1755-0998.2010.02855.x">https://doi.org/10.1111/j.1755-0998.2010.02855.x</a>.
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<div id="ref-Taberlet2012-pf" class="csl-entry" role="doc-biblioentry">
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Taberlet, Pierre, Eric Coissac, Mehrdad Hajibabaei, and Loren H Rieseberg. 2012. <span>“<span>Environmental DNA</span>.”</span> <em>Molecular Ecology</em> 21 (8): 1789–93. <a href="https://doi.org/10.1111/j.1365-294X.2012.05542.x">https://doi.org/10.1111/j.1365-294X.2012.05542.x</a>.
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<div id="ref-Thomsen2012-au" class="csl-entry" role="doc-biblioentry">
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Thomsen, Philip Francis, Jos Kielgast, Lars L Iversen, Carsten Wiuf, Morten Rasmussen, M Thomas P Gilbert, Ludovic Orlando, and Eske Willerslev. 2012. <span>“<span class="nocase">Monitoring endangered freshwater biodiversity using environmental DNA</span>.”</span> <em>Molecular Ecology</em> 21 (11): 2565–73. <a href="https://doi.org/10.1111/j.1365-294X.2011.05418.x">https://doi.org/10.1111/j.1365-294X.2011.05418.x</a>.
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<div id="ref-Valentini2009-ay" class="csl-entry" role="doc-biblioentry">
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||||
Valentini, Alice, Christian Miquel, Muhammad Ali Nawaz, Eva Bellemain, Eric Coissac, François Pompanon, Ludovic Gielly, et al. 2009. <span>“<span class="nocase">New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach</span>.”</span> <em>Molecular Ecology Resources</em> 9 (1): 51–60. <a href="https://doi.org/10.1111/j.1755-0998.2008.02352.x">https://doi.org/10.1111/j.1755-0998.2008.02352.x</a>.
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Yoccoz, N G, K A Bråthen, L Gielly, J Haile, M E Edwards, T Goslar, H Von Stedingk, et al. 2012. <span>“<span class="nocase">DNA from soil mirrors plant taxonomic and growth form diversity</span>.”</span> <em>Molecular Ecology</em> 21 (15): 3647–55. <a href="https://doi.org/10.1111/j.1365-294X.2012.05545.x">https://doi.org/10.1111/j.1365-294X.2012.05545.x</a>.
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<p>Five sequence formats are accepted for input files. <em>Fasta</em> (<a href="formats.html#sec-fasta"><span>Section 3.2</span></a>) and <em>Fastq</em> (<a href="formats.html#sec-fastq"><span>Section 3.3</span></a>) are the main ones, EMBL and Genbank allow the use of flat files produced by these two international databases. The last one, ecoPCR, is maintained for compatibility with previous <em>OBITools</em> and allows to read <em>ecoPCR</em> outputs as sequence files.</p>
|
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|
||||
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|
||||
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|
||||
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|
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|
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<p>Several encoding schemes have been proposed for quality scores in <em>Fastq</em> format. Currently, <em>OBITools</em> considers Sanger encoding as the standard. For reasons of compatibility with older datasets produced with <em>Solexa</em> sequencers, it is possible, by using the following option, to force the use of the corresponding quality encoding scheme when reading these older files.</p>
|
||||
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|
||||
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|
||||
</div>
|
||||
</li>
|
||||
</ul>
|
||||
</li>
|
||||
</ul>
|
||||
</div>
|
||||
</nav>
|
||||
<!-- margin-sidebar -->
|
||||
<div id="quarto-margin-sidebar" class="sidebar margin-sidebar">
|
||||
<nav id="TOC" role="doc-toc" class="toc-active">
|
||||
<h2 id="toc-title">Table of contents</h2>
|
||||
|
||||
<ul>
|
||||
<li><a href="#biosequence" id="toc-biosequence" class="nav-link active" data-scroll-target="#biosequence">BioSequence</a>
|
||||
<ul class="collapse">
|
||||
<li><a href="#creating-new-instances" id="toc-creating-new-instances" class="nav-link" data-scroll-target="#creating-new-instances">Creating new instances</a></li>
|
||||
<li><a href="#end-of-life-of-a-biosequence-instance" id="toc-end-of-life-of-a-biosequence-instance" class="nav-link" data-scroll-target="#end-of-life-of-a-biosequence-instance">End of life of a <code>BioSequence</code> instance</a></li>
|
||||
<li><a href="#accessing-to-the-elements-of-a-sequence" id="toc-accessing-to-the-elements-of-a-sequence" class="nav-link" data-scroll-target="#accessing-to-the-elements-of-a-sequence">Accessing to the elements of a sequence</a></li>
|
||||
<li><a href="#the-annotations-of-a-sequence" id="toc-the-annotations-of-a-sequence" class="nav-link" data-scroll-target="#the-annotations-of-a-sequence">The annotations of a sequence</a></li>
|
||||
</ul></li>
|
||||
<li><a href="#the-sequence-iterator" id="toc-the-sequence-iterator" class="nav-link" data-scroll-target="#the-sequence-iterator">The sequence iterator</a>
|
||||
<ul class="collapse">
|
||||
<li><a href="#basic-usage-of-a-sequence-iterator" id="toc-basic-usage-of-a-sequence-iterator" class="nav-link" data-scroll-target="#basic-usage-of-a-sequence-iterator">Basic usage of a sequence iterator</a></li>
|
||||
<li><a href="#the-pipable-functions" id="toc-the-pipable-functions" class="nav-link" data-scroll-target="#the-pipable-functions">The <code>Pipable</code> functions</a></li>
|
||||
<li><a href="#the-teeable-functions" id="toc-the-teeable-functions" class="nav-link" data-scroll-target="#the-teeable-functions">The <code>Teeable</code> functions</a></li>
|
||||
</ul></li>
|
||||
</ul>
|
||||
</nav>
|
||||
</div>
|
||||
<!-- main -->
|
||||
<main class="content" id="quarto-document-content">
|
||||
|
||||
<header id="title-block-header" class="quarto-title-block default">
|
||||
<div class="quarto-title">
|
||||
<h1 class="title d-none d-lg-block">The GO <em>OBITools</em> library</h1>
|
||||
</div>
|
||||
|
||||
|
||||
|
||||
<div class="quarto-title-meta">
|
||||
|
||||
|
||||
|
||||
|
||||
</div>
|
||||
|
||||
|
||||
</header>
|
||||
|
||||
<section id="biosequence" class="level2">
|
||||
<h2 class="anchored" data-anchor-id="biosequence">BioSequence</h2>
|
||||
<p>The <code>BioSequence</code> class is used to represent biological sequences. It allows for storing : - the sequence itself as a <code>[]byte</code> - the sequencing quality score as a <code>[]byte</code> if needed - an identifier as a <code>string</code> - a definition as a <code>string</code> - a set of <em>(key, value)</em> pairs in a <code>map[sting]interface{}</code></p>
|
||||
<p>BioSequence is defined in the obiseq module and is included using the code</p>
|
||||
<div class="sourceCode" id="cb1"><pre class="sourceCode go code-with-copy"><code class="sourceCode go"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a><span class="kw">import</span> <span class="op">(</span></span>
|
||||
<span id="cb1-2"><a href="#cb1-2" aria-hidden="true" tabindex="-1"></a> <span class="st">"git.metabarcoding.org/lecasofts/go/obitools/pkg/obiseq"</span></span>
|
||||
<span id="cb1-3"><a href="#cb1-3" aria-hidden="true" tabindex="-1"></a><span class="op">)</span></span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<section id="creating-new-instances" class="level3">
|
||||
<h3 class="anchored" data-anchor-id="creating-new-instances">Creating new instances</h3>
|
||||
<p>To create new instance, use</p>
|
||||
<ul>
|
||||
<li><code>MakeBioSequence(id string, sequence []byte, definition string) obiseq.BioSequence</code></li>
|
||||
<li><code>NewBioSequence(id string, sequence []byte, definition string) *obiseq.BioSequence</code></li>
|
||||
</ul>
|
||||
<p>Both create a <code>BioSequence</code> instance, but when the first one returns the instance, the second returns a pointer on the new instance. Two other functions <code>MakeEmptyBioSequence</code>, and <code>NewEmptyBioSequence</code> do the same job but provide an uninitialized objects.</p>
|
||||
<ul>
|
||||
<li><code>id</code> parameters corresponds to the unique identifier of the sequence. It mist be a string constituted of a single word (not containing any space).</li>
|
||||
<li><code>sequence</code> is the DNA sequence itself, provided as a <code>byte</code> array (<code>[]byte</code>).</li>
|
||||
<li><code>definition</code> is a <code>string</code>, potentially empty, but usualy containing a sentence explaining what is that sequence.</li>
|
||||
</ul>
|
||||
<div class="sourceCode" id="cb2"><pre class="sourceCode go code-with-copy"><code class="sourceCode go"><span id="cb2-1"><a href="#cb2-1" aria-hidden="true" tabindex="-1"></a><span class="kw">import</span> <span class="op">(</span></span>
|
||||
<span id="cb2-2"><a href="#cb2-2" aria-hidden="true" tabindex="-1"></a> <span class="st">"git.metabarcoding.org/lecasofts/go/obitools/pkg/obiseq"</span></span>
|
||||
<span id="cb2-3"><a href="#cb2-3" aria-hidden="true" tabindex="-1"></a><span class="op">)</span></span>
|
||||
<span id="cb2-4"><a href="#cb2-4" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb2-5"><a href="#cb2-5" aria-hidden="true" tabindex="-1"></a><span class="kw">func</span> main<span class="op">()</span> <span class="op">{</span></span>
|
||||
<span id="cb2-6"><a href="#cb2-6" aria-hidden="true" tabindex="-1"></a> myseq <span class="op">:=</span> obiseq<span class="op">.</span>NewBiosequence<span class="op">(</span></span>
|
||||
<span id="cb2-7"><a href="#cb2-7" aria-hidden="true" tabindex="-1"></a> <span class="st">"seq_GH0001"</span><span class="op">,</span></span>
|
||||
<span id="cb2-8"><a href="#cb2-8" aria-hidden="true" tabindex="-1"></a> bytes<span class="op">.</span>FromString<span class="op">(</span><span class="st">"ACGTGTCAGTCG"</span><span class="op">),</span></span>
|
||||
<span id="cb2-9"><a href="#cb2-9" aria-hidden="true" tabindex="-1"></a> <span class="st">"A short test sequence"</span><span class="op">,</span></span>
|
||||
<span id="cb2-10"><a href="#cb2-10" aria-hidden="true" tabindex="-1"></a> <span class="op">)</span></span>
|
||||
<span id="cb2-11"><a href="#cb2-11" aria-hidden="true" tabindex="-1"></a><span class="op">}</span></span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<p>When formated as fasta the parameters correspond to the following schema</p>
|
||||
<pre><code>>id definition containing potentially several words
|
||||
sequence</code></pre>
|
||||
</section>
|
||||
