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[DESCRIPTION]
<p>The 16S protocol detailed here is designed to amplify prokaryotes (bacteria and archaea) using paired-end 16S community sequencing on the Illumina platform. Primers 515F-806R target the V4 region of the 16S SSU rRNA.</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>

[GUIDELINES]
<p><strong>16S amplification primers </strong></p>
<p> </p>
<p>The current primers have been modified from the original 515F-806R primer pair (Caporaso et al., 2011) in the following ways:</p>
<p> </p>
<ol>
<li>Barcodes are now on the forward primer of the new 515FB-806RB primer pair. This enables the usage of various reverse primer constructs to obtain longer amplicons (tested on 806RB and 926R).</li>
<li>Degeneracy was added to both the forward and reverse primers to remove known biases against Crenarachaeota/Thaumarchaeota (515FB, also called 515F-Y, Parada et al., 2016) and the marine and freshwater Alphaproteobacterial clade SAR11 (806RB, Apprill et al., 2015).</li>
</ol>
<p>The primer sequences without linker, pad, barcode, or adapter are as follows:</p>
<p> </p>
<ul>
<li>Current, 2015-present (fwd-barcoded: 515FB-806RB): <br /><code>FWD:GTGYCAGCMGCCGCGGTAA; REV:GGACTACNVGGGTWTCTAAT</code></li>
<li>Original, pre-2015 (rev-barcoded: 515F-806R): <br /><code>FWD:GTGCCAGCMGCCGCGGTAA; REV:GGACTACHVGGGTWTCTAAT</code></li>
</ul>
<p> </p>
<p>Note: Studies in the <a href="https://qiita.ucsd.edu/" target="_blank" rel="noopener noreferrer">Qiita</a> database will have <code>library_construction_protocol</code> </p>
<p>as <code>515f/806rbc</code> if original primers or <code>515fbc/806r</code> if current primers (“bc” stands for barcode).</p>
<p> </p>
<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> for more information. Primers and primer constructs were designed by Greg Caporaso (2011, 2012). Modifications to primer degeneracy were done by the labs of Jed Furhman (Parada et al., 2016) and Amy Apprill (Apprill et al., 2015). Forward-barcoded constructs were redesigned by Walters et al. (2016) based upon the original constructs generated by Caporaso et al. (2012).</p>
<p> </p>
<ul>
<li><a href="ftp://ftp.microbio.me/emp/protocols/primers/515FB_806RB_Nov2016.xlsx" target="_blank" rel="noopener noreferrer">Illumina 16S Primer Constructs (shorter: 515FB–806RB)</a></li>
<li><a href="ftp://ftp.microbio.me/emp/protocols/primers/515FB_926R_Nov2016.xlsx" target="_blank" rel="noopener noreferrer">Illumina 16S Primer Constructs (longer: 515FB–926R)</a></li>
</ul>
<p> </p>
<p><strong>515FB forward primer, barcoded:</strong></p>
<p>Field descriptions (space-delimited):</p>
<ol>
<li>5′ Illumina adapter</li>
<li>Golay barcode</li>
<li>Forward primer pad</li>
<li>Forward primer linker</li>
<li>Forward primer (515FB)</li>
</ol>
<p><code>AATGATACGGCGACCACCGAGATCTACACGCT XXXXXXXXXXXX TATGGTAATT GT GTGYCAGCMGCCGCGGTAA</code></p>
<p> </p>
<p><strong>806RB reverse primer:</strong></p>
<p>Field descriptions (space-delimited):</p>
<ol>
<li>Reverse complement of 3′ Illumina adapter</li>
<li>Reverse primer pad</li>
<li>Reverse primer linker</li>
<li>Reverse primer (806RB)</li>
</ol>
<p><code>CAAGCAGAAGACGGCATACGAGAT AGTCAGCCAG CC GGACTACNVGGGTWTCTAAT</code></p>
<p> </p>
<p> </p>
<p><strong>PCR reaction mixture </strong></p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/us3fw26.png" width="231" height="189" data-src="https://s3.amazonaws.com/pr-journal/us3fw26.png" data-ofn="Screen Shot 2018-03-15 at 10.44.13 PM.png" /></p>
<p> </p>
<p>Notes: </p>
<ul>
<li>PCR-grade water from Sigma (cat. no. W3500) or MoBio (cat. no. 17000-11)</li>
<li>Platinum Hot Start PCR Master Mix (2x) from ThermoFisher (cat. no. 13000014)</li>
<li>Final master mix concentration in 1x reaction: 0.8x</li>
<li>Final primer concentration in 1x reaction: 0.2 µM</li>
</ul>
<p> </p>
<p><strong>Thermocycler conditions </strong></p>
<ul>
<li>Primers: 16S V4 515f-806rB</li>
<li>Amplicon size: ~390 bp</li>
<li>Cycle times are longer for 384-well thermocyclers.</li>
</ul>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/us4fw26.png" width="373" height="190" data-src="https://s3.amazonaws.com/pr-journal/us4fw26.png" data-ofn="Screen Shot 2018-03-15 at 10.46.01 PM.png" /></p>
<p> </p>
<p> </p>
<p><strong>16 S sequencing primers </strong></p>
<p> </p>
<p><strong>Read 1 sequencing primer: </strong></p>
<p>Field descriptions (space-delimited):</p>
<ol>
<li>Forward primer pad</li>
<li>Forward primer linker</li>
<li>Forward primer</li>
</ol>
<p><code>TATGGTAATT GT GTGYCAGCMGCCGCGGTAA</code></p>
<p> </p>
<p><strong>Read 2 sequencing primer: </strong></p>
<p>Field descriptions (space-delimited):</p>
<ol>
<li>Reverse primer pad</li>
<li>Reverse primer linker</li>
<li>Reverse primer</li>
</ol>
<p><code>AGTCAGCCAG CC GGACTACNVGGGTWTCTAAT</code></p>
<p> </p>
<p> </p>
<p><strong>References </strong></p>
<ul>
<li>Apprill, A., McNally, S., Parsons, R. &amp; Weber, L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol 75, 129–137 (2015). <a href="http://doi.org/10.3354/ame01753" target="_blank" rel="noopener noreferrer">doi:10.3354/ame01753</a></li>
<li>Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci USA 108, 4516–4522 (2011). <a href="http://doi.org/10.1073/pnas.1000080107" target="_blank" rel="noopener noreferrer">doi:10.1073/pnas.1000080107</a></li>
<li>Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J (2012). <a href="http://doi.org/10.1038/ismej.2012.8" target="_blank" rel="noopener noreferrer">doi:10.1038/ismej.2012.8</a></li>
<li>Parada, A. E., Needham, D. M. &amp; Fuhrman, J. A. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol 18, 1403–1414 (2016). <a href="http://doi.org/10.1111/1462-2920.13023" target="_blank" rel="noopener noreferrer">doi:10.1111/1462-2920.13023</a></li>
<li>Walters, W. et al. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys. mSystems 1, 915 (2016). <a href="http://doi.org/10.1128/mSystems.00009-15" target="_blank" rel="noopener noreferrer">doi:10.1128/mSystems.00009-15</a></li>
</ul>

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