TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Double digested Restriction-site Associated DNA (ddRAD) sequencing is a powerful approach for identifying and analyzing genome-wide SNP variation. Many studies have now used ddRAD protocols for population genetic studies. Here we have adapted the protocol from Peterson (2012) for Ion Torrent sequencing to produce a significantly streamlined workflow capable of having fully sequenced ddRAD libraries in two days. A reduced number of steps for producing a ddRAD library is achieved through the use of a unidirectional double digestion-ligation reaction with adaptors having fusion barcodes in the 5' and 3' ends. This also allows for the immediate pooling of sets of compatible barcoded samples for downstream processing. The A-adaptor contains the standard Ion Torrent barcodes and a key sequence, while the P1-adaptor contains a divergent Y-adaptor with a paired-end code for increased multiplexing. The described system and adaptors are compatible with the SbfI, PstI and NsiI restriction endonucleases in the A adaptor, while the P1-adaptor has compatible overhangs with NdeI or MseI, and, MspI or HpaII. The later two restriction enzymes were chosen as isoschizomers for their differentially sensitivity to CpG methylation, thus allowing the use of this protocol for epigenetic ddRAD profiling at genomic loci with 5-methylcytosine and 5-hydroxymethylcytosine modifications. This protocol takes an advantage in that the Ion Torrent platform has scalability with different sequencing chip sizes, and that the protocol has a range of compatible restriction endonucleases with different motif lengths. This ensures a versatile, cost-effective and flexible method to which you can tune the number of ddRAD loci being profiled, for both small and large genomes, with relative speed and with the ability to observe the performance from small pilot scale reactions.</p>

[BEFORE_START]
<p><strong>Order single stranded oligo nucleotides:</strong></p>
<p> </p>
<p>Since the Y adaptors are used more frequently they can be ordered in the scale of 200 nmol (high purity). Y adaptors are designated as P1 by Ion Torrent from Thermofisher.</p>
<p> </p>
<p>The following oligos permit the production of adaptors which have compatible overhangs for both <em>MspI and HpaII </em>(4 bp recognition sequences).</p>
<p> </p>
<p><strong>P1-Y adaptors for <em>MspI</em> and <em>HpaII</em> provided without a combinatorial multiplex code and paired-end sequencing (5' to 3'):</strong></p>
<p> </p>
<table>
<tbody>
<tr>
<td>ddRAD-PE-Y-MspI-top</td>
<td>CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAG</td>
</tr>
<tr>
<td>ddRAD-PE-Y-MspI-bottom</td>
<td>C*G*CTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>Or P1-Y adaptors with a multiplex code and paired-end sequencing:</strong></p>
<p> </p>
<p>eg. a depiction of the ddRAD divergent Y adaptor used in this study.</p>
<p> </p>
<p>               <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/qcjd7f6.jpg" data-src="https://s3.amazonaws.com/pr-journal/qcjd7f6.jpg" data-ofn="Y-Nde-PEcode1" /></p>
<p>Yellow/green - divergent Y adaptor</p>
<p>Yellow - P1 amplification primer binding site</p>
<p>Blue/grey - 5 nt combinatorial tag</p>
<p>Red - Nt.BbvCI nicking site for paired-end sequencing</p>
<p>* protected phosphorothioate bonds</p>
<p> </p>
<table width="805">
<tbody>
<tr style="height: 20px;">
<td style="height: 20px;" width="240">ddRAD-PEcode-1-Y-MspI_top</td>
<td style="height: 20px;" width="565">CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCACGCAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-1-Y-MspI_bottom</td>
<td style="height: 20px;">C*G*CTGCGTGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-2-Y-MspI_top</td>
<td style="height: 20px;">CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCTACGAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-2-Y-MspI_bottom</td>
<td style="height: 20px;">C*G*CTCGTAGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-3-Y-MspI_top</td>
<td style="height: 20px;">CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCGTAGAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-3-Y-MspI_bottom</td>
<td style="height: 20px;">C*G*CTCTACGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-4-Y-MspI_top</td>
<td style="height: 20px;">CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCGATACG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-4-Y-MspI_bottom</td>
<td style="height: 20px;">C*G*CGTATCGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-5-Y-MspI_top</td>
<td style="height: 20px;">CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCTATATG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-5-Y-MspI_bottom</td>
