TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when <em>a priori</em> sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al. method: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS) method. The details about how each approach performs can be found in the citation shown below.</p>
<p>This protocol explains how to prepare each type of GBS library, with comments on expected results and controls needed to confirm each step worked.</p>

[BEFORE_START]
<p><strong>NOTES</strong></p>
<p> </p>
<ol>
<li>Dissolve the barcoded adaptor oligonucleotides (plus and minus strand) in 1X TE pH 7.5 to a final concentration of 20 pmol/μL. Dissolve the common adaptor oligonucleotides to 500 pmol/μL. Assign one person to look after these stocks to prevent cross contamination between wells and tubes.</li>
<li>Include digestion and ligation controls processed in parallel to your experiment to confirm those steps worked. For restriction enzymes that cut frequently, Lambda DNA and/or pBluescript are good controls. Do an <em>in silico</em> digestion to evaluate the desired enzyme or enzyme combination.</li>
<li>If you are planning a stdGBS experiment with a rare restriction enzyme, consider a double digest GBS approach (Poland JA, Brown PJ, Sorrells ME, Jannink J-L (2012) Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach. PLoS ONE 7: e32253.)</li>
<li>You need <strong>high molecular DNA fragments &gt;20 kbp</strong>, without RNA, regardless if you are performing a stdGBS or rtGBS experiment.</li>
<li>For stdGBS normalize your genomic DNA samples to the same concentration value, for example 100 ng/µL, to speed up the pipetting. Aim for a rather concentrated DNA solution to avoid carrying too much liquid into the restriction digestion reaction, especially if the DNA has been dissolved in TE buffer. Up to 20 μL of DNA in TE buffer can be safely used in a restriction digestion, in a final volume of 50 μL.</li>
<li>Please note the number of cycles in the PCR profile was <u>optimized for the BamH I </u>adaptor set used for a kiwifruit pilot study. You need to optimize the number of cycles for your own plant/restriction enzyme combination. Evaluate 12-20 or 25 cycles, in increments of 2. Analyze your amplified libraries in a Fragment Analyzer, or a BioAnalyzer.</li>
<li>Size selection can be done by gel extraction, but is labor intensive. Run the amplicons in a 3% Agarose gel. Make this gel mixture with 0.5 g Standard Agarose, and 2.5 g High resolution Agarose, in 100 mL 1X TAE. Run the gel at 140 V for 30 min. Stain the gel with SybrSafe, and cut the desired block under blue light.</li>
<li>The rtGBS protocol requires a modified common oligonucletide. When ordering your barcoded oligonucleotides, include the GBS-C-N oligo: CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNN, at 200 nmol, cartridge purified, delivered as a dry pellet.</li>
<li>When <strong>pipetting dense solutions</strong>, like the ligation or PCR master mixes, prime the pipette tips by drawing and expelling the liquid slowly, one time. Multichannel pipettes (and robotic stations) allow you to select the speed of this operation. Also, if the required volume is 47.5 µL, set the pipette to 46.7 µL, and don’t dip the tips too far into the solution. The amount of carry over solution outside the tip will compensate for the “missing” µL in the settings.</li>
</ol>
<p> </p>
<p><strong>MATERIALS</strong></p>
<p> </p>
<p>Consumables</p>
<p> </p>
<ul>
<li>1 mL deep well plate (NUNC cat# 260252) or 2 mL deep well plate (NUNC cat#  278752)</li>
<li>1.5 mL screw-capped tubes, sterile</li>
<li>Falcon tubes, 15 mL</li>
<li>Disposable reservoirs</li>
<li>Aluminium tape (NUNC cat#  232698, or similar). For PCR reactions and long term storage</li>
<li>Clear packaging tape (48 mm wide, for example: Snell). For temporary plate sealing</li>
<li>Cutter (retractable blade, general purpose)</li>
<li>PCR plates (Thermo-Fast AB-0600)</li>
<li>FrameStar® 96 Well Semi Skirted PCR (Cut Corner A12, 4titute cat# 4ti-0770/C). These plates are used for the Fragment Analyzer only</li>
<li>2 L plastic box (21 x 7.5 x 14.4 cm). Mark on the side of the box the water level at 1.5 L and always fill it up to this mark. By doing this small step you will be saving 30-60 min at the cool down annealing steps since small changes in water volume translate to a long/short wait.</li>
<li>Paper towels, low lint</li>
</ul>
<p> </p>
<p>Equipment and kits</p>
<p> </p>
<ul>
<li>Water bath at 65°C</li>
<li>Bench top centrifuge with plate rotor (max speed 4000 rpm)</li>
<li>Bench top microcentrifuge (for 1.5 mL tubes)</li>
<li>Vortex with plate adaptor</li>
<li><a href="https://www.aati-us.com/instruments/fragment-analyzer/" target="_blank">Fragment Analyzer</a>, High Sensitivity genomic DNA kit (1000 reactions, DNF-488) and High Sensitivity NGS (1000 reactions, DNF-474)</li>
<li>Qubit and High Sensitivity dsDNA kit</li>
<li>Horizontal gel electrophoresis unit with power supply</li>