<section id="end-of-life-of-a-biosequence-instance" class="level3">
|
||||
<h3 class="anchored" data-anchor-id="end-of-life-of-a-biosequence-instance">End of life of a <code>BioSequence</code> instance</h3>
|
||||
<p>When an instance of <code>BioSequence</code> is no longer in use, it is normally taken over by the GO garbage collector. If you know that an instance will never be used again, you can, if you wish, call the <code>Recycle</code> method on it to store the allocated memory elements in a <code>pool</code> to limit the allocation effort when many sequences are being handled. Once the recycle method has been called on an instance, you must ensure that no other method is called on it.</p>
|
||||
</section>
|
||||
<section id="accessing-to-the-elements-of-a-sequence" class="level3">
|
||||
<h3 class="anchored" data-anchor-id="accessing-to-the-elements-of-a-sequence">Accessing to the elements of a sequence</h3>
|
||||
<p>The different elements of an <code>obiseq.BioSequence</code> must be accessed using a set of methods. For the three main elements provided during the creation of a new instance methodes are :</p>
|
||||
<ul>
|
||||
<li><code>Id() string</code></li>
|
||||
<li><code>Sequence() []byte</code></li>
|
||||
<li><code>Definition() string</code></li>
|
||||
</ul>
|
||||
<p>It exists pending method to change the value of these elements</p>
|
||||
<ul>
|
||||
<li><code>SetId(id string)</code></li>
|
||||
<li><code>SetSequence(sequence []byte)</code></li>
|
||||
<li><code>SetDefinition(definition string)</code></li>
|
||||
</ul>
|
||||
<div class="sourceCode" id="cb4"><pre class="sourceCode go code-with-copy"><code class="sourceCode go"><span id="cb4-1"><a href="#cb4-1" aria-hidden="true" tabindex="-1"></a><span class="kw">import</span> <span class="op">(</span></span>
|
||||
<span id="cb4-2"><a href="#cb4-2" aria-hidden="true" tabindex="-1"></a> <span class="st">"fmt"</span></span>
|
||||
<span id="cb4-3"><a href="#cb4-3" aria-hidden="true" tabindex="-1"></a> <span class="st">"git.metabarcoding.org/lecasofts/go/obitools/pkg/obiseq"</span></span>
|
||||
<span id="cb4-4"><a href="#cb4-4" aria-hidden="true" tabindex="-1"></a><span class="op">)</span></span>
|
||||
<span id="cb4-5"><a href="#cb4-5" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb4-6"><a href="#cb4-6" aria-hidden="true" tabindex="-1"></a><span class="kw">func</span> main<span class="op">()</span> <span class="op">{</span></span>
|
||||
<span id="cb4-7"><a href="#cb4-7" aria-hidden="true" tabindex="-1"></a> myseq <span class="op">:=</span> obiseq<span class="op">.</span>NewBiosequence<span class="op">(</span></span>
|
||||
<span id="cb4-8"><a href="#cb4-8" aria-hidden="true" tabindex="-1"></a> <span class="st">"seq_GH0001"</span><span class="op">,</span></span>
|
||||
<span id="cb4-9"><a href="#cb4-9" aria-hidden="true" tabindex="-1"></a> bytes<span class="op">.</span>FromString<span class="op">(</span><span class="st">"ACGTGTCAGTCG"</span><span class="op">),</span></span>
|
||||
<span id="cb4-10"><a href="#cb4-10" aria-hidden="true" tabindex="-1"></a> <span class="st">"A short test sequence"</span><span class="op">,</span></span>
|
||||
<span id="cb4-11"><a href="#cb4-11" aria-hidden="true" tabindex="-1"></a> <span class="op">)</span></span>
|
||||
<span id="cb4-12"><a href="#cb4-12" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb4-13"><a href="#cb4-13" aria-hidden="true" tabindex="-1"></a> fmt<span class="op">.</span>Println<span class="op">(</span>myseq<span class="op">.</span>Id<span class="op">())</span></span>
|
||||
<span id="cb4-14"><a href="#cb4-14" aria-hidden="true" tabindex="-1"></a> myseq<span class="op">.</span>SetId<span class="op">(</span><span class="st">"SPE01_0001"</span><span class="op">)</span></span>
|
||||
<span id="cb4-15"><a href="#cb4-15" aria-hidden="true" tabindex="-1"></a> fmt<span class="op">.</span>Println<span class="op">(</span>myseq<span class="op">.</span>Id<span class="op">())</span></span>
|
||||
<span id="cb4-16"><a href="#cb4-16" aria-hidden="true" tabindex="-1"></a><span class="op">}</span></span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<section id="different-ways-for-accessing-an-editing-the-sequence" class="level4">
|
||||
<h4 class="anchored" data-anchor-id="different-ways-for-accessing-an-editing-the-sequence">Different ways for accessing an editing the sequence</h4>
|
||||
<p>If <code>Sequence()</code>and <code>SetSequence(sequence []byte)</code> methods are the basic ones, several other methods exist.</p>
|
||||
<ul>
|
||||
<li><code>String() string</code> return the sequence directly converted to a <code>string</code> instance.</li>
|
||||
<li>The <code>Write</code> method family allows for extending an existing sequence following the buffer protocol.
|
||||
<ul>
|
||||
<li><code>Write(data []byte) (int, error)</code> allows for appending a byte array on 3’ end of the sequence.</li>
|
||||
<li><code>WriteString(data string) (int, error)</code> allows for appending a <code>string</code>.</li>
|
||||
<li><code>WriteByte(data byte) error</code> allows for appending a single <code>byte</code>.</li>
|
||||
</ul></li>
|
||||
</ul>
|
||||
<p>The <code>Clear</code> method empties the sequence buffer.</p>
|
||||
<div class="sourceCode" id="cb5"><pre class="sourceCode go code-with-copy"><code class="sourceCode go"><span id="cb5-1"><a href="#cb5-1" aria-hidden="true" tabindex="-1"></a><span class="kw">import</span> <span class="op">(</span></span>
|
||||
<span id="cb5-2"><a href="#cb5-2" aria-hidden="true" tabindex="-1"></a> <span class="st">"fmt"</span></span>
|
||||
<span id="cb5-3"><a href="#cb5-3" aria-hidden="true" tabindex="-1"></a> <span class="st">"git.metabarcoding.org/lecasofts/go/obitools/pkg/obiseq"</span></span>
|
||||
<span id="cb5-4"><a href="#cb5-4" aria-hidden="true" tabindex="-1"></a><span class="op">)</span></span>
|
||||
<span id="cb5-5"><a href="#cb5-5" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb5-6"><a href="#cb5-6" aria-hidden="true" tabindex="-1"></a><span class="kw">func</span> main<span class="op">()</span> <span class="op">{</span></span>
|
||||
<span id="cb5-7"><a href="#cb5-7" aria-hidden="true" tabindex="-1"></a> myseq <span class="op">:=</span> obiseq<span class="op">.</span>NewEmptyBiosequence<span class="op">()</span></span>
|
||||
<span id="cb5-8"><a href="#cb5-8" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb5-9"><a href="#cb5-9" aria-hidden="true" tabindex="-1"></a> myseq<span class="op">.</span>WriteString<span class="op">(</span><span class="st">"accc"</span><span class="op">)</span></span>
|
||||
<span id="cb5-10"><a href="#cb5-10" aria-hidden="true" tabindex="-1"></a> myseq<span class="op">.</span>WriteByte<span class="op">(</span><span class="dt">byte</span><span class="op">(</span><span class="ch">'c'</span><span class="op">))</span></span>
|
||||
<span id="cb5-11"><a href="#cb5-11" aria-hidden="true" tabindex="-1"></a> fmt<span class="op">.</span>Println<span class="op">(</span>myseq<span class="op">.</span>String<span class="op">())</span></span>
|
||||
<span id="cb5-12"><a href="#cb5-12" aria-hidden="true" tabindex="-1"></a><span class="op">}</span></span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</section>
|
||||
<section id="sequence-quality-scores" class="level4">
|
||||
<h4 class="anchored" data-anchor-id="sequence-quality-scores">Sequence quality scores</h4>
|
||||
<p>Sequence quality scores cannot be initialized at the time of instance creation. You must use dedicated methods to add quality scores to a sequence.</p>
|
||||
<p>To be coherent the length of both the DNA sequence and que quality score sequence must be equal. But assessment of this constraint is realized. It is of the programmer responsability to check that invariant.</p>
|
||||
<p>While accessing to the quality scores relies on the method <code>Quality() []byte</code>, setting the quality need to call one of the following method. They run similarly to their sequence dedicated conterpart.</p>
|
||||
<ul>
|
||||
<li><code>SetQualities(qualities Quality)</code></li>
|
||||
<li><code>WriteQualities(data []byte) (int, error)</code></li>
|
||||
<li><code>WriteByteQualities(data byte) error</code></li>
|
||||
</ul>
|
||||
<p>In a way analogous to the <code>Clear</code> method, <code>ClearQualities()</code> empties the sequence of quality scores.</p>
|
||||
</section>
|
||||
</section>
|
||||
<section id="the-annotations-of-a-sequence" class="level3">
|
||||
<h3 class="anchored" data-anchor-id="the-annotations-of-a-sequence">The annotations of a sequence</h3>
|
||||
<p>A sequence can be annotated with attributes. Each attribute is associated with a value. An attribute is identified by its name. The name of an attribute consists of a character string containing no spaces or blank characters. Values can be of several types.</p>
|
||||
<ul>
|
||||
<li>Scalar types:
|
||||
<ul>
|
||||
<li>integer</li>
|
||||
<li>numeric</li>
|
||||
<li>character</li>
|
||||
<li>boolean</li>
|
||||
</ul></li>
|
||||
<li>Container types:
|
||||
<ul>
|
||||
<li>vector</li>
|
||||
<li>map</li>
|
||||
</ul></li>
|
||||
</ul>
|
||||
<p>Vectors can contain any type of scalar. Maps are compulsorily indexed by strings and can contain any scalar type. It is not possible to have nested container type.</p>
|
||||
<p>Annotations are stored in an object of type <code>bioseq.Annotation</code> which is an alias of <code>map[string]interface{}</code>. This map can be retrieved using the <code>Annotations() Annotation</code> method. If no annotation has been defined for this sequence, the method returns an empty map. It is possible to test an instance of <code>BioSequence</code> using its <code>HasAnnotation() bool</code> method to see if it has any annotations associated with it.</p>
|
||||
<ul>
|
||||
<li>GetAttribute(key string) (interface{}, bool)</li>
|
||||
</ul>
|
||||
</section>
|
||||
</section>
|
||||
<section id="the-sequence-iterator" class="level2">
|
||||
<h2 class="anchored" data-anchor-id="the-sequence-iterator">The sequence iterator</h2>
|
||||
<p>The pakage <em>obiter</em> provides an iterator mecanism for manipulating sequences. The main class provided by this package is <code>obiiter.IBioSequence</code>. An <code>IBioSequence</code> iterator provides batch of sequences.</p>
|
||||
<section id="basic-usage-of-a-sequence-iterator" class="level3">
|
||||
<h3 class="anchored" data-anchor-id="basic-usage-of-a-sequence-iterator">Basic usage of a sequence iterator</h3>
|
||||