<td style="height: 20px;">C*G*CATATAGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-6-Y-MspI_top</td>
<td style="height: 20px;">CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCTCGTAG</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">ddRAD-PEcode-6-Y-MspI_bottom</td>
<td style="height: 20px;">C*G*CTACGAGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p>The following oligos permit the production of adaptors which have compatible overhangs for both <em>NdeI</em> (a 6 bp recognition sequence), and <em>MseI</em> (a 4 bp recognition sequence).</p>
<p> </p>
<p><strong>P1-Y adaptors for <em>NdeI</em> and <em>MseI </em>overhangs provided without a multiplex code and paired-end sequencing:</strong> </p>
<p> </p>
<table>
<tbody>
<tr>
<td>
<p>ddRAD-PE-Y-NdeI-top</p>
</td>
<td>CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGC</td>
</tr>
<tr>
<td>ddRAD-PE-Y-NdeI-bottom</td>
<td>T*A*GCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>Or P1-Y adaptors for <em>NdeI</em> and <em>MseI</em> overhangs with a multiplex code and paired-end sequencing:</strong></p>
<p> </p>
<table width="805">
<tbody>
<tr>
<td width="240">ddRAD-PEcode-1-Y-NdeI-top</td>
<td width="565">CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCTCGCTC</td>
</tr>
<tr>
<td>ddRAD-PEcode-1-Y-NdeI-bottom</td>
<td>T*A*GAGCGAGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr>
<td>ddRAD-PEcode-2-Y-NdeI-top</td>
<td>CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCGACTAC</td>
</tr>
<tr>
<td>ddRAD-PEcode-2-Y-NdeI-bottom</td>
<td>T*A*GTAGTCGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr>
<td>ddRAD-PEcode-3-Y-NdeI-top</td>
<td>CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCGTCAGC</td>
</tr>
<tr>
<td>ddRAD-PEcode-3-Y-NdeI-bottom</td>
<td>T*A*GCTGACGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr>
<td>ddRAD-PEcode-4-Y-NdeI-top</td>
<td>CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCATACGC</td>
</tr>
<tr>
<td>ddRAD-PEcode-4-Y-NdeI-bottom</td>
<td>T*A*GCGTATGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr>
<td>ddRAD-PEcode-5-Y-NdeI-top</td>
<td>CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCTACATC</td>
</tr>
<tr>
<td>ddRAD-PEcode-5-Y-NdeI-bottom</td>
<td>T*A*GATGTAGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
<tr>
<td>ddRAD-PEcode-6-Y-NdeI-top</td>
<td>CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGCTCTTCCGATCTCCTCAGCATCGTC</td>
</tr>
<tr>
<td>ddRAD-PEcode-6-Y-NdeI-bottom</td>
<td>T*A*GACGATGCTGAGGAGATCGGAAGAGCATCCAACTTGAAG</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>Barcoded A adaptors compatible with <em>SbfI</em>, <em>PstI</em> and <em>NsiI </em>overhangs</strong></p>
<p> </p>
<p>A nucleotide g is placed between the barcode adaptor and the overhang to ensure a unidirectional ligation permitting the use of <em>SbfI</em>, <em>PstI</em> or <em>NsiI</em> in a restriction and ligation reaction. This nucleotide can be trimmed from the overhang tag for bioinformatic purposes.</p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/qchd7f6.jpg" data-src="https://s3.amazonaws.com/pr-journal/qchd7f6.jpg" data-ofn="SbfI-13.JPG" /></p>
<p>* represents protected bonds. Overhangs and restriction sites (also appearing in the double stranded regions of adaptors), are protected internally with phosphorothioate bonds.</p>
<p> </p>
<table width="805">
<tbody>
<tr>
<td width="240">ddRAD-SbfI-13-top</td>
<td width="565">CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTAACGGACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-13-bottom</td>
<td>cATCGTCCGTTAGACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-14-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTTGGAGTGTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-14-bottom</td>
<td>cATCGACACTCCAACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-15-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTAGAGGTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-15-bottom</td>
<td>cATCGACCTCTAGACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-16-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTGGATGACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-16-bottom</td>
<td>cATCGTCATCCAGATGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-17-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTATTCGTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-17-bottom</td>
<td>cATCGACGAATAGACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-18-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGAGGCAATTGCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-18-bottom</td>
<td>cATCGCAATTGCCTCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-19-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTTAGTCGGACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-19-bottom</td>
<td>cATCGTCCGACTAACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-20-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGCAGATCCAT*C*GATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-20-bottom</td>
<td>cAT*C*GATGGATCTGCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-21-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGCAATTACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-21-bottom</td>
<td>cATCGTAATTGCGACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-22-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCGAGACGCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-22-bottom</td>
<td>cATCGCGTCTCGAACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-23-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTGCCACGAACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-23-bottom</td>
<td>cATCGTTCGTGGCACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-24-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGAACCTCATTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-24-bottom</td>
<td>cATCGAATGAGGTTCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-25-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGCCTGAGATACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-25-bottom</td>
<td>cATCGTATCTCAGGCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-26-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTTACAACCTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-26-bottom</td>
<td>cATCGAGGTTGTAACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-27-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGAACCATCCGCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-27-bottom</td>
<td>cATCGCGGATGGTTCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-28-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGATC*C*GGAAT*C*GATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-28-bottom</td>
<td>cAT*C*GATTC*C*GGATCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-29-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGACCACTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-29-bottom</td>
<td>cATCGAGTGGTCGACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-30-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGCGAGGTTAT*C*GATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-30-bottom</td>
<td>cAT*C*GATAACCTCGCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-31-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTCCAAGCTGCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-31-bottom</td>
<td>cATCGCAGCTTGGACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-32-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTTACACACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-32-bottom</td>
<td>cATCGTGTGTAAGACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-33-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCTCATTGAACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-33-bottom</td>
<td>cATCGTTCAATGAGAACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-34-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGCATCGTTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-34-bottom</td>
<td>cATCGAACGATGCGACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-35-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTAAGCCATTGTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-35-bottom</td>
<td>cATCGACAATGGCTTACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-36-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGAAGGAATCGTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-36-bottom</td>
<td>cATCGACGATTCCTTCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-37-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGCTTGAGAATGTCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-37-bottom</td>
<td>cATCGACATTCTCAAGCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-38-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTGGAGGACGGACGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-38-bottom</td>
<td>cATCGTCCGTCCTCCACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-39-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTAACAATCGGCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-39-bottom</td>
<td>cATCGCCGATTGTTACTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-40-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGCTGACATAAT*C*GATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-40-bottom</td>
<td>cAT*C*GATTATGTCAGCTGAGTCGGAGACACGCAGGGATGAGATGG*T*T</td>
</tr>
<tr>
<td>ddRAD-SbfI-41-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCCACTTCGCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-41-bottom</td>
