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[DESCRIPTION]
<span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><strong>Motivation:</strong> An important step of "metagenomics" analysis is the assembly of multiple genomes from mixed sequence reads of multiple species in a microbial community. Most conventional pipelines employ a single-genome assembler with carefully optimized parameters and post-process the resulting scaffolds to correct assembly errors. Limitations of the use of a single-genome assembler for </span><em style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">de novo</em><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"> metagenome assembly are that highly conserved sequences shared between different species often causes chimera contigs, and sequences of highly abundant species are likely mis-identified as repeats in a single genome.<br /><br /><strong>Methods:</strong> </span><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">We modified and extended a single-genome and de Bruijn-graph based assembler, </span><a href="http://www.ebi.ac.uk/~zerbino/velvet/" target="_blank">Velvet</a><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">, for </span><em style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">de novo </em><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">metagenome assembly. Our fundamental ideas are first decomposing de Bruijn graph constructed from mixed short reads into individual sub-graphs and second building scaffolds based on every decomposed de Bruijn sub-graph as isolate species genome.<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bmzc2w6.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bmyc2w6.png" data-ofn="fig1.png" /></span><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><br /><br /><br /></span>

[GUIDELINES]
<strong>What is improved?<br /><br />Longer N50 size: </strong><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">On artificially generated simulation datasets of short sequence reads, it is proven that MetaVelvet assembled metegenomic read data with significantly longer N50 sizes than single-genome assemblers (</span><a href="http://www.ebi.ac.uk/~zerbino/velvet/" target="_blank">Velvet</a><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"> and </span><a href="http://soap.genomics.org.cn/soapdenovo.html" target="_blank">SOAPdenovo</a><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">) and another meta-genome assembler (</span><a href="http://i.cs.hku.hk/~alse/hkubrg/projects/metaidba/" target="_blank">MetaIDBA</a><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">) as shown in the table below.<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bm3c2w6.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bm2c2w6.png" data-ofn="fig2.png" /><br /><br /><strong>Improved gene prediction:</strong> </span><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">We applied Velvet and MetaVelvet to real metagenomic dataset (SRR041654 and SRR041655; human gut microbiome data downloaded from </span><a href="http://www.ncbi.nlm.nih.gov/sra" target="_blank">NCBI sequence read archive</a><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">). Then, </span><a href="http://metagene.cb.k.u-tokyo.ac.jp/" target="_blank">MetaGeneAnnotator</a> <span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">was used to predict a gene set of the microbiome. From the results, it is demonstrated that MetaVelvet can efficiently avoid chimeric scaffolds, resulting in longer scaffolds, increased number of predicted genes, and decreased fraction of partial genes.<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bm4c2w6.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bm4c2w6.png" data-ofn="fig3.png" /><br /><br /><strong>Widely applicable to metagenomic analyses:</strong> </span><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">We also compared the results of metagenomic analyses, such as taxonomic content analysis, functional composition analysis, and pathway mapping analysis, between Velvet and MetaVelvet. The results demonstrate that taxonomic content can be estimated more clearly when based on the MetaVelvet scaffolds than when based on the Velvet scaffolds as shown in the figure below left (the number of genes that cannot be assigned to phylum-level taxonomy is substantially decreased). Moreover, the result of functional composition analysis also shows that MetaVelvet is more appropriate for metagenomics purpose than Velvet as shown in the figure below right (the number of genes assigned to any functional category is improved).<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bm5c2w6.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bm5c2w6.png" data-ofn="fig4.png" /><br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bm6c2w6.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bm6c2w6.png" data-ofn="fig5.png" /><br /><br /><strong>Source Code (Release Notes)<br /></strong></span>
<p style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">The following source codes is freely available under <a href="http://www.gnu.org/licenses/gpl.html" target="_blank">GNU General Public License</a>.</p>
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<li>MetaVelvet-v1.2.01 (May.29,2012) <br />- enable to use paired-end information in a graph decomposition step. <br />- three options are newly added in order to specify how to use paired-end connections for graph decomposition: -valid_connections, -noise_connections, and -use_connections. <br />- for details, please see <a href="http://metavelvet.dna.bio.keio.ac.jp/MV.html#advanced-topic-3" target="_blank">Advanced topic 3: how to use paired-end information for graph decompositoin</a>.</li>