<p>Many functions, among them functions reading sequences from a text file, return a <code>IBioSequence</code> iterator. The iterator class provides two main methods:</p>
|
||||
<ul>
|
||||
<li><code>Next() bool</code></li>
|
||||
<li><code>Get() obiiter.BioSequenceBatch</code></li>
|
||||
</ul>
|
||||
<p>The <code>Next</code> method moves the iterator to the next value, while the <code>Get</code> method returns the currently pointed value. Using them, it is possible to loop over the data as in the following code chunk.</p>
|
||||
<div class="sourceCode" id="cb6"><pre class="sourceCode go code-with-copy"><code class="sourceCode go"><span id="cb6-1"><a href="#cb6-1" aria-hidden="true" tabindex="-1"></a><span class="kw">import</span> <span class="op">(</span></span>
|
||||
<span id="cb6-2"><a href="#cb6-2" aria-hidden="true" tabindex="-1"></a> <span class="st">"git.metabarcoding.org/lecasofts/go/obitools/pkg/obiformats"</span></span>
|
||||
<span id="cb6-3"><a href="#cb6-3" aria-hidden="true" tabindex="-1"></a><span class="op">)</span></span>
|
||||
<span id="cb6-4"><a href="#cb6-4" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb6-5"><a href="#cb6-5" aria-hidden="true" tabindex="-1"></a><span class="kw">func</span> main<span class="op">()</span> <span class="op">{</span></span>
|
||||
<span id="cb6-6"><a href="#cb6-6" aria-hidden="true" tabindex="-1"></a> mydata <span class="op">:=</span> obiformats<span class="op">.</span>ReadFastSeqFromFile<span class="op">(</span><span class="st">"myfile.fasta"</span><span class="op">)</span></span>
|
||||
<span id="cb6-7"><a href="#cb6-7" aria-hidden="true" tabindex="-1"></a> </span>
|
||||
<span id="cb6-8"><a href="#cb6-8" aria-hidden="true" tabindex="-1"></a> <span class="cf">for</span> mydata<span class="op">.</span>Next<span class="op">()</span> <span class="op">{</span></span>
|
||||
<span id="cb6-9"><a href="#cb6-9" aria-hidden="true" tabindex="-1"></a> data <span class="op">:=</span> mydata<span class="op">.</span>Get<span class="op">()</span></span>
|
||||
<span id="cb6-10"><a href="#cb6-10" aria-hidden="true" tabindex="-1"></a> <span class="co">//</span></span>
|
||||
<span id="cb6-11"><a href="#cb6-11" aria-hidden="true" tabindex="-1"></a> <span class="co">// Whatever you want to do with the data chunk</span></span>
|
||||
<span id="cb6-12"><a href="#cb6-12" aria-hidden="true" tabindex="-1"></a> <span class="co">//</span></span>
|
||||
<span id="cb6-13"><a href="#cb6-13" aria-hidden="true" tabindex="-1"></a> <span class="op">}</span></span>
|
||||
<span id="cb6-14"><a href="#cb6-14" aria-hidden="true" tabindex="-1"></a><span class="op">}</span></span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<p>An <code>obiseq.BioSequenceBatch</code> instance is a set of sequences stored in an <code>obiseq.BioSequenceSlice</code> and a sequence number. The number of sequences in a batch is not defined. A batch can even contain zero sequences, if for example all sequences initially included in the batch have been filtered out at some stage of their processing.</p>
|
||||
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|
||||
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|
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|
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<h3 class="anchored" data-anchor-id="the-teeable-functions">The <code>Teeable</code> functions</h3>
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|
||||
<li><a href="#the-fasta-and-fastq-annotations-format" id="toc-the-fasta-and-fastq-annotations-format" class="nav-link" data-scroll-target="#the-fasta-and-fastq-annotations-format"><span class="toc-section-number">5.2</span> The Fasta and Fastq annotations format</a></li>
|
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|
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<h1 class="title d-none d-lg-block"><span class="chapter-number">5</span> <span class="chapter-title">Controling OBITools outputs</span></h1>
|
||||
</div>
|
||||
|
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|
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|
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||||
</header>
|
||||
|
||||
<section id="specifying-output-format" class="level2" data-number="5.1">
|
||||
<h2 data-number="5.1" class="anchored" data-anchor-id="specifying-output-format"><span class="header-section-number">5.1</span> Specifying output format</h2>
|
||||
<p>Only two output sequence formats are supported by OBITools, Fasta and Fastq. Fastq is used when output sequences are associated with quality information. Otherwise, Fasta is the default format. However, it is possible to force the output format by using one of the following two options. Forcing the use of Fasta results in the loss of quality information. Conversely, when the Fastq format is forced with sequences that have no quality data, dummy qualities set to 40 for each nucleotide are added.</p>
|
||||
<ul>
|
||||
<li><code>--fasta-output</code> Read data following the ecoPCR output format.</li>
|
||||
<li><code>--fastq-output</code> Read data following the EMBL flatfile format.</li>
|
||||
</ul>
|
||||
<p>OBITools allows multiple input files to be specified for a single command.</p>
|
||||
<ul>
|
||||
<li><code>--no-order</code> When several input files are provided, indicates that there is no order among them. (default: false). Using such option can increase a lot the processing of the data.</li>
|
||||
</ul>
|
||||
</section>
|
||||
<section id="the-fasta-and-fastq-annotations-format" class="level2" data-number="5.2">
|
||||
<h2 data-number="5.2" class="anchored" data-anchor-id="the-fasta-and-fastq-annotations-format"><span class="header-section-number">5.2</span> The Fasta and Fastq annotations format</h2>
|
||||
<p>OBITools extend the <a href="#the-fasta-sequence-format">Fasta</a> and <a href="#the-fastq-sequence-format">Fastq</a> formats by introducing a format for the title lines of these formats allowing to annotate every sequence. While the previous version of OBITools used an <em>ad-hoc</em> format for these annotation, this new version introduce the usage of the standard JSON format to store them.</p>
|
||||
<p>On input, OBITools automatically recognize the format of the annotations, but two options allows to force the parsing following one of them. You should normally not need to use these options.</p>
|
||||
<ul>
|
||||
<li><p><code>--input-OBI-header</code> FASTA/FASTQ title line annotations follow OBI format. (default: false)</p></li>
|
||||
<li><p><code>--input-json-header</code> FASTA/FASTQ title line annotations follow json format. (default: false)</p></li>
|
||||
</ul>
|
||||
<p>On output, by default annotation are formatted using the new JSON format. For compatibility with previous version of OBITools and with external scripts and software, it is possible to force the usage of the previous OBITools format.</p>
|
||||
<ul>
|
||||
<li><p><code>--output-OBI-header|-O</code> output FASTA/FASTQ title line annotations follow OBI format. (default: false)</p></li>
|
||||
<li><p><code>--output-json-header</code> output FASTA/FASTQ title line annotations follow json format. (default: false)</p></li>
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<h1 class="quarto-secondary-nav-title"><span class="chapter-number">3</span> <span class="chapter-title">OBITools V4 Tutorial</span></h1>
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<span class="sidebar-item-text sidebar-link text-start">The GO *OBITools* library</span>
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<h2 id="toc-title">Table of contents</h2>
|
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|
||||
<ul>
|
||||
<li><a href="#wolves-diet-based-on-dna-metabarcoding" id="toc-wolves-diet-based-on-dna-metabarcoding" class="nav-link active" data-scroll-target="#wolves-diet-based-on-dna-metabarcoding"><span class="toc-section-number">3.1</span> Wolves’ diet based on DNA metabarcoding</a></li>
|
||||
<li><a href="#step-by-step-analysis" id="toc-step-by-step-analysis" class="nav-link" data-scroll-target="#step-by-step-analysis"><span class="toc-section-number">3.2</span> Step by step analysis</a>
|
||||
<ul class="collapse">
|
||||
<li><a href="#recover-full-sequence-reads-from-forward-and-reverse-partial-reads" id="toc-recover-full-sequence-reads-from-forward-and-reverse-partial-reads" class="nav-link" data-scroll-target="#recover-full-sequence-reads-from-forward-and-reverse-partial-reads"><span class="toc-section-number">3.2.1</span> Recover full sequence reads from forward and reverse partial reads</a></li>
|
||||
<li><a href="#remove-unaligned-sequence-records" id="toc-remove-unaligned-sequence-records" class="nav-link" data-scroll-target="#remove-unaligned-sequence-records"><span class="toc-section-number">3.2.2</span> Remove unaligned sequence records</a></li>
|
||||
<li><a href="#assign-each-sequence-record-to-the-corresponding-samplemarker-combination" id="toc-assign-each-sequence-record-to-the-corresponding-samplemarker-combination" class="nav-link" data-scroll-target="#assign-each-sequence-record-to-the-corresponding-samplemarker-combination"><span class="toc-section-number">3.2.3</span> Assign each sequence record to the corresponding sample/marker combination</a></li>
|
||||
<li><a href="#dereplicate-reads-into-uniq-sequences" id="toc-dereplicate-reads-into-uniq-sequences" class="nav-link" data-scroll-target="#dereplicate-reads-into-uniq-sequences"><span class="toc-section-number">3.2.4</span> Dereplicate reads into uniq sequences</a></li>
|
||||
<li><a href="#denoise-the-sequence-dataset" id="toc-denoise-the-sequence-dataset" class="nav-link" data-scroll-target="#denoise-the-sequence-dataset"><span class="toc-section-number">3.2.5</span> Denoise the sequence dataset</a></li>
|
||||
<li><a href="#taxonomic-assignment-of-sequences" id="toc-taxonomic-assignment-of-sequences" class="nav-link" data-scroll-target="#taxonomic-assignment-of-sequences"><span class="toc-section-number">3.2.6</span> Taxonomic assignment of sequences</a></li>
|
||||
<li><a href="#assign-each-sequence-to-a-taxon" id="toc-assign-each-sequence-to-a-taxon" class="nav-link" data-scroll-target="#assign-each-sequence-to-a-taxon"><span class="toc-section-number">3.2.7</span> Assign each sequence to a taxon</a></li>
|
||||
<li><a href="#generate-the-final-result-table" id="toc-generate-the-final-result-table" class="nav-link" data-scroll-target="#generate-the-final-result-table"><span class="toc-section-number">3.2.8</span> Generate the final result table</a></li>
