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[GUIDELINES]
<span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">In order to decompose a complecated metagenomic de Bruijn graph into simpler subgraphs, MetaVelvet searches the following X-structure, i.e., chimeric nodes have two incoming edges and two outgoing edges.<br /><br /><img id="s-mce-img" class="s-mce-img" style="display: block; margin-left: auto; margin-right: auto;" src="https://s3.amazonaws.com/pr-journal/bngc2w6.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bnfc2w6.png" data-ofn="fig7.png" /><br /></span>
<p style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">Then, MetaVelvet split the X-structures when the following conditions are satisfied: </p>
<ul style="padding-left: 60px; color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">
<li>Primary-IN and Primary-OUT nodes are classified into the same peak.</li>
<li>Secondary-IN and Secondary-OUT nodes are classified into the same peak.</li>
<li>Chimeric node has a coverage value mostly equal (within 50% difference by default) to the average between the sum of coverage values of the two origin nodes of incoming edges (Primary-IN and Secondary-IN), and the sum of the two destination nodes of outgoing edges (Primary-OUT and Secondary-OUT).</li>
</ul>
<br 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;">In order to accurately avoid chimeric contigs/scaffolds caused by subgraph decomposition, we added the following two conditions (&gt;= 1.2.01):</span>
<ul style="padding-left: 60px; color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">
<li>The number of <em>consistent paired-end connections</em> is equal to or larger than a certain value. This cutoff value can be specified by the <strong>-valid_connections</strong> option (default: 1).</li>
<li>The number of <em>inconsistent paired-end connections</em> is equal to or smaller than a certain value. This cutoff value can be specified by the <strong>-noise_connections</strong> option (default: 0).</li>
<li>For lower compatibility, users can turn off the paired-end conditions by the <strong>-use_connections</strong> option.</li>
<li></li>
</ul>
<span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">Here, we denote paired-end connections between Primary-IN and Primary-OUT or between Secondary-IN and Secondary-OUT as </span><em style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">consistent paired-end connections</em><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">. We also denote paired-end connections between Primary-IN and Secondary-OUT or between Secondary-IN and Primary-OUT as </span><em style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">inconsistent paired-end connections</em><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">. </span><br style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;" /><br 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;">Based upon our accuracy evaluation, </span><strong style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">-valid_connections 1 -noise_connections 0</strong><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"> is an appropriate setting when very similar species co-exist in an environment (MetaVelvet can efficiently avoid misassemblies). Otherwise, </span><strong style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">-valid_connections 0 -noise_connections 0</strong><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"> is a more appropriate setting (MetaVelvet can achieve a greater contig/scaffold N50 while avoiding misassemblies).</span><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><br /><br /></span><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 style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;">Frequently Asked Questions<br /><br style="box-sizing: border-box;" /></strong></span></span>
<ul>
<li>Q: meta-velveth is not generated in the new version (&gt;= 1.1.01). Is it problem? <br />A: 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 meta-velveth. Instead, please use velveth, velvetg, and meta-velvetg.</li>
</ul>
<br />
<ul>
<li>Q: When only one coverage peak is detected (or manually input), is there any difference between MetaVelvet and Velvet algorithms? <br />A: There is no substantial difference. In such cases, meta-velvetg moves to "single-peak mode" and graph splitting functions in meta-velvetg is not called. Instead of graph splitting functions, standard velvet functions are called in such cases.</li>
</ul>
<br />
<ul>
<li>Q: What's the difference in working procedures between velvetg, meta-velvetg (&lt;= 0.3.1), and meta-velvetg(&gt;= 1.1.01)? <br />A: The following is the working procedure of velvetg, meta-velvetg (&lt;= 0.3.1), and meta-velvetg (&gt;= 1.1.01): <br /><br />    velvetg &amp; meta-velvetg(&lt;=0.3.1) : <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 />    meta-velvetg (&gt;= 1.1.01): <br />        Load Sequences &amp; Roadmaps &amp; Graph2 file <br />        -&gt; Generate meta-velvetg.contigs.fa and meta-velvetg.LastGraph </li>
</ul>
<br />
<ul>
<li>Q: Is version compatibility between Velvet and MetaVelvet fully tested? <br />A: 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 style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;"><br />Troubleshooting<br style="box-sizing: border-box;" /><br style="box-sizing: border-box;" /></strong><strong style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;">Trouble:</strong><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;"> </span><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;">When drawing</span><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;"> </span><em style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;">k</em><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;">-mer coverage histogram (as in the</span><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;"> </span><a href="http://metavelvet.dna.bio.keio.ac.jp/MV.html#advanced-topic-1" target="_blank">"Advanced topics 1"</a><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;"> </span><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;">section), the following warning messages is appeared:<br /><br /></span></span></span>
<blockquote style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; quotes: none; background-color: #f6f6f4;">&gt; weighted.hist(data$shot1_cov,data$lgth,breaks=seq(0,200,1)) <br style="box-sizing: border-box;" />Warning messages: <br style="box-sizing: border-box;" />1: In min(x, na.rm = na.rm) : <br style="box-sizing: border-box;" />no non-missing arguments to min; returning Inf <br style="box-sizing: border-box;" />2: In max(x, na.rm = na.rm) : <br style="box-sizing: border-box;" />no non-missing arguments to max; returning -Inf <br style="box-sizing: border-box;" />3: In weighted.hist(data$shot1_cov, data$lgth, breaks = seq(0, 200, : <br style="box-sizing: border-box;" />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 style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;"><br />Solution:</strong><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;"> </span><span style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; background-color: #f6f6f4;">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></span>
<blockquote style="box-sizing: border-box; margin: 0px; padding: 0px; border: 0px; font-stretch: inherit; vertical-align: baseline; quotes: none; background-color: #f6f6f4;"><br />$ head -n 1 meta-velvetg.Graph2-stats.txt \ <br style="box-sizing: border-box;" />        &gt; meta-velvetg.Graph2-stats.rmInf.txt <br style="box-sizing: border-box;" />$ perl -ne '{print $_ unless /Inf/;}' \ <br style="box-sizing: border-box;" />        meta-velvetg.Graph2-stats.txt \ <br style="box-sizing: border-box;" />        &gt;&gt; meta-velvetg.Graph2-stats.rmInf.txt <br style="box-sizing: border-box;" />$ R <br style="box-sizing: border-box;" />&gt; library(plotrix) <br style="box-sizing: border-box;" />&gt; data = read.table("meta-velvetg.Graph2-stats.rmInf.txt", header=TRUE) <br style="box-sizing: border-box;" />&gt; weighted.hist(data$shot1_cov,data$lgth,breaks=seq(0,200,1)) </blockquote>

[STEPS]