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[DESCRIPTION]
<p> </p>
<p><strong><u>Background Information</u></strong></p>
<ul>
<li>High throughput amplicon sequencing is an extremely sensitive technique. The lab environment is full of airborne bacterial and fungal cells as well as bacterial and fungal genomic DNA from cultures, and so extreme care must be taken to keep reactions uncontaminated and in preventing cross-contamination.</li>
<li>This protocol was modified from the IBEST Geomics Core at University of Idaho.  It utilizes a 2-step PCR, where the first PCR amplifies the specific region of interest (e.g., ITS or 16S V4), and the second PCR adds adapters and barcodes to these amplicons. The target specific primers include (in addition to the standard primer sequence) the universal sequences CS1 and CS2, which provide the bases used in the second PCR to extend the amplicons with Illumina adapters and sample-identification barcodes.</li>
<li>Illumina adapters and sample-identification barcodes are not included in the region-specific primers used in PCR1. This allows for maximum flexibility in choosing which primers to use and provides the ability to swap out targets, or include multiple targets in the same sequencing reaction, without needing to purchase a large number of barcoded target specific primers.</li>
<li>Barcodes are included in both adapters, therefor a pair of barcodes is used to uniquely identify each sample during bioinformatic processing. This allows us to use 32 barcode pairs to uniquely identify 1024 samples (32 x 32).</li>
<li>Phase-shifted primers are used to introduce 1, 2, and/or 3 additional bases to allow for the maximum amount of “sequence diversity” in the first 4 bases. This is important because of how the Illumina sequencers detect clusters on the flowcell during sequencing. If sequences are too similar (as is often the case with amplicons), then sequencing yields will be much lower than expected. 2-bp linkers and phase-shifting bases were chosen to have low identity between primers and the target sequence.</li>
<li>Tagged primers were ordered from IDT (standard desalting; not HPLC-purified), re-suspended at 100 μM using a new bottle of molecular grade water in the PCR-free hood, and mixed in equimolar concentration (250 μl each).  Working stocks were diluted to 50 μM for PCR1.</li>
</ul>

[GUIDELINES]
<p><strong><u>General Lab Considerations when working with any high-throughput sequencing protocol</u></strong></p>
<ul>
<li>Gloves must be worn at all times.  Change gloves frequently and decontaminate often with DNA Away.  This is especially critical when working with more than one sample pool, in order to avoid cross-contamination.</li>
<li>Pipetting must be done extremely carefully to minimize the risk of aerosols that can easily cause contamination between samples.</li>
</ul>
<ul>
<li>Prior to opening any tubes or plates, centrifuge DNAs and PCR products for 1 minute at high speed to minimize cross-contamination. </li>
</ul>
<ul>
<li>Use only unopened, sterile, aerosol-resistant pipette tips (filter tips) to minimize contamination of the pipette shaft and your samples.  Clean the body of the pipettes regularly with DNA Away. </li>
<li>Discard pipette tips after each use to avoid cross-contamination. Eject pipette tips carefully to prevent aerosol formation or other splashing.</li>
</ul>
<ul>
<li>PCR1 set-up must be done in designated pre-PCR hood <em>only</em>, away from laboratory areas where all other PCR or cloning work is done. Prior to each PCR set-up, decontaminate all pipettes and surfaces in the hood with DNA Away and 30 min. UV light. </li>
<li>Work with PCR1 and PCR2 amplicons must be done in a separate hood from pre-PCR preparation.  If at all possible, use different gloves, pipettes, boxes of tips, bags of plastics, plate lids, etc. for handling any amplified DNA.</li>
<li>Remember to always clean the hood and any equipment with DNA Away and decontaminate where possible with UV for 30 minutes prior to use to avoid cross-contamination. Reminder: high throughput amplicons sequencing is <em>extremely</em> sensitive.</li>
</ul>
<ul>
<li>The pipettes used need to be calibrated annually.</li>
</ul>

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