|
||||
<li><a href="#looking-at-the-data-in-r" id="toc-looking-at-the-data-in-r" class="nav-link" data-scroll-target="#looking-at-the-data-in-r"><span class="toc-section-number">3.2.9</span> Looking at the data in R</a></li>
|
||||
</ul></li>
|
||||
</ul>
|
||||
</nav>
|
||||
</div>
|
||||
<!-- main -->
|
||||
<main class="content" id="quarto-document-content">
|
||||
|
||||
<header id="title-block-header" class="quarto-title-block default">
|
||||
<div class="quarto-title">
|
||||
<h1 class="title d-none d-lg-block"><span class="chapter-number">3</span> <span class="chapter-title">OBITools V4 Tutorial</span></h1>
|
||||
</div>
|
||||
|
||||
|
||||
|
||||
<div class="quarto-title-meta">
|
||||
|
||||
|
||||
|
||||
|
||||
</div>
|
||||
|
||||
|
||||
</header>
|
||||
|
||||
<p>Here is a short tutorial on how to analyze DNA metabarcoding data produced on Illumina sequencers using:</p>
|
||||
<ul>
|
||||
<li>the OBITools</li>
|
||||
<li>some basic Unix commands</li>
|
||||
</ul>
|
||||
<section id="wolves-diet-based-on-dna-metabarcoding" class="level2" data-number="3.1">
|
||||
<h2 data-number="3.1" class="anchored" data-anchor-id="wolves-diet-based-on-dna-metabarcoding"><span class="header-section-number">3.1</span> Wolves’ diet based on DNA metabarcoding</h2>
|
||||
<p>The data used in this tutorial correspond to the analysis of four wolf scats, using the protocol published in <span class="citation" data-cites="Shehzad2012-pn">Shehzad et al. (<a href="references.html#ref-Shehzad2012-pn" role="doc-biblioref">2012</a>)</span> for assessing carnivore diet. After extracting DNA from the faeces, the DNA amplifications were carried out using the primers <code>TTAGATACCCCACTATGC</code> and <code>TAGAACAGGCTCCTCTAG</code> amplifiying the <em>12S-V5</em> region <span class="citation" data-cites="Riaz2011-gn">(<a href="references.html#ref-Riaz2011-gn" role="doc-biblioref">Riaz et al. 2011</a>)</span>, together with a wolf blocking oligonucleotide.</p>
|
||||
<p>The complete data set can be downloaded here: <a href="wolf_diet.tgz">the tutorial dataset</a></p>
|
||||
<p>Once the data file is downloaded, using a UNIX terminal unarchive the data from the <code>tgz</code> file.</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb1"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a><span class="fu">tar</span> zxvf wolf_diet.tgz</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>That command create a new directory named <code>wolf_data</code> containing every required data files:</p>
|
||||
<ul>
|
||||
<li><p><code>fastq <fastq></code> files resulting of aGA IIx (Illumina) paired-end (2 x 108 bp) sequencing assay of DNA extracted and amplified from four wolf faeces:</p>
|
||||
<ul>
|
||||
<li><code>wolf_F.fastq</code></li>
|
||||
<li><code>wolf_R.fastq</code></li>
|
||||
</ul></li>
|
||||
<li><p>the file describing the primers and tags used for all samples sequenced:</p>
|
||||
<ul>
|
||||
<li><code>wolf_diet_ngsfilter.txt</code> The tags correspond to short and specific sequences added on the 5' end of each primer to distinguish the different samples</li>
|
||||
</ul></li>
|
||||
<li><p>the file containing the reference database in a fasta format:</p>
|
||||
<ul>
|
||||
<li><code>db_v05_r117.fasta</code> This reference database has been extracted from the release 117 of EMBL using <code>obipcr</code></li>
|
||||
</ul></li>
|
||||
</ul>
|
||||
<div class="cell">
|
||||
|
||||
</div>
|
||||
<p>To not mix raw data and processed data a new directory called <code>results</code> is created.</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb2"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb2-1"><a href="#cb2-1" aria-hidden="true" tabindex="-1"></a><span class="fu">mkdir</span> results</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
</section>
|
||||
<section id="step-by-step-analysis" class="level2" data-number="3.2">
|
||||
<h2 data-number="3.2" class="anchored" data-anchor-id="step-by-step-analysis"><span class="header-section-number">3.2</span> Step by step analysis</h2>
|
||||
<section id="recover-full-sequence-reads-from-forward-and-reverse-partial-reads" class="level3" data-number="3.2.1">
|
||||
<h3 data-number="3.2.1" class="anchored" data-anchor-id="recover-full-sequence-reads-from-forward-and-reverse-partial-reads"><span class="header-section-number">3.2.1</span> Recover full sequence reads from forward and reverse partial reads</h3>
|
||||
<p>When using the result of a paired-end sequencing assay with supposedly overlapping forward and reverse reads, the first step is to recover the assembled sequence.</p>
|
||||
<p>The forward and reverse reads of the same fragment are <em>at the same line position</em> in the two fastq files obtained after sequencing. Based on these two files, the assembly of the forward and reverse reads is done with the <code>obipairing</code> utility that aligns the two reads and returns the reconstructed sequence.</p>
|
||||
<p>In our case, the command is:</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb3"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb3-1"><a href="#cb3-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obipairing</span> <span class="at">--min-identity</span><span class="op">=</span>0.8 <span class="dt">\</span></span>
|
||||
<span id="cb3-2"><a href="#cb3-2" aria-hidden="true" tabindex="-1"></a> <span class="at">--min-overlap</span><span class="op">=</span>10 <span class="dt">\</span></span>
|
||||
<span id="cb3-3"><a href="#cb3-3" aria-hidden="true" tabindex="-1"></a> <span class="at">-F</span> wolf_data/wolf_F.fastq <span class="dt">\</span></span>
|
||||
<span id="cb3-4"><a href="#cb3-4" aria-hidden="true" tabindex="-1"></a> <span class="at">-R</span> wolf_data/wolf_R.fastq <span class="dt">\</span></span>
|
||||
<span id="cb3-5"><a href="#cb3-5" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/wolf.fastq </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>The <code>--min-identity</code> and <code>--min-overlap</code> options allow discarding sequences with low alignment quality. If after the aligment, the overlaping parts of the reads is shorter than 10 base pairs or the similarity over this aligned region is below 80% of identity, in the output file, the forward and reverse reads are not aligned but concatenated, and the value of the <code>mode</code> attribute in the sequence header is set to <code>joined</code> instead of <code>alignment</code>.</p>
|
||||
</section>
|
||||
<section id="remove-unaligned-sequence-records" class="level3" data-number="3.2.2">
|
||||
<h3 data-number="3.2.2" class="anchored" data-anchor-id="remove-unaligned-sequence-records"><span class="header-section-number">3.2.2</span> Remove unaligned sequence records</h3>
|
||||
<p>Unaligned sequences (:py<code class="interpreted-text" role="mod">mode=joined</code>) cannot be used. The following command allows removing them from the dataset:</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb4"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb4-1"><a href="#cb4-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obigrep</span> <span class="at">-p</span> <span class="st">'annotations.mode != "join"'</span> <span class="dt">\</span></span>
|
||||
<span id="cb4-2"><a href="#cb4-2" aria-hidden="true" tabindex="-1"></a> results/wolf.fastq <span class="op">></span> results/wolf.ali.fastq</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>The <code>-p</code> requires a go like expression. <code>annotations.mode != "join"</code> means that if the value of the <code>mode</code> annotation of a sequence is different from <code>join</code>, the corresponding sequence record will be kept.</p>
|
||||
<p>The first sequence record of <code>wolf.ali.fastq</code> can be obtained using the following command line:</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb5"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb5-1"><a href="#cb5-1" aria-hidden="true" tabindex="-1"></a><span class="fu">head</span> <span class="at">-n</span> 4 results/wolf.ali.fastq</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>The folling piece of code appears on thew window of tour terminal.</p>
|
||||
<pre><code>@HELIUM_000100422_612GNAAXX:7:108:5640:3823#0/1 {"ali_dir":"left","ali_length":62,"mode":"alignment","pairing_mismatches":{"(T:26)->(G:13)":62,"(T:34)->(G:18)":48},"score":484,"score_norm":0.968,"seq_a_single":46,"seq_ab_match":60,"seq_b_single":46}
|
||||
ccgcctcctttagataccccactatgcttagccctaaacacaagtaattaatataacaaaattgttcgccagagtactaccggcaatagcttaaaactcaaaggacttggcggtgctttatacccttctagaggagcctgttctaaggaggcgg
|
||||
+
|
||||
CCCCCCCBCCCCCCCCCCCCCCCCCCCCCCBCCCCCBCCCCCCC<CcCccbe[`F`accXV<TA\RYU\\ee_e[XZ[XEEEEEEEEEE?EEEEEEEEEEDEEEEEEECCCCCCCCCCCCCCCCCCCCCCCACCCCCACCCCCCCCCCCCCCCC</code></pre>
|
||||
</section>
|
||||
<section id="assign-each-sequence-record-to-the-corresponding-samplemarker-combination" class="level3" data-number="3.2.3">
|
||||
<h3 data-number="3.2.3" class="anchored" data-anchor-id="assign-each-sequence-record-to-the-corresponding-samplemarker-combination"><span class="header-section-number">3.2.3</span> Assign each sequence record to the corresponding sample/marker combination</h3>
|
||||
<p>Each sequence record is assigned to its corresponding sample and marker using the data provided in a text file (here <code>wolf_diet_ngsfilter.txt</code>). This text file contains one line per sample, with the name of the experiment (several experiments can be included in the same file), the name of the tags (for example: <code>aattaac</code> if the same tag has been used on each extremity of the PCR products, or <code>aattaac:gaagtag</code> if the tags were different), the sequence of the forward primer, the sequence of the reverse primer, the letter <code>T</code> or <code>F</code> for sample identification using the forward primer and tag only or using both primers and both tags, respectively (see <code>obimultiplex</code> for details).</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb7"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb7-1"><a href="#cb7-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obimultiplex</span> <span class="at">-t</span> wolf_data/wolf_diet_ngsfilter.txt <span class="dt">\</span></span>
|
||||
<span id="cb7-2"><a href="#cb7-2" aria-hidden="true" tabindex="-1"></a> <span class="at">-u</span> results/unidentified.fastq <span class="dt">\</span></span>
