TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The 18S protocol detailed here is designed to amplify eukaryotes broadly with a focus on microbial eukaryotic lineages. The primers target the 18S SSU rRNA and are based on those of Amaral-Zettler et al. (2009). The constructs are designed to be used with the Illumina platform.</p>
<p> </p>
<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>
<p>The outlines of the protocol are the same as the 16S protocol, but different primers, PCR conditions, and sequencing primers are used. In addition, we have designed a blocking primer that reduces the amplification of vertebrate host DNA to be used on host-associated samples, especially those that have a low eukaryotic biomass. Blocking primer strategy is based on Vestheim et al. (2008).</p>

[GUIDELINES]
<p><strong>Ordering primers </strong></p>
<p> </p>
<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="&quot;https://press.igsb.anl.gov/earthmicrobiome/?page_" target="_blank" rel="noopener noreferrer">Primer Ordering and Resuspension</a>. Primer constructs were designed by Laura Wegener Parfrey.</p>
<ul>
<li><a href="ftp://ftp.microbio.me/emp/protocols/primers/Euk1391f_EukBr_Nov2016.xlsx" target="_blank" rel="noopener noreferrer">Illumina 18S Primer Constructs (Euk1391f-EukBr)</a></li>
</ul>
<p> </p>
<p><strong>Illumina_Euk_1391f forward primer: </strong></p>
<p>Field descriptions (space-delimited): </p>
<ol>
<li>5′ Illumina adapter</li>
<li>Forward primer pad</li>
<li>Forward primer linker</li>
<li>Forward primer (1391f)</li>
</ol>
<p><code>AATGATACGGCGACCACCGAGATCTACAC TATCGCCGTT CG GTACACACCGCCCGTC</code></p>
<p><code></code></p>
<p> </p>
<p><strong>Illumina_EukBr reverse primer, barcoded: </strong><code></code></p>
<ol>
<li>Reverse complement of 3′ Illumina adapter</li>
<li>Golay barcode</li>
<li>Reverse primer pad</li>
<li>Reverse primer linker</li>
<li>Reverse primer (EukBr)</li>
</ol>
<p><code>CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCAGTCAG CA TGATCCTTCTGCAGGTTCACCTAC</code></p>
<p> </p>
<p><strong>Mammal_block_I-short_1391f mammal blocking primer:</strong></p>
<p>The mammal blocking primer is to be used when there is a high probability of picking up host genomic DNA. The C3 spacer (/3SpC3/) is a chemical modification that prevents extension during the PCR. Please note that the use of blocking primer reduces the number of host sequences detected but does not completely eliminate them. Thus remaining host sequences should also be filtered out during the analysis phase. We have found blocking primers to be particularly useful for host-associated samples with a low biomass of eukaryotic DNA. Note: Sequence is formatted for ordering from IDT.</p>
<p> </p>
<p><code>GCCCGTCGCTACTACCGATTGG/ideoxyI//ideoxyI//ideoxyI//ideoxyI//ideoxyI/TTAGTGAGGCCCT/3SpC3/</code></p>
<p> </p>
<p> </p>
<p><strong>PCR reaction mixture </strong></p>
<p> </p>
<p>No blocking primer</p>
<p><img src="https://s3.amazonaws.com/pr-journal/us7fw26.png" width="253" height="205" data-src="https://s3.amazonaws.com/pr-journal/us7fw26.png" data-ofn="Screen Shot 2018-03-15 at 11.08.01 PM.png" /></p>
<p> </p>
<p>With blocking primer </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/us8fw26.png" width="254" height="234" data-src="https://s3.amazonaws.com/pr-journal/us8fw26.png" data-ofn="Screen Shot 2018-03-15 at 11.08.44 PM.png" /></p>
<p> </p>
<p>Notes:</p>
<ul>
<li>PCR-grade water from Sigma (<a href="https://www.sigmaaldrich.com/catalog/product/sigma/w3500?lang=en&amp;region=US" target="_blank" rel="noopener noreferrer">cat. no. W3500</a>) or MoBio (<a href="https://mobio.com/products/lab-supplies/certified-water/pcr-water-certified-dna-free.html" target="_blank" rel="noopener noreferrer">cat. no. 17000-11</a>)</li>
<li>Platinum Hot Start PCR Master Mix (2x) from ThermoFisher (<a href="http://www.thermofisher.com/order/catalog/product/13000014" target="_blank" rel="noopener noreferrer">cat. no. 13000014</a>)</li>
<li>Final master mix concentration in 1x reaction: 0.8x</li>
<li>Final primer concentration in 1x reaction: 0.2 µM</li>
<li>Final blocking primer concentration in 1x reaction: 1.6 µM</li>
</ul>
<p> </p>
<p><strong>Thermocycler conditions </strong></p>
<p> </p>
<p><strong>No blocking primer: </strong></p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/us9fw26.png" width="256" height="214" data-src="https://s3.amazonaws.com/pr-journal/us9fw26.png" data-ofn="Screen Shot 2018-03-15 at 11.10.33 PM.png" /></p>
<p> </p>
<p><strong>With blocking primer: </strong></p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/utafw26.png" width="257" height="245" data-src="https://s3.amazonaws.com/pr-journal/utafw26.png" data-ofn="Screen Shot 2018-03-15 at 11.11.16 PM.png" /></strong></p>
<p> </p>
<p> </p>
<p><strong>18S sequencing primers </strong></p>
<p> </p>
<p><strong>Euk_illumina_read_seq_primer:</strong></p>
<p>Field descriptions (space-delimited):</p>
<ol>
<li>Reverse primer pad</li>
<li>Reverse primer linker</li>
<li>Reverse primer (EukBr)</li>
</ol>
<p><code>AGTCAGTCAG CA TGATCCTTCTGCAGGTTCACCTAC</code></p>
<p> </p>
<p><strong>Euk_illumina_index_seq_primer:</strong></p>
<p>Field description (space-delimited):</p>
<ol>
<li>Reverse complement of reverse primer (EukBr)</li>
<li>Reverse complement of reverse primer linker</li>
<li>Reverse complement of reverse primer pad</li>
</ol>
<p><code>GTAGGTGAACCTGCAGAAGGATCA TG CTGACTGACT</code></p>
<p> </p>
<p> </p>
<p><strong>References </strong></p>
<ul>
<li>Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W. &amp; Huse, S. M. A method for studying protistan diversity using massively parallel sequencing of V9 hyper variable regions of small-subunit ribosomal RNA Genes. PLoS ONE 4, e6372 (2009).</li>
<li>Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J (2012). doi:10.1038/ismej.2012.8</li>
<li>Vestheim, H. &amp; Jarman, S. N. Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs. Frontiers in Zoology 2008 5:1 5, 12 (2008).</li>
</ul>

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