<li>Image capture equipment (for example, GelDoc, LSA3000, etc)</li>
<li>Multichannel pipettes (1-10 µL, 5-200 µL, 50-1000 µL) or liquid handling robotic station with appropriate multichannel tools and tips, for example Biomek series, Hamilton, Integra’s Viaflo, etc</li>
<li>PCR machine</li>
<li>Timer</li>
</ul>
<p> </p>
<p>Reagents</p>
<p> </p>
<ul>
<li>Sterile deionised water, 100 mL</li>
<li>Restriction enzyme set from NEB, Invitrogen, Fermentas, etc.</li>
<li>Lambda DNA (Life Technologies cat# 25250010)</li>
<li>Uncut pBluescript or any other cloning vector like pUC19 often included as control in competent cells kits. A BAC clone could also be used as control, especially for rare cutting restriction enzymes.</li>
<li>1X TE pH 7.5 (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) sterile, 100 mL</li>
<li>Blue TE buffer (1X TE pH 7.5, 0.0025% xylene cyanol FF), 50 mL</li>
<li>10 mM Tris-HCl pH 8, sterile, 50 mL</li>
<li>25 mM MgCl<sub>2</sub> sterile, 2 mL</li>
<li>T4 DNA ligase and 5X T4 DNA ligase buffer (Life technologies)</li>
<li>20 mg/mL Dextran Blue (dissolved in sterile deionised water, Sigma Aldrich cat# D5751), 1 mL</li>
<li>3 M sodium acetate pH 5.2, 50 mL</li>
<li>Isopropanol</li>
<li>100% absolute ethanol, 100 mL</li>
<li>70% ethanol (freshly made), 100 mL</li>
<li>AccuPrime Taq DNA polymerase High Fidelity 5 U/µL and 10X AccuPrime High Fidelity Buffer I and II (Invitrogen 12346094)</li>
<li>Mineral oil (Sigma-Aldrich cat # M5904-500ML)</li>
<li>Platinum <em>Pfx</em> DNA polymerase 2.5 U/µL (Invitrogen 11708039)</li>
<li>1.2 M Trehalose, dissolved in sterile deionized water and filtered through 0.2 or 0.4 µm</li>
<li>10X <em>Pfx</em> reaction buffer homemade: 200 mM Tris-HCl, 100 mM KCl, 100 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, pH 8, sterile. <strong><em>Note</em></strong><em>: I have not tried the 10X buffer and enhancer solutions supplied with the Platinum Pfx DNA polymerase (Invitrogen 11708039). It should work, but do a preliminary test.</em></li>
<li>50 mM MgSO<sub>4</sub> (use the stock provided with either DNA polymerase)</li>
<li>2 mM dNTP (dilution prepared in deionized sterile water from 100 mM stocks)</li>
<li>1% agarose gel in 1X TAE buffer, 100 mL, plus 1 L of 1X TAE running buffer</li>
<li>1 kb plus DNA ladder (Invitrogen) or similar</li>
<li>SybrSafe (Invitrogen)</li>
<li>Ampure XP magnetic beads (Beckman Coulter) and appropriate magnet stand</li>
<li>85% ethanol, 50 mL freshly made</li>
</ul>
<p> </p>
<p><strong>ACKNOWLEDGEMENTS</strong></p>
<p> </p>
<p>Thanks to all the people that have asked me to train them on how to prepare GBS libraries: David Ashton, Lorna Barron, Maud Bomert, Bastienne Brauksiepe, Paul Datson, Ken Fischer, Angela Fleming, Nikolai Macnee, Caroline Mitchell, Liz Popowski, Igor Ruza, Jibrahn Tahir, Melinda  Zhang and Qiong Zhang. With their help I have improved the description of every step.</p>

[GUIDELINES]
<p>This protocol explains how to prepare GBS libraries from intact (standard GBS (stdGBS)) and from amplified DNA (random tagged GBS (rtGBS)).</p>
<p> </p>
<p>The stdGBS protocol described below differs from Elshire et al. in the following steps:</p>
<p> </p>
<ul>
<li>One microgram of genomic DNA of each plant was used for the restriction digestion.</li>
<li>The restriction enzyme was selected based on the RADsites test_iPlant.xlsx (see file uploaded).</li>
<li>Adaptors are annealed according to Ko et al. (Ko W-Y, David R M, Akashi H (2003) J Mol Evol (2003) 57:562–573 DOI: 10.1007/s00239-003-2510-x).</li>
<li>The adaptor ligation step was done after digestion, without drying out the DNA/adaptor mixture.</li>
<li>A high fidelity enzyme was used for amplification (AccuPrime Taq DNA polymerase High Fidelity, Life Technologies).</li>
<li>The amplification of the library was done on each individual plant before pooling.</li>
<li>The GBS adaptors and primers were designed by <a href="http://www.deenabio.com/services/gbs-adapters." target="_blank">Deena Bioinformatics</a>.</li>
<li>All oligonucleotides were ordered from BioSearch Technologies. The common adaptors and PCR primers were ordered at 200 nmol scale, cartridge purified, dry pellets, in tubes. The barcoded adaptors were ordered normalised at 5 nmol, purified by desalting, dry pellets, in plates.</li>
</ul>
<p>The rtGBS protocol is ilustrated with the following diagram:</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/qr9eupe.jpg" data-src="https://s3.amazonaws.com/pr-journal/qr8eupe.jpg" data-ofn="rtGBS diagram.jpg" /></p>
<p> </p>
<p>The stdGBS and rtGBS methods share most of the steps described below, with some modifications: the source of the starting material, and the omission of the common barcoded adaptor in rtGBS:</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/qy8eupe.jpg" data-src="https://s3.amazonaws.com/pr-journal/qy7eupe.jpg" data-ofn="Outline of stdGBS and rtGBS protocol steps.jpg" /></p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.