|
||||
<span id="cb7-3"><a href="#cb7-3" aria-hidden="true" tabindex="-1"></a> results/wolf.ali.fastq <span class="dt">\</span></span>
|
||||
<span id="cb7-4"><a href="#cb7-4" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/wolf.ali.assigned.fastq</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>This command creates two files:</p>
|
||||
<ul>
|
||||
<li><code>unidentified.fastq</code> containing all the sequence records that were not assigned to a sample/marker combination</li>
|
||||
<li><code>wolf.ali.assigned.fastq</code> containing all the sequence records that were properly assigned to a sample/marker combination</li>
|
||||
</ul>
|
||||
<p>Note that each sequence record of the <code>wolf.ali.assigned.fastq</code> file contains only the barcode sequence as the sequences of primers and tags are removed by the <code>obimultiplex</code> program. Information concerning the experiment, sample, primers and tags is added as attributes in the sequence header.</p>
|
||||
<p>For instance, the first sequence record of <code>wolf.ali.assigned.fastq</code> is:</p>
|
||||
<pre><code>@HELIUM_000100422_612GNAAXX:7:108:5640:3823#0/1_sub[28..127] {"ali_dir":"left","ali_length":62,"direction":"direct","experiment":"wolf_diet","forward_match":"ttagataccccactatgc","forward_mismatches":0,"forward_primer":"ttagataccccactatgc","forward_tag":"gcctcct","mode":"alignment","pairing_mismatches":{"(T:26)->(G:13)":35,"(T:34)->(G:18)":21},"reverse_match":"tagaacaggctcctctag","reverse_mismatches":0,"reverse_primer":"tagaacaggctcctctag","reverse_tag":"gcctcct","sample":"29a_F260619","score":484,"score_norm":0.968,"seq_a_single":46,"seq_ab_match":60,"seq_b_single":46}
|
||||
ttagccctaaacacaagtaattaatataacaaaattgttcgccagagtactaccggcaatagcttaaaactcaaaggacttggcggtgctttataccctt
|
||||
+
|
||||
CCCBCCCCCBCCCCCCC<CcCccbe[`F`accXV<TA\RYU\\ee_e[XZ[XEEEEEEEEEE?EEEEEEEEEEDEEEEEEECCCCCCCCCCCCCCCCCCC</code></pre>
|
||||
</section>
|
||||
<section id="dereplicate-reads-into-uniq-sequences" class="level3" data-number="3.2.4">
|
||||
<h3 data-number="3.2.4" class="anchored" data-anchor-id="dereplicate-reads-into-uniq-sequences"><span class="header-section-number">3.2.4</span> Dereplicate reads into uniq sequences</h3>
|
||||
<p>The same DNA molecule can be sequenced several times. In order to reduce both file size and computations time, and to get easier interpretable results, it is convenient to work with unique <em>sequences</em> instead of <em>reads</em>. To <em>dereplicate</em> such <em>reads</em> into unique <em>sequences</em>, we use the <code>obiuniq</code> command.</p>
|
||||
<table class="table">
|
||||
<colgroup>
|
||||
<col style="width: 86%">
|
||||
</colgroup>
|
||||
<tbody>
|
||||
<tr class="odd">
|
||||
<td>Definition: Dereplicate reads into unique sequences</td>
|
||||
</tr>
|
||||
<tr class="even">
|
||||
<td><ol type="1">
|
||||
<li>compare all the reads in a data set to each other</li>
|
||||
<li>group strictly identical reads together</li>
|
||||
<li>output the sequence for each group and its count in the original dataset (in this way, all duplicated reads are removed)</li>
|
||||
</ol>
|
||||
<p>Definition adapted from <span class="citation" data-cites="Seguritan2001-tg">Seguritan and Rohwer (<a href="references.html#ref-Seguritan2001-tg" role="doc-biblioref">2001</a>)</span></p></td>
|
||||
</tr>
|
||||
</tbody>
|
||||
</table>
|
||||
<p>For dereplication, we use the <code>obiuniq</code> command with the <code>-m sample</code>. The <code>-m sample</code> option is used to keep the information of the samples of origin for each uniquesequence.</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb9"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb9-1"><a href="#cb9-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obiuniq</span> <span class="at">-m</span> sample <span class="dt">\</span></span>
|
||||
<span id="cb9-2"><a href="#cb9-2" aria-hidden="true" tabindex="-1"></a> results/wolf.ali.assigned.fastq <span class="dt">\</span></span>
|
||||
<span id="cb9-3"><a href="#cb9-3" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/wolf.ali.assigned.uniq.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>Note that <code>obiuniq</code> returns a fasta file.</p>
|
||||
<p>The first sequence record of <code>wolf.ali.assigned.uniq.fasta</code> is:</p>
|
||||
<pre><code>>HELIUM_000100422_612GNAAXX:7:93:6991:1942#0/1_sub[28..126] {"ali_dir":"left","ali_length":63,"count":1,"direction":"reverse","experiment":"wolf_diet","forward_match":"ttagataccccactatgc","forward_mismatches":0,"forward_primer":"ttagataccccactatgc","forward_tag":"gaatatc","merged_sample":{"26a_F040644":1},"mode":"alignment","pairing_mismatches":{"(A:10)->(G:34)":76,"(C:06)->(A:34)":58},"reverse_match":"tagaacaggctcctctag","reverse_mismatches":0,"reverse_primer":"tagaacaggctcctctag","reverse_tag":"gaatatc","score":730,"score_norm":0.968,"seq_a_single":45,"seq_ab_match":61,"seq_b_single":45}
|
||||
ttagccctaaacataaacattcaataaacaagaatgttcgccagagaactactagcaaca
|
||||
gcctgaaactcaaaggacttggcggtgctttatatccct</code></pre>
|
||||
<p>The run of <code>obiuniq</code> has added two key=values entries in the header of the fasta sequence:</p>
|
||||
<ul>
|
||||
<li><code class="interpreted-text" role="mod">"merged_sample":{"29a_F260619":1}</code>: this sequence have been found once in a single sample called <strong>29a_F260619</strong></li>
|
||||
<li><code>"count":1</code> : the total count for this sequence is <span class="math inline">\(1\)</span></li>
|
||||
</ul>
|
||||
<p>To keep only these two attributes, we can use the <code>obiannotate</code> command:</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb11"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb11-1"><a href="#cb11-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obiannotate</span> <span class="at">-k</span> count <span class="at">-k</span> merged_sample <span class="dt">\</span></span>
|
||||
<span id="cb11-2"><a href="#cb11-2" aria-hidden="true" tabindex="-1"></a> results/wolf.ali.assigned.uniq.fasta <span class="dt">\</span></span>
|
||||
<span id="cb11-3"><a href="#cb11-3" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/wolf.ali.assigned.simple.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>The first five sequence records of <code>wolf.ali.assigned.simple.fasta</code> become:</p>
|
||||
<pre><code>>HELIUM_000100422_612GNAAXX:7:26:18930:11105#0/1_sub[28..127] {"count":1,"merged_sample":{"29a_F260619":1}}
|
||||
ttagccctaaacacaagtaattaatataacaaaatwattcgcyagagtactacmggcaat
|
||||
agctyaaarctcamagrwcttggcggtgctttataccctt
|
||||
>HELIUM_000100422_612GNAAXX:7:58:5711:11399#0/1_sub[28..127] {"count":1,"merged_sample":{"29a_F260619":1}}
|
||||
ttagccctaaacacaagtaattaatataacaaaattattcgccagagtwctaccgssaat
|
||||
agcttaaaactcaaaggactgggcggtgctttataccctt
|
||||
>HELIUM_000100422_612GNAAXX:7:100:15836:9304#0/1_sub[28..127] {"count":1,"merged_sample":{"29a_F260619":1}}
|
||||
ttagccctaaacatagataattacacaaacaaaattgttcaccagagtactagcggcaac
|
||||
agcttaaaactcaaaggacttggcggtgctttataccctt
|
||||
>HELIUM_000100422_612GNAAXX:7:55:13242:9085#0/1_sub[28..126] {"count":4,"merged_sample":{"26a_F040644":4}}
|
||||
ttagccctaaacataaacattcaataaacaagagtgttcgccagagtactactagcaaca
|
||||
gcctgaaactcaaaggacttggcggtgctttacatccct
|
||||
>HELIUM_000100422_612GNAAXX:7:86:8429:13723#0/1_sub[28..127] {"count":7,"merged_sample":{"15a_F730814":5,"29a_F260619":2}}
|
||||
ttagccctaaacacaagtaattaatataacaaaattattcgccagagtactaccggcaat
|
||||
agcttaaaactcaaaggactcggcggtgctttataccctt</code></pre>
|
||||
</section>
|
||||
<section id="denoise-the-sequence-dataset" class="level3" data-number="3.2.5">
|
||||
<h3 data-number="3.2.5" class="anchored" data-anchor-id="denoise-the-sequence-dataset"><span class="header-section-number">3.2.5</span> Denoise the sequence dataset</h3>
|
||||
<p>To have a set of sequences assigned to their corresponding samples does not mean that all sequences are <em>biologically</em> meaningful i.e. some of these sequences can contains PCR and/or sequencing errors, or chimeras.</p>
|
||||
<section id="tag-the-sequences-for-pcr-errors-sequence-variants" class="level4 unnumbered">
|
||||
<h4 class="unnumbered anchored" data-anchor-id="tag-the-sequences-for-pcr-errors-sequence-variants">Tag the sequences for PCR errors (sequence variants)</h4>
|
||||
<p>The <code>obiclean</code> program tags sequence variants as potential error generated during PCR amplification. We ask it to keep the <span class="title-ref">head</span> sequences (<code>-H</code> option) that are sequences which are not variants of another sequence with a count greater than 5% of their own count (<code>-r 0.05</code> option).</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb13"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb13-1"><a href="#cb13-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obiclean</span> <span class="at">-s</span> sample <span class="at">-r</span> 0.05 <span class="at">-H</span> <span class="dt">\</span></span>
|
||||
<span id="cb13-2"><a href="#cb13-2" aria-hidden="true" tabindex="-1"></a> results/wolf.ali.assigned.simple.fasta <span class="dt">\</span></span>
|
||||
<span id="cb13-3"><a href="#cb13-3" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/wolf.ali.assigned.simple.clean.fasta </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>One of the sequence records of <code>wolf.ali.assigned.simple.clean.fasta</code> is:</p>
|
||||
<pre><code>>HELIUM_000100422_612GNAAXX:7:66:4039:8016#0/1_sub[28..127] {"count":17,"merged_sample":{"13a_F730603":17},"obiclean_head":true,"obiclean_headcount":1,"obiclean_internalcount":0,"obi
|
||||
clean_samplecount":1,"obiclean_singletoncount":0,"obiclean_status":{"13a_F730603":"h"},"obiclean_weight":{"13a_F730603":25}}
|
||||
ctagccttaaacacaaatagttatgcaaacaaaactattcgccagagtactaccggcaac
|
||||
agcccaaaactcaaaggacttggcggtgcttcacaccctt</code></pre>
|
||||
<p>To remove such sequences as much as possible, we first discard rare sequences and then rsequence variants that likely correspond to artifacts.</p>
|
||||
</section>
|
||||
<section id="get-some-statistics-about-sequence-counts" class="level4 unnumbered">
|
||||
<h4 class="unnumbered anchored" data-anchor-id="get-some-statistics-about-sequence-counts">Get some statistics about sequence counts</h4>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb15"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb15-1"><a href="#cb15-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obicount</span> results/wolf.ali.assigned.simple.clean.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<div class="cell-output cell-output-stdout">
