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[DESCRIPTION]
<p>The ITS protocol detailed here is designed to amplify fungal microbial eukaryotic lineages using paired-end community sequencing on the Illumina platform with primers ITS1f-ITS2 (EMP.ITSkabir).</p>

[BEFORE_START]
<p>For running these libraries on the MiSeq and HiSeq, please make sure you read the supplementary methods of Caporaso et al. (2012). You will need to make your sample more complex by adding 5-10% PhiX to your run.</p>
<p> </p>
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[GUIDELINES]
<h3>Ordering primers</h3>
<p>The primer sequences in this protocol are always listed in the 5′ -&gt; 3′ orientation. This is the orientation that should be used for ordering. See the page <a href="http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/primer-ordering-and-resuspension/" target="_blank" rel="noopener noreferrer">Primer Ordering and Resuspension</a> for more information. Primer constructs were designed by Dylan Smith and Kabir Peay.</p>
<p> </p>
<ul>
<li><a href="ftp://ftp.microbio.me/emp/protocols/primers/ITS1f_ITS2_Nov2016.xlsx" target="_blank" rel="noopener noreferrer">Illumina ITS Primer Constructs (ITS1f-ITS2)</a></li>
</ul>
<p> </p>
<p><strong>Note</strong>: Unlike the 16S and 18S sequencing primers, the ITS sequencing primers have additional 3′ bases beyond the PCR primers, in order to match the melting temperature of the Illumina adapters. The forward sequencing primer has 19 and the reverse sequencing primer has 15 additional 3′ bases; therefore the amplicon sequences will begin 19 bp (forward read) and 15 bp (reverse read) after the PCR primers. </p>
<p> </p>
<h4>EMP.ITSkabir forward primer (ITS1f)</h4>
<p>Field descriptions (space-delimited):</p>
<ol>
<li>5′ Illumina adapter</li>
<li>Forward primer linker</li>
<li>Forward primer (ITS1f; Note: This is 38 bp upstream of ITS1 from White et al., 1990.)</li>
</ol>
<p> </p>
<p><code>AATGATACGGCGACCACCGAGATCTACAC GG CTTGGTCATTTAGAGGAAGTAA</code></p>
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<h4> </h4>
<h4>EMP.ITSkabir reverse primer (ITS2), barcoded</h4>
<p>Field descriptions (space-delimited):</p>
<ol>
<li>Reverse complement of 3′ Illumina adapter</li>
<li>Golay barcode</li>
<li>Reverse primer linker</li>
<li>Reverse primer (ITS2; Note: This is identical to ITS2 from White et al., 1990.)</li>
</ol>
<p> </p>
<p><code>CAAGCAGAAGACGGCATACGAGAT NNNNNNNNNN CG GCTGCGTTCTTCATCGATGC</code></p>
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<p> <strong>PCR reaction mixtures</strong></p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/vy5fw26.png" width="247" height="207" data-src="https://s3.amazonaws.com/pr-journal/vy5fw26.png" data-ofn="Screen Shot 2018-04-13 at 4.02.44 PM.png" /></p>
<p style="text-align: left;"><code></code></p>
<p style="text-align: left;"> </p>
<p style="text-align: left;"><strong>Notes</strong>:</p>
<ul>
<li style="text-align: left;">PCR-grade water from Sigma (cat. no. W3500) or MoBio (cat. no. 17000-11)</li>
<li style="text-align: left;">Platinum Hot Start PCR Master Mix (2x) from ThermoFisher (cat. no. 13000014)</li>
<li style="text-align: left;">Final master mix concentration in 1x reaction: 0.8x</li>
<li style="text-align: left;">Final primer concentration in 1x reaction: 0.2 µM</li>
</ul>
<p> </p>
<p><strong>Thermocycler conditions </strong></p>
<p><strong>ITS amplification </strong></p>
<ul>
<li style="text-align: left;">Primers: ITS1f-ITS2</li>
<li style="text-align: left;">Amplicon size: ~230 bp</li>
</ul>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/vy6fw26.png" width="254" height="227" data-src="https://s3.amazonaws.com/pr-journal/vy6fw26.png" data-ofn="Screen Shot 2018-04-13 at 4.05.23 PM.png" /></p>
<h3>  </h3>
<h3>References</h3>
<div>
<div>
<ul>
<li>Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S. M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J. A., Smith, G., &amp; Knight, R. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621–1624. <a href="http://doi.org/10.1038/ismej.2012.8" target="_blank" rel="noopener noreferrer">http://doi.org/10.1038/ismej.2012.8</a></li>
<li>Gardes, M., &amp; Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts. Molecular Ecology, 2(2), 113–118. <a href="http://doi.org/10.1111/j.1365-294X.1993.tb00005.x" target="_blank" rel="noopener noreferrer">http://doi.org/10.1111/j.1365-294X.1993.tb00005.x</a></li>
<li>Smith, D. P., &amp; Peay, K. G. (2014). Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing. PLoS ONE, 9(2), e90234–e90234. <a href="http://doi.org/10.1371/journal.pone.0090234" target="_blank" rel="noopener noreferrer">http://doi.org/10.1371/journal.pone.0090234</a></li>
<li>Walters, W., Hyde, E. R., Berg-Lyons, D., Ackermann, G., Humphrey, G., Parada, A., Gilbert, J. A., Jansson, J. K., Caporaso, J. G., Fuhrman, J. A., Apprill, A., &amp; Knight, R. (2016). Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. mSystems, 1(1), e00009–15. <a href="http://doi.org/10.1128/mSystems.00009-15" target="_blank" rel="noopener noreferrer">http://doi.org/10.1128/mSystems.00009-15</a></li>
<li>White, T. J., Bruns, T., Lee, S., &amp; Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols: a guide to methods and applications (pp. 315–322). New York: Academic Press.</li>
</ul>
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