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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>
<section id="aims-of-obitools" class="level2">
<h2 class="anchored" data-anchor-id="aims-of-obitools">Aims of <em>OBITools</em></h2>
<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>
<div id="refs" class="references csl-bib-body hanging-indent" role="doc-bibliography" style="display: none">
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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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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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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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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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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">
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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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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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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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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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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