|
||||
<pre><code>time="2023-02-23T18:43:26+01:00" level=info msg="Appending results/wolf.ali.assigned.simple.clean.fasta file\n"
|
||||
2749 36409 273387</code></pre>
|
||||
</div>
|
||||
</div>
|
||||
<p>The dataset contains <span class="math inline">\(4313\)</span> sequences variant corresponding to 42452 sequence reads. Most of the variants occur only a single time in the complete dataset and are usualy named <em>singletons</em></p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb17"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb17-1"><a href="#cb17-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obigrep</span> <span class="at">-p</span> <span class="st">'sequence.Count() == 1'</span> results/wolf.ali.assigned.simple.clean.fasta <span class="dt">\</span></span>
|
||||
<span id="cb17-2"><a href="#cb17-2" aria-hidden="true" tabindex="-1"></a> <span class="kw">|</span> <span class="ex">obicount</span></span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<div class="cell-output cell-output-stdout">
|
||||
<pre><code>time="2023-02-23T18:43:26+01:00" level=info msg="Appending results/wolf.ali.assigned.simple.clean.fasta file\n"
|
||||
time="2023-02-23T18:43:26+01:00" level=info msg="Reading sequences from stdin in guessed\n"
|
||||
time="2023-02-23T18:43:26+01:00" level=info msg="On output use JSON headers"
|
||||
2623 2623 261217</code></pre>
|
||||
</div>
|
||||
</div>
|
||||
<p>In that dataset sigletons corresponds to <span class="math inline">\(3511\)</span> variants.</p>
|
||||
<p>Using <em>R</em> and the <code>ROBIFastread</code> package able to read headers of the fasta files produced by <em>OBITools</em>, we can get more complete statistics on the distribution of occurrencies.</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb19"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb19-1"><a href="#cb19-1" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(ROBIFastread)</span>
|
||||
<span id="cb19-2"><a href="#cb19-2" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(ggplot2)</span>
|
||||
<span id="cb19-3"><a href="#cb19-3" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb19-4"><a href="#cb19-4" aria-hidden="true" tabindex="-1"></a>seqs <span class="ot"><-</span> <span class="fu">read_obifasta</span>(<span class="st">"results/wolf.ali.assigned.simple.clean.fasta"</span>,<span class="at">keys=</span><span class="st">"count"</span>)</span>
|
||||
<span id="cb19-5"><a href="#cb19-5" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb19-6"><a href="#cb19-6" aria-hidden="true" tabindex="-1"></a><span class="fu">ggplot</span>(<span class="at">data =</span> seqs, <span class="at">mapping=</span><span class="fu">aes</span>(<span class="at">x =</span> count)) <span class="sc">+</span></span>
|
||||
<span id="cb19-7"><a href="#cb19-7" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_histogram</span>(<span class="at">bins=</span><span class="dv">100</span>) <span class="sc">+</span></span>
|
||||
<span id="cb19-8"><a href="#cb19-8" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_y_sqrt</span>() <span class="sc">+</span></span>
|
||||
<span id="cb19-9"><a href="#cb19-9" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_x_sqrt</span>() <span class="sc">+</span></span>
|
||||
<span id="cb19-10"><a href="#cb19-10" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_vline</span>(<span class="at">xintercept =</span> <span class="dv">10</span>, <span class="at">col=</span><span class="st">"red"</span>, <span class="at">lty=</span><span class="dv">2</span>) <span class="sc">+</span></span>
|
||||
<span id="cb19-11"><a href="#cb19-11" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlab</span>(<span class="st">"number of occurrencies of a variant"</span>) </span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<div class="cell-output-display">
|
||||
<p><img src="tutorial_files/figure-html/unnamed-chunk-9-1.png" class="img-fluid" width="672"></p>
|
||||
</div>
|
||||
</div>
|
||||
<p>In a similar way it is also possible to plot the distribution of the sequence length.</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb20"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb20-1"><a href="#cb20-1" aria-hidden="true" tabindex="-1"></a><span class="fu">ggplot</span>(<span class="at">data =</span> seqs, <span class="at">mapping=</span><span class="fu">aes</span>(<span class="at">x =</span> <span class="fu">nchar</span>(sequence))) <span class="sc">+</span></span>
|
||||
<span id="cb20-2"><a href="#cb20-2" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_histogram</span>() <span class="sc">+</span></span>
|
||||
<span id="cb20-3"><a href="#cb20-3" aria-hidden="true" tabindex="-1"></a> <span class="fu">scale_y_log10</span>() <span class="sc">+</span></span>
|
||||
<span id="cb20-4"><a href="#cb20-4" aria-hidden="true" tabindex="-1"></a> <span class="fu">geom_vline</span>(<span class="at">xintercept =</span> <span class="dv">80</span>, <span class="at">col=</span><span class="st">"red"</span>, <span class="at">lty=</span><span class="dv">2</span>) <span class="sc">+</span></span>
|
||||
<span id="cb20-5"><a href="#cb20-5" aria-hidden="true" tabindex="-1"></a> <span class="fu">xlab</span>(<span class="st">"sequence lengths in base pair"</span>)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<div class="cell-output-display">
|
||||
<p><img src="tutorial_files/figure-html/unnamed-chunk-10-1.png" class="img-fluid" width="672"></p>
|
||||
</div>
|
||||
</div>
|
||||
</section>
|
||||
<section id="keep-only-the-sequences-having-a-count-greater-or-equal-to-10-and-a-length-shorter-than-80-bp" class="level4 unnumbered">
|
||||
<h4 class="unnumbered anchored" data-anchor-id="keep-only-the-sequences-having-a-count-greater-or-equal-to-10-and-a-length-shorter-than-80-bp">Keep only the sequences having a count greater or equal to 10 and a length shorter than 80 bp</h4>
|
||||
<p>Based on the previous observation, we set the cut-off for keeping sequences for further analysis to a count of 10. To do this, we use the <code class="interpreted-text" role="doc">obigrep <scripts/obigrep></code> command. The <code>-p 'count>=10'</code> option means that the <code>python</code> expression :py<code class="interpreted-text" role="mod">count>=10</code> must be evaluated to :py<code class="interpreted-text" role="mod">True</code> for each sequence to be kept. Based on previous knowledge we also remove sequences with a length shorter than 80 bp (option -l) as we know that the amplified 12S-V5 barcode for vertebrates must have a length around 100bp.</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb21"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb21-1"><a href="#cb21-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obigrep</span> <span class="at">-l</span> 80 <span class="at">-p</span> <span class="st">'sequence.Count() >= 10'</span> results/wolf.ali.assigned.simple.clean.fasta <span class="dt">\</span></span>
|
||||
<span id="cb21-2"><a href="#cb21-2" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/wolf.ali.assigned.simple.clean.c10.l80.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>The first sequence record of <code>results/wolf.ali.assigned.simple.clean.c10.l80.fasta</code> is:</p>
|
||||
<pre><code>>HELIUM_000100422_612GNAAXX:7:22:2603:18023#0/1_sub[28..127] {"count":12182,"merged_sample":{"15a_F730814":7559,"29a_F260619":4623},"obiclean_head":true,"obiclean_headcount":2,"obiclean_internalcount":0,"obiclean_samplecount":2,"obiclean_singletoncount":0,"obiclean_status":{"15a_F730814":"h","29a_F260619":"h"},"obiclean_weight":{"15a_F730814":9165,"29a_F260619":6275}}
|
||||
ttagccctaaacacaagtaattaatataacaaaattattcgccagagtactaccggcaat
|
||||
agcttaaaactcaaaggacttggcggtgctttataccctt</code></pre>
|
||||
<p>At that time in the data cleanning we have conserved :</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb23"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb23-1"><a href="#cb23-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obicount</span> results/wolf.ali.assigned.simple.clean.c10.l80.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<div class="cell-output cell-output-stdout">
|
||||
<pre><code>time="2023-02-23T18:43:28+01:00" level=info msg="Appending results/wolf.ali.assigned.simple.clean.c10.l80.fasta file\n"
|
||||
26 31337 2585</code></pre>
|
||||
</div>
|
||||
</div>
|
||||
</section>
|
||||
</section>
|
||||
<section id="taxonomic-assignment-of-sequences" class="level3" data-number="3.2.6">
|
||||
<h3 data-number="3.2.6" class="anchored" data-anchor-id="taxonomic-assignment-of-sequences"><span class="header-section-number">3.2.6</span> Taxonomic assignment of sequences</h3>
|
||||
<p>Once denoising has been done, the next step in diet analysis is to assign the barcodes to the corresponding species in order to get the complete list of species associated to each sample.</p>
|
||||
<p>Taxonomic assignment of sequences requires a reference database compiling all possible species to be identified in the sample. Assignment is then done based on sequence comparison between sample sequences and reference sequences.</p>
|
||||
<section id="download-the-taxonomy" class="level4 unnumbered">
|
||||
<h4 class="unnumbered anchored" data-anchor-id="download-the-taxonomy">Download the taxonomy</h4>
|
||||
<p>It is always possible to download the complete taxonomy from NCBI using the following commands.</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb25"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb25-1"><a href="#cb25-1" aria-hidden="true" tabindex="-1"></a><span class="fu">mkdir</span> TAXO</span>
|
||||
<span id="cb25-2"><a href="#cb25-2" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> TAXO</span>
|
||||
<span id="cb25-3"><a href="#cb25-3" aria-hidden="true" tabindex="-1"></a><span class="ex">curl</span> http://ftp.ncbi.nih.gov/pub/taxonomy/taxdump.tar.gz <span class="dt">\</span></span>
|
||||
<span id="cb25-4"><a href="#cb25-4" aria-hidden="true" tabindex="-1"></a> <span class="kw">|</span> <span class="fu">tar</span> <span class="at">-zxvf</span> <span class="at">-</span></span>
|
||||
<span id="cb25-5"><a href="#cb25-5" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> ..</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>For people have a low speed internet connection, a copy of the <code>taxdump.tar.gz</code> file is provided in the wolf_data directory. The NCBI taxonomy is dayly updated, but the one provided here is ok for running this tutorial.</p>