<td>cATCGCGAAGTGGAACTGAGTCGGAGACACGCAGGGATGAGATGGT*T*</td>
</tr>
<tr>
<td>ddRAD-SbfI-42-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGAGCACGAAT*C*GATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-42-bottom</td>
<td>cATC*G*ATTCGTGCTCTGAGTCGGAGACACGCAGGGATGAGATGGT*T*</td>
</tr>
<tr>
<td>ddRAD-SbfI-43-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGCTTGACACCGCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-43-bottom</td>
<td>cATCGCGGTGTCAAGCTGAGTCGGAGACACGCAGGGATGAGATGGT*T*</td>
</tr>
<tr>
<td>ddRAD-SbfI-44-top</td>
<td>CCATCTCATCCCTGCGTGTCTCCGACTCAGTTGGAGGCCAGCGATgTGCA</td>
</tr>
<tr>
<td>ddRAD-SbfI-44-bottom</td>
<td>cATCGCTGGCCTCCAACTGAGTCGGAGACACGCAGGGATGAGATGGT*T*</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>ddRAD library amplification and enrichment primers:</strong></p>
<p> </p>
<table style="width: 546px;">
<tbody>
<tr>
<td style="width: 203px;">A-amp</td>
<td style="width: 331px;">CCATCTCATCCCTGCGTGTCTCCGACTCAG</td>
</tr>
<tr>
<td style="width: 203px;">P1-amp</td>
<td style="width: 331px;">CCACTACGCCTCCGCTTTCCTCTCTATG</td>
</tr>
<tr>
<td style="width: 203px;">paired-end_sequencing_primer</td>
<td style="width: 331px;">C*C*A*T*CTCATCCCTGCGTGTCTCCGAC</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>qPCR primers and probe:</strong></p>
<p> </p>
<table style="width: 546px;">
<tbody>
<tr>
<td style="width: 202px;">Ion_left</td>
<td style="width: 332px;">CCACTACGCCTCCGCTTT</td>
</tr>
<tr>
<td style="width: 202px;">Ion_right</td>
<td style="width: 332px;">ATCTCATCCCTGCGTGTCTC</td>
</tr>
<tr>
<td style="width: 202px;">Ion_probe_5'FAM, 3'BHQ1</td>
<td style="width: 332px;">/56-FAM/CTCTATGGGCAGTCGGTGAT/3BHQ_1/</td>
</tr>
</tbody>
</table>

[GUIDELINES]
<p><strong>General guidelines:</strong></p>
<p>Set up all ddRAD digestion-ligation reactions in an amplification free environment. Use appropriate pipetting practices to minimize barcode cross contamination. A post-amplification laboratory area should be used for processing all amplified libraries.</p>
<p> </p>
<p><strong>Tuning the number of genomic loci being sequenced:</strong></p>
<p>An <em>in silico</em> digestion (eg. <a href="http://bioinformatics.psb.ugent.be/webtools/aflpinsilico/" target="_blank">Rombauts et al., 2003</a>; <a href="http://www.bioinformatics.org/sms2/rest_digest.html" target="_blank">bioinformatics.org</a>, or <a href="http://biopython.org/" target="_blank">biopython.org</a> using the <a href="http://biopython.org/DIST/docs/cookbook/Restriction.html" target="_blank">Bio.Restriction</a> package), of a suitable reference genome, can be used to guide and tune the number of genomic loci being sequenced and to examine allelic dropout. Plastid and mitochondrial sequences can also be used for predicting the number of organellar genome sequences also being sampled, so as to either include or minimize these sequences from the variant discovery. Thus an <em>in silico</em> digestion can be used to select enzymes that are most appropriate for ddRAD sequencing, and to scale and determine the expected frequency of cutting within the genome. Thus this utility can be used to fine tune the number of fragments, together with the number of individuals being sequenced for use on the different Ion Torrent chips. Note that the adapters comprise a total of 103 bp and this length must be added to total size of the targeted fragment length during an in silico size simulation (e.g. for sequence lengths of 100-300 bp, a range of 203-403 bp would be selected).</p>
<p> </p>
<p><strong>Magnetic bead-based DNA purification and separation:</strong></p>
<p>This protocol requires the use of rare earth magnets for bead-based DNA purification and separation. Use individual 1.5 ml Eppendorf magnetic racks, a magnetic strip for PCR strips (8 well), and/or a 96-well PCR magnetic plate for separation. Care should be taken with the use of these magnets to prevent personal injury.</p>
<p> </p>
<p> </p>
<p><strong>References:</strong></p>
<ol>
<li><strong>Dabney, J. and Meyer, M.</strong> (2012). Length and GC-biases during sequencing library amplification: A comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. Biotechniques 52: 87–94.</li>
<li><strong>Peterson, B.K., Weber, J.N., Kay, E.H., Fisher, H.S., and Hoekstra, H.E.</strong> (2012). Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS One 7: e37135.</li>
<li><strong>Rombauts, S., Van De Peer, Y., and Rouzé, P.</strong> (2003). <a href=" http://bioinformatics.psb.ugent.be/webtools/aflpinsilico/" target="_blank">AFLPinSilico</a>, simulating AFLP fingerprints. Bioinformatics 19: 776–7 </li>
</ol>
<p> </p>
<p> </p>

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