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<li>MetaVelvet-v1.1.01 (Oct.19,2011) <br />- this version is based upon Velvet 1.1.06. <br />- fix a frequent bug: no peak detection error. <br />- fix a frequent bug: infinite loop error. <br />- modify the graph split algorithm: an exception for "knock-turn" loops. <br />- modify the graph split algorithm: split priority is now based on peak IDs rather than node IDs. </li>
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<li>MetaVelvet-v0.3 (Jan.14,2011) <br />- this version is based upon Velvet 0.7.62.</li>
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<p style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">Source codes at these versions can be downloaded from <a href="http://metavelvet.dna.bio.keio.ac.jp/src" target="_blank">here</a>.<br /><br /></p>
<span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><strong>Requirements (&gt;=1.1.01)<br /></strong></span></span>
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<li>Required libraries: <br />
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<li>zlib library (&gt;= 1.2.3) - zlib library is also included in the Velvet distribution.</li>
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<li>Required programs: <br />
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<li>velveth (&gt;= 1.1.06) - included in the Velvet distribution.</li>
<li>velvetg (&gt;= 1.1.06) - included in the Velvet distribution.</li>
<li>g++ (&gt;= 4.3.2) - for compile.</li>
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<li>Recommened environment: <br />
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<li>OS : standard 64bit Linux (It can in theory function on a 32bit environment, but not recommended).</li>
<li>RAM: at least 12GB (more is no luxury).</li>
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<span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><br /><strong>Frequently Asked Questions<br /></strong></span></span>
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<li><strong>Q:</strong> <strong>meta-velveth</strong> is not generated in the new version (&gt;= 1.1.01). Is it problem? <br /><strong>A:</strong> This is not problem. The usage of MetaVelvet is changed when the version 1.1.01 is released, and the new version does not include <strong>meta-velveth</strong>. Instead, please use <strong>velveth</strong>, <strong>velvetg</strong>, and <strong>meta-velvetg</strong>.</li>
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<li><strong>Q:</strong> When only one coverage peak is detected (or manually input), is there any difference between MetaVelvet and Velvet algorithms? <br /><strong>A:</strong> There is no substantial difference. In such cases, <strong>meta-velvetg</strong> moves to "single-peak mode" and graph splitting functions in <strong>meta-velvetg</strong> is not called. Instead of graph splitting functions, standard velvet functions are called in such cases.</li>
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<li><strong>Q:</strong> What's the difference in working procedures between <strong>velvetg</strong>, <strong>meta-velvetg</strong> (&lt;= 0.3.1), and <strong>meta-velvetg</strong>(&gt;= 1.1.01)? </li>
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<li><strong>A:</strong> The following is the working procedure of <strong>velvetg</strong>, <strong>meta-velvetg</strong> (&lt;= 0.3.1), and <strong>meta-velvetg</strong> (&gt;= 1.1.01): <br /><br />    <strong>velvetg &amp; meta-velvetg(&lt;=0.3.1) : </strong><br />        Load Sequences &amp; Roadmaps file <br />        -&gt; Generate PreGraph file <br />        -&gt; Generate Graph or Graph2 file <br />        -&gt; Generate contigs.fa and LastGraph <br /><br />    <strong>meta-velvetg (&gt;= 1.1.01): </strong><br />        Load Sequences &amp; Roadmaps &amp; Graph2 file <br />        -&gt; Generate meta-velvetg.contigs.fa and meta-velvetg.LastGraph </li>
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<li><strong>Q:</strong> Is version compatibility between Velvet and MetaVelvet fully tested? <br /><strong>A:</strong> Version compatibility between Velvet-1.0.06 and MetaVelvet-1.1.01 is fully tested.</li>
</ul>
<span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><strong><br />Troubleshooting<br /><br /></strong><strong>Trouble:</strong> When drawing <em>k</em>-mer coverage histogram (as in the <a href="http://metavelvet.dna.bio.keio.ac.jp/MV.html#advanced-topic-1" target="_blank">"Advanced topics 1"</a> section), the following warning messages is appeared:</span></span>
<blockquote>&gt; weighted.hist(data$shot1_cov,data$lgth,breaks=seq(0,200,1)) <br />Warning messages: <br />1: In min(x, na.rm = na.rm) : <br />no non-missing arguments to min; returning Inf <br />2: In max(x, na.rm = na.rm) : <br />no non-missing arguments to max; returning -Inf <br />3: In weighted.hist(data$shot1_cov, data$lgth, breaks = seq(0, 200, : <br />Areas will not relate to frequencies </blockquote>
<span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><strong>Solution:</strong> This warning (error) is caused by "Inf" values in the Graph2 node stats. Accordingly, by removing "Inf" values from the Graph2 stats, the error is resolved:</span></span>
<blockquote>$ head -n 1 meta-velvetg.Graph2-stats.txt \ <br />        &gt; meta-velvetg.Graph2-stats.rmInf.txt <br />$ perl -ne '{print $_ unless /Inf/;}' \ <br />        meta-velvetg.Graph2-stats.txt \ <br />        &gt;&gt; meta-velvetg.Graph2-stats.rmInf.txt <br />$ R <br />&gt; library(plotrix) <br />&gt; data = read.table("meta-velvetg.Graph2-stats.rmInf.txt", header=TRUE) <br />&gt; weighted.hist(data$shot1_cov,data$lgth,breaks=seq(0,200,1)) </blockquote>
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