|
||||
<p>To build the TAXO directory from the provided <code>taxdump.tar.gz</code>, you need to execute the following commands</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb26"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb26-1"><a href="#cb26-1" aria-hidden="true" tabindex="-1"></a><span class="fu">mkdir</span> TAXO</span>
|
||||
<span id="cb26-2"><a href="#cb26-2" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> TAXO</span>
|
||||
<span id="cb26-3"><a href="#cb26-3" aria-hidden="true" tabindex="-1"></a><span class="fu">tar</span> zxvf wolf_data/taxdump.tar.gz </span>
|
||||
<span id="cb26-4"><a href="#cb26-4" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> ..</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
</section>
|
||||
<section id="build-a-reference-database" class="level4 unnumbered">
|
||||
<h4 class="unnumbered anchored" data-anchor-id="build-a-reference-database">Build a reference database</h4>
|
||||
<p>One way to build the reference database is to use the <code>obipcr</code> program to simulate a PCR and extract all sequences from a general purpose DNA database such as genbank or EMBL that can be amplified <em>in silico</em> by the two primers (here <strong>TTAGATACCCCACTATGC</strong> and <strong>TAGAACAGGCTCCTCTAG</strong>) used for PCR amplification.</p>
|
||||
<p>The two steps to build this reference database would then be</p>
|
||||
<ol type="1">
|
||||
<li><p>Today, the easiest database to download is <em>Genbank</em>. But this will take you more than a day and occupy more than half a terabyte on your hard drive. In the <code>wolf_data</code> directory, a shell script called <code>download_gb.sh</code> is provided to perform this task. It requires that the programs <code>wget2</code> and <code>curl</code> are available on your computer.</p></li>
|
||||
<li><p>Use <code>obipcr</code> to simulate amplification and build a reference database based on the putatively amplified barcodes and their recorded taxonomic information.</p></li>
|
||||
</ol>
|
||||
<p>As these steps can take a long time (about a day for the download and an hour for the PCR), we already provide the reference database produced by the following commands so you can skip its construction. Note that as the Genbank and taxonomic database evolve frequently, if you run the following commands you may get different results.</p>
|
||||
<section id="download-the-sequences" class="level5 unnumbered">
|
||||
<h5 class="unnumbered anchored" data-anchor-id="download-the-sequences">Download the sequences</h5>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb27"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb27-1"><a href="#cb27-1" aria-hidden="true" tabindex="-1"></a><span class="fu">mkdir</span> genbank</span>
|
||||
<span id="cb27-2"><a href="#cb27-2" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> genbank</span>
|
||||
<span id="cb27-3"><a href="#cb27-3" aria-hidden="true" tabindex="-1"></a><span class="ex">../wolf_data/install_gb.sh</span></span>
|
||||
<span id="cb27-4"><a href="#cb27-4" aria-hidden="true" tabindex="-1"></a><span class="bu">cd</span> ..</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>DO NOT RUN THIS COMMAND EXCEPT IF YOU ARE REALLY CONSIENT OF THE TIME AND DISK SPACE REQUIRED.</p>
|
||||
</section>
|
||||
<section id="use-obipcr-to-simulate-an-in-silico-pcr" class="level5 unnumbered">
|
||||
<h5 class="unnumbered anchored" data-anchor-id="use-obipcr-to-simulate-an-in-silico-pcr">Use obipcr to simulate an in silico` PCR</h5>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb28"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb28-1"><a href="#cb28-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obipcr</span> <span class="at">-t</span> TAXO <span class="at">-e</span> 3 <span class="at">-l</span> 50 <span class="at">-L</span> 150 <span class="dt">\ </span></span>
|
||||
<span id="cb28-2"><a href="#cb28-2" aria-hidden="true" tabindex="-1"></a> <span class="ex">--forward</span> TTAGATACCCCACTATGC <span class="dt">\</span></span>
|
||||
<span id="cb28-3"><a href="#cb28-3" aria-hidden="true" tabindex="-1"></a> <span class="at">--reverse</span> TAGAACAGGCTCCTCTAG <span class="dt">\</span></span>
|
||||
<span id="cb28-4"><a href="#cb28-4" aria-hidden="true" tabindex="-1"></a> <span class="at">--no-order</span> <span class="dt">\</span></span>
|
||||
<span id="cb28-5"><a href="#cb28-5" aria-hidden="true" tabindex="-1"></a> genbank/Release-251/gb<span class="pp">*</span>.seq.gz</span>
|
||||
<span id="cb28-6"><a href="#cb28-6" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/v05.pcr.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>Note that the primers must be in the same order both in <code>wolf_diet_ngsfilter.txt</code> and in the <code>obipcr</code> command. The part of the path indicating the <em>Genbank</em> release can change. Please check in your genbank directory the exact name of your release.</p>
|
||||
</section>
|
||||
<section id="clean-the-database" class="level5 unnumbered">
|
||||
<h5 class="unnumbered anchored" data-anchor-id="clean-the-database">Clean the database</h5>
|
||||
<ol type="1">
|
||||
<li>filter sequences so that they have a good taxonomic description at the species, genus, and family levels (<code>obigrep</code> command command below).</li>
|
||||
<li>remove redundant sequences (<code>obiuniq</code> command below).</li>
|
||||
<li>ensure that the dereplicated sequences have a taxid at the family level (<code>obigrep</code> command below).</li>
|
||||
<li>ensure that sequences each have a unique identification (<code>obiannotate</code> command below)</li>
|
||||
</ol>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb29"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb29-1"><a href="#cb29-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obigrep</span> <span class="at">-t</span> TAXO <span class="dt">\</span></span>
|
||||
<span id="cb29-2"><a href="#cb29-2" aria-hidden="true" tabindex="-1"></a> <span class="at">--require-rank</span> species <span class="dt">\</span></span>
|
||||
<span id="cb29-3"><a href="#cb29-3" aria-hidden="true" tabindex="-1"></a> <span class="at">--require-rank</span> genus <span class="dt">\</span></span>
|
||||
<span id="cb29-4"><a href="#cb29-4" aria-hidden="true" tabindex="-1"></a> <span class="at">--require-rank</span> family <span class="dt">\</span></span>
|
||||
<span id="cb29-5"><a href="#cb29-5" aria-hidden="true" tabindex="-1"></a> results/v05.ecopcr <span class="op">></span> results/v05_clean.fasta</span>
|
||||
<span id="cb29-6"><a href="#cb29-6" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb29-7"><a href="#cb29-7" aria-hidden="true" tabindex="-1"></a><span class="ex">obiuniq</span> <span class="at">-c</span> taxid <span class="dt">\</span></span>
|
||||
<span id="cb29-8"><a href="#cb29-8" aria-hidden="true" tabindex="-1"></a> results/v05_clean.fasta <span class="dt">\</span></span>
|
||||
<span id="cb29-9"><a href="#cb29-9" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/v05_clean_uniq.fasta</span>
|
||||
<span id="cb29-10"><a href="#cb29-10" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb29-11"><a href="#cb29-11" aria-hidden="true" tabindex="-1"></a><span class="ex">obirefidx</span> <span class="at">-t</span> TAXO results/v05_clean_uniq.fasta <span class="dt">\</span></span>
|
||||
<span id="cb29-12"><a href="#cb29-12" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/v05_clean_uniq.indexed.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<div class="warning">
|
||||
<div class="title">
|
||||
<p>Warning</p>
|
||||
</div>
|
||||
<p>From now on, for the sake of clarity, the following commands will use the filenames of the files provided with the tutorial. If you decided to run the last steps and use the files you have produced, you'll have to use <code>results/v05_clean_uniq.indexed.fasta</code> instead of <code>wolf_data/db_v05_r117.indexed.fasta</code>.</p>
|
||||
</div>
|
||||
</section>
|
||||
</section>
|
||||
</section>
|
||||
<section id="assign-each-sequence-to-a-taxon" class="level3" data-number="3.2.7">
|
||||
<h3 data-number="3.2.7" class="anchored" data-anchor-id="assign-each-sequence-to-a-taxon"><span class="header-section-number">3.2.7</span> Assign each sequence to a taxon</h3>
|
||||
<p>Once the reference database is built, taxonomic assignment can be carried out using the <code>obitag</code> command.</p>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb30"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb30-1"><a href="#cb30-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obitag</span> <span class="at">-t</span> TAXO <span class="at">-R</span> wolf_data/db_v05_r117.indexed.fasta <span class="dt">\</span></span>
|
||||
<span id="cb30-2"><a href="#cb30-2" aria-hidden="true" tabindex="-1"></a> results/wolf.ali.assigned.simple.clean.c10.l80.fasta <span class="dt">\</span></span>
|
||||
<span id="cb30-3"><a href="#cb30-3" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/wolf.ali.assigned.simple.clean.c10.l80.taxo.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>The <code>obitag</code> adds several attributes in the sequence record header, among them:</p>
|
||||
<ul>
|
||||
<li>obitag_bestmatch=ACCESSION where ACCESSION is the id of hte sequence in the reference database that best aligns to the query sequence;</li>
|
||||
<li>obitag_bestid=FLOAT where FLOAT*100 is the percentage of identity between the best match sequence and the query sequence;</li>
|
||||
<li>taxid=TAXID where TAXID is the final assignation of the sequence by <code>obitag</code></li>
|
||||
<li>scientific_name=NAME where NAME is the scientific name of the assigned taxid.</li>
|
||||
</ul>
|
||||
<p>The first sequence record of <code>wolf.ali.assigned.simple.clean.c10.l80.taxo.fasta</code> is:</p>
|
||||
