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[DESCRIPTION]
<p>ezRAD is from the family of RAD (restriction site associated DNA) techniques that cuts up the DNA (either by sonication or restriction enzymes) and looks at large areas of the genome, as apposed to the whole genome. RADseq data often forms stacks of DNA on the same loci (vertical coverage of the genome). These stacks are then used to find SNPs (single-nucleotide polymorphisms). The interesting difference with ezRAD compared to other RAD techniques is that it utilizes a frequent cutting enzyme and so the data produced not only forms vertical stacks but also horizontal coverage across other regions of the genome. This makes the data more tricky to analyse, but it does mean that with this technique we can easily annotate our loci and get almost 100% of the mitochondrial genome, ribosomes and histones (i.e. high copy regions).</p>

[BEFORE_START]
<p><strong><u>Introduction</u></strong></p>
<p>One of the hardest things with the RADSeq protocols is knowing how much you 'can get away with' and still get good data! This protocol will go through every step to try and help you get genomic data from non-model organisms. Determining from gels what to do/how to salvage your samples and determining how much DNA you really have to have to get it through the Illumina or Kapa kits and still pass the quality control steps at the end and get good data. Our test species were cnidarians, which are notoriously difficult to work with, we've also worked with sponges, echinoderms, crustaceans, molluscs, fish etc. So we hope this protocol is useful to many people who work on a range of organisms.</p>
<p><strong><u>What’s possible?</u></strong></p>
<p>Each sequencing system is different, but the library output is the same. For sequencing we currently use the <strong>MiSeq</strong>, which can run 1 flow cell with 2 lanes, one of which is a control lane. On the remaining lane we run <strong>8 libraries</strong> to maximize coverage and reduce costs. Primarily we choose to use the <strong>MiSeq</strong> because with the current reagent kits it can sequence <strong>300bp paired-end reads</strong> (i.e. 600bp total), which is considerably longer than the <strong>HiSeq</strong>, which only sequences <strong>150bp paired end reads</strong>. However, the HiSeq does have its benefits in that you can load more samples per lane and get considerably more reads per library, you pick what you need/want based on your question.</p>
<p> </p>
<p>One of the main differences between ezRAD and other RADSeq techniques is that you can end up not only with stacks (vertical coverage), which can be used to call SNPs, but also considerable horizontal coverage. This horizontal coverage means that we can get exceptionally long contigs, resulting often in the resolution of a large percentage, if not all, of the mitochondrial genome amongst other regions (e.g. histones, ribosomes). These long contigs also mean we can identify them and their location in the genome, making ezRAD a very versatile technique.</p>
<p> </p>
<p>Almost anything can be successfully made into a library. With ezRAD each library can either consist of one individual or a pool of individuals. The latter requires more work, but the amount of DNA required is less per extraction. There are many kits on the market designed to generate libraries. Illumina's two main DNA kits include a <strong>PCR-free</strong> one which requires <strong>1μg of input DNA</strong> and the <strong>Nano kit</strong>, which only need <strong>100-200ng.</strong> Both kits come in low-throughput (24 individual indices) and high-throughput (96 dual indices). Alternatively, there is also the <strong>Kapa Hyper Prep kit</strong>, which only needs <strong>1ng input DNA</strong>, is cheaper and quicker than the Illumina kits; however, it does require that you buy the adapters and beads separately; more on this later. </p>
<p> </p>
<p>I’ve tried to write this protocol so that almost anyone can make libraries, but I also didn’t want it to be a tome, so this protocol is aimed at people with at least some molecular bench work experience.</p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>First things first there are 2 exceptionally important things to do throughout the protocol:</strong></span></p>
<ol>
<li>Make sure that before any step, where your DNA has been sitting in a fridge or freezer, that you thoroughly mix and briefly spin down the sample. I can’t stress how important this is! It will affect your gels, quantitation, pooling etc.</li>
<li>Watch your pipetting, literally! This is especially important when using the multiwall pipettes as some tips can loosen. There are often markers on pipette tips, use them. Get to know what 1 or 2µl looks like, it will improve your lab work.</li>
</ol>
<p><span style="text-decoration: underline;"><strong>Hyperlinks</strong></span></p>
<p>If the hyperlinks aren't working in this online version please try them in the pdf copy.</p>
<p> </p>
<p><span style="text-decoration: underline;"><strong>Any mistakes?</strong></span></p>
<p>This protocol is always changing to move with the times, so if you notice any mistakes or issues, please feel free to let us know so we can improve it. It is by no means an infallible protocol.  </p>
<p> </p>
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<p><span style="text-decoration: underline;"><strong>Disclaimer </strong></span></p>
<p>This a not-for-profit protocol and is designed to be used for educational purposes only. Whilst we endeavour to ensure that the information in this protocol is correct, we do not warrant its completeness or accuracy and we cannot be be held responsible for variable results in library preparations made whilst following this protocol. Any opinions given in this protocol are not those of the University of Hawaii or The Hawaii Institute of Marine Biology. </p>
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[GUIDELINES]
<p><strong><u>Table of Contents</u></strong></p>
<p><strong>Step 1</strong>: Extractions    </p>
<p><strong>Step 2</strong>: Are your extractions ok?     </p>
<p><strong>Step 3</strong>: DNA quantitation     </p>
<p><strong>Step 4</strong>: Pooling samples (not necessary if doing single libraries)  </p>
<p><strong>Step 5</strong>: Choose your library preparation kit </p>
<p><strong>Step 6</strong>: Concentrating your DNA     </p>
<p><strong>Step 7</strong>: Bead clean-up           </p>
<p><strong>Step 8</strong>: Digest</p>
<p><strong>Step 9</strong>: Post-digest clean-up and checks      </p>
<p><strong>Step 10</strong>: Make libraries!       </p>
<p><strong>Illumina TruSeq DNA library protocol      </strong></p>
<p>1) End-repair-Illumina          </p>
<p>2) Size selection-Illumina     </p>
<p>3) Adenylate 3’ Ends (A-tailing) -Illumina  </p>
<p>4) Adapter ligation-Illumina </p>
<p>5) Post ligation clean-up-Illumina    </p>
<p>6) Enrich DNA fragments- Nano kit only-Illumina </p>
<p>7) Post-amplification clean-up-Illumina      </p>
<p><strong>KAPA Hyper Prep DNA library protocol  </strong></p>
<p>1) End-repair and A-tailing-Kapa     </p>
<p>2) Adapter ligation-Kapa      </p>
<p>3) Post ligation Clean-up-Kapa        </p>
<p>4) Size selection (for 350-700bp insert) -Kapa        </p>
<p>5) Library amplification-Kapa          </p>
<p>6) Post-amplification clean-up-Kapa</p>
<p><strong>Step 11</strong>: Validate your libraries        </p>
<p><strong>Appendix 1</strong>: Setting up a lab to do ezRAD  </p>
<p><strong>Appendix 2</strong>: Removed from this protocol, but still in old versions</p>
<p><strong>Appendix 3</strong>: Illumina adapter sequences     </p>
<p><strong>Appendix 4</strong>: Illumina experienced user version      </p>
<p><strong>Appendix 5</strong>: KAPA experienced user version          </p>
<p> </p>

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