<div class="sourceCode" id="cb31"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb31-1"><a href="#cb31-1" aria-hidden="true" tabindex="-1"></a><span class="op">></span>HELIUM_000100422_612GNAAXX:7:81:18704:12346#0/1_sub[28..126] <span class="dt">{</span><span class="st">"count"</span><span class="dt">:88</span><span class="op">,</span><span class="st">"merged_sample"</span><span class="dt">:{</span><span class="st">"26a_F040644"</span><span class="dt">:88}</span><span class="op">,</span><span class="st">"obiclean_head"</span><span class="dt">:true</span><span class="op">,</span><span class="st">"obiclean_headcount"</span><span class="dt">:1</span><span class="op">,</span><span class="st">"obiclean_internalcount"</span><span class="dt">:0</span><span class="op">,</span><span class="st">"obiclean_samplecount"</span><span class="dt">:1</span><span class="op">,</span><span class="st">"obiclean_singletoncount"</span><span class="dt">:0</span><span class="op">,</span><span class="st">"obiclean_status"</span><span class="dt">:{</span><span class="st">"26a_F040644"</span><span class="dt">:</span><span class="st">"h"</span><span class="dt">}</span><span class="op">,</span><span class="st">"obiclean_weight"</span><span class="dt">:{</span><span class="st">"26a_F040644"</span><span class="dt">:208}</span><span class="op">,</span><span class="st">"obitag_bestid"</span><span class="dt">:0.9207920792079208</span><span class="op">,</span><span class="st">"obitag_bestmatch"</span><span class="dt">:</span><span class="st">"AY769263"</span><span class="op">,</span><span class="st">"obitag_difference"</span><span class="dt">:8</span><span class="op">,</span><span class="st">"obitag_match_count"</span><span class="dt">:1</span><span class="op">,</span><span class="st">"obitag_rank"</span><span class="dt">:</span><span class="st">"clade"</span><span class="op">,</span><span class="st">"scientific_name"</span><span class="dt">:</span><span class="st">"Boreoeutheria"</span><span class="op">,</span><span class="st">"taxid"</span><span class="dt">:1437010}</span></span>
|
||||
<span id="cb31-2"><a href="#cb31-2" aria-hidden="true" tabindex="-1"></a><span class="ex">ttagccctaaacataaacattcaataaacaagaatgttcgccagaggactactagcaata</span></span>
|
||||
<span id="cb31-3"><a href="#cb31-3" aria-hidden="true" tabindex="-1"></a><span class="ex">gcttaaaactcaaaggacttggcggtgctttatatccct</span></span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</section>
|
||||
<section id="generate-the-final-result-table" class="level3" data-number="3.2.8">
|
||||
<h3 data-number="3.2.8" class="anchored" data-anchor-id="generate-the-final-result-table"><span class="header-section-number">3.2.8</span> Generate the final result table</h3>
|
||||
<p>Some unuseful attributes can be removed at this stage.</p>
|
||||
<ul>
|
||||
<li>obiclean_head</li>
|
||||
<li>obiclean_headcount</li>
|
||||
<li>obiclean_internalcount</li>
|
||||
<li>obiclean_samplecount</li>
|
||||
<li>obiclean_singletoncount</li>
|
||||
</ul>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb32"><pre class="sourceCode bash code-with-copy"><code class="sourceCode bash"><span id="cb32-1"><a href="#cb32-1" aria-hidden="true" tabindex="-1"></a><span class="ex">obiannotate</span> <span class="at">--delete-tag</span><span class="op">=</span>obiclean_head <span class="dt">\</span></span>
|
||||
<span id="cb32-2"><a href="#cb32-2" aria-hidden="true" tabindex="-1"></a> <span class="at">--delete-tag</span><span class="op">=</span>obiclean_headcount <span class="dt">\</span></span>
|
||||
<span id="cb32-3"><a href="#cb32-3" aria-hidden="true" tabindex="-1"></a> <span class="at">--delete-tag</span><span class="op">=</span>obiclean_internalcount <span class="dt">\</span></span>
|
||||
<span id="cb32-4"><a href="#cb32-4" aria-hidden="true" tabindex="-1"></a> <span class="at">--delete-tag</span><span class="op">=</span>obiclean_samplecount <span class="dt">\</span></span>
|
||||
<span id="cb32-5"><a href="#cb32-5" aria-hidden="true" tabindex="-1"></a> <span class="at">--delete-tag</span><span class="op">=</span>obiclean_singletoncount <span class="dt">\</span></span>
|
||||
<span id="cb32-6"><a href="#cb32-6" aria-hidden="true" tabindex="-1"></a> results/wolf.ali.assigned.simple.clean.c10.l80.taxo.fasta <span class="dt">\</span></span>
|
||||
<span id="cb32-7"><a href="#cb32-7" aria-hidden="true" tabindex="-1"></a> <span class="op">></span> results/wolf.ali.assigned.simple.clean.c10.l80.taxo.ann.fasta</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
</div>
|
||||
<p>The first sequence record of <code>wolf.ali.assigned.simple.c10.l80.clean.taxo.ann.fasta</code> is then:</p>
|
||||
<pre><code>>HELIUM_000100422_612GNAAXX:7:84:16335:5083#0/1_sub[28..126] {"count":96,"merged_sample":{"26a_F040644":11,"29a_F260619":85},"obiclean_status":{"26a_F040644":"s","29a_F260619":"h"},"obiclean_weight":{"26a_F040644":14,"29a_F260619":110},"obitag_bestid":0.9595959595959596,"obitag_bestmatch":"AC187326","obitag_difference":4,"obitag_match_count":1,"obitag_rank":"subspecies","scientific_name":"Canis lupus familiaris","taxid":9615}
|
||||
ttagccctaaacataagctattccataacaaaataattcgccagagaactactagcaaca
|
||||
gattaaacctcaaaggacttggcagtgctttatacccct</code></pre>
|
||||
</section>
|
||||
<section id="looking-at-the-data-in-r" class="level3" data-number="3.2.9">
|
||||
<h3 data-number="3.2.9" class="anchored" data-anchor-id="looking-at-the-data-in-r"><span class="header-section-number">3.2.9</span> Looking at the data in R</h3>
|
||||
<div class="cell">
|
||||
<div class="sourceCode cell-code" id="cb34"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb34-1"><a href="#cb34-1" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(ROBIFastread)</span>
|
||||
<span id="cb34-2"><a href="#cb34-2" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(vegan)</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<div class="cell-output cell-output-stderr">
|
||||
<pre><code>Le chargement a nécessité le package : permute</code></pre>
|
||||
</div>
|
||||
<div class="cell-output cell-output-stderr">
|
||||
<pre><code>Le chargement a nécessité le package : lattice</code></pre>
|
||||
</div>
|
||||
<div class="cell-output cell-output-stderr">
|
||||
<pre><code>This is vegan 2.6-4</code></pre>
|
||||
</div>
|
||||
<div class="sourceCode cell-code" id="cb38"><pre class="sourceCode r code-with-copy"><code class="sourceCode r"><span id="cb38-1"><a href="#cb38-1" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(magrittr)</span>
|
||||
<span id="cb38-2"><a href="#cb38-2" aria-hidden="true" tabindex="-1"></a> </span>
|
||||
<span id="cb38-3"><a href="#cb38-3" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb38-4"><a href="#cb38-4" aria-hidden="true" tabindex="-1"></a>diet_data <span class="ot"><-</span> <span class="fu">read_obifasta</span>(<span class="st">"results/wolf.ali.assigned.simple.clean.c10.l80.taxo.fasta"</span>) </span>
|
||||
<span id="cb38-5"><a href="#cb38-5" aria-hidden="true" tabindex="-1"></a>diet_data <span class="sc">%<>%</span> <span class="fu">extract_features</span>(<span class="st">"obitag_bestmatch"</span>,<span class="st">"obitag_rank"</span>,<span class="st">"scientific_name"</span>,<span class="st">'taxid'</span>)</span>
|
||||
<span id="cb38-6"><a href="#cb38-6" aria-hidden="true" tabindex="-1"></a></span>
|
||||
<span id="cb38-7"><a href="#cb38-7" aria-hidden="true" tabindex="-1"></a>diet_tab <span class="ot"><-</span> <span class="fu">extract_readcount</span>(diet_data,<span class="at">key=</span><span class="st">"obiclean_weight"</span>)</span>
|
||||
<span id="cb38-8"><a href="#cb38-8" aria-hidden="true" tabindex="-1"></a>diet_tab</span></code><button title="Copy to Clipboard" class="code-copy-button"><i class="bi"></i></button></pre></div>
|
||||
<div class="cell-output cell-output-stdout">
|
||||
<pre><code>4 x 26 sparse Matrix of class "dgCMatrix"</code></pre>
|
||||
</div>
|
||||
<div class="cell-output cell-output-stderr">
|
||||
<pre><code> [[ suppressing 26 column names 'HELIUM_000100422_612GNAAXX:7:72:17638:8081#0/1_sub[28..126]', 'HELIUM_000100422_612GNAAXX:7:17:3675:13316#0/1_sub[28..126]', 'HELIUM_000100422_612GNAAXX:7:58:11419:17203#0/1_sub[28..126]' ... ]]</code></pre>
|
||||
</div>
|
||||
<div class="cell-output cell-output-stdout">
|
||||
<pre><code>
|
||||
26a_F040644 15 17 481 . . . 208 . . . . . 14 31 . . 43 52 14 72
|
||||
13a_F730603 . . 19 1 . 15 . 22 . . 20 25 . . . 8409 . . . .
|
||||
15a_F730814 . . 5 . . . . . . . . . . . . . . . . .
|
||||
29a_F260619 . . 1 13 25 . . . 16 44 . . . . 391 . . . 110 .
|
||||
|
||||
26a_F040644 88 18 . . 12830 .
|
||||
13a_F730603 . . 9 . . .
|
||||
15a_F730814 . . 4 . . 9165
|
||||
29a_F260619 . . . 353 . 5884</code></pre>
|
||||
</div>
|
||||
</div>
|
||||
<dl>
|
||||
<dt>This file contains 26 sequences. You can deduce the diet of each sample:</dt>
|
||||
<dd>
|
||||
<ul>
|
||||
<li>13a_F730603: Cervus elaphus</li>
|
||||
<li>15a_F730814: Capreolus capreolus</li>
|
||||
<li>26a_F040644: Marmota sp. (according to the location, it is Marmota marmota)</li>
|
||||
<li>29a_F260619: Capreolus capreolus</li>
|
||||
</ul>
|
||||
</dd>
|
||||
</dl>
|
||||
<p>Note that we also obtained a few wolf sequences although a wolf-blocking oligonucleotide was used.</p>
|
||||
|
||||
|
||||
<div id="refs" class="references csl-bib-body hanging-indent" role="doc-bibliography" style="display: none">
|
||||
<div id="ref-Riaz2011-gn" class="csl-entry" role="doc-biblioentry">
|
||||
Riaz, Tiayyba, Wasim Shehzad, Alain Viari, François Pompanon, Pierre Taberlet, and Eric Coissac. 2011. <span>“<span class="nocase">ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis</span>.”</span> <em>Nucleic Acids Research</em> 39 (21): e145. <a href="https://doi.org/10.1093/nar/gkr732">https://doi.org/10.1093/nar/gkr732</a>.
|
||||
</div>
|
||||
<div id="ref-Seguritan2001-tg" class="csl-entry" role="doc-biblioentry">
|
||||
Seguritan, V, and F Rohwer. 2001. <span>“<span class="nocase">FastGroup: a program to dereplicate libraries of 16S rDNA sequences</span>.”</span> <em>BMC Bioinformatics</em> 2 (October): 9. <a href="https://doi.org/10.1186/1471-2105-2-9">https://doi.org/10.1186/1471-2105-2-9</a>.
|
||||
</div>
|
||||
<div id="ref-Shehzad2012-pn" class="csl-entry" role="doc-biblioentry">
|
||||
Shehzad, Wasim, Tiayyba Riaz, Muhammad A Nawaz, Christian Miquel, Carole Poillot, Safdar A Shah, Francois Pompanon, Eric Coissac, and Pierre Taberlet. 2012. <span>“<span class="nocase">Carnivore diet analysis based on next-generation sequencing: Application to the leopard cat (Prionailurus bengalensis) in Pakistan</span>.”</span> <em>Molecular Ecology</em> 21 (8): 1951–65. <a href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-294X.2011.05424.x">https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-294X.2011.05424.x</a>.
|
||||
</div>
|
||||
</div>
|
||||
</section>
|
||||
</section>
|
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