TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was developed with the aim of sequencing the GC rich part of heterogeneous genomes with the Illumina technology. To this aim, fragmented genomic DNA is denaturized up to 90°C before library preparation. This procedure enriches the library in GC-rich fragments and leads to a substantial increase in average GC content of sequence reads.</p>

[GUIDELINES]
<p>Reagents:</p>
<p><strong>Illumina library protocol is an adaptation to publication Meyer and Kircher 2010 (Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb Protoc 2010:pdb.prot5448. doi: 10.1101/pdb.prot5448).</strong></p>
<p>DNeasy Blood and Tissue Kit (Qiagen)</p>
<p>Agencourt AMPure XP beads (Beckman Coulter, France S.AS)</p>
<p>10X Tango buffer (Thermo Fisher Scientific, Waltham, MA, USA)</p>
<p>25mM dNTPs (New England Biolabs, Ipswich, MA, USA)</p>
<p>100mM dATP (New England Biolabs)</p>
<p>T4 DNA polymerase (New England Biolabs)</p>
<p>T4 polynucleotide Kinase (New England Biolabs)</p>
<p>T4 DNA ligase (New England Biolabs)</p>
<p>Bst polymerase large fragment (New England Biolabs)</p>
<p>2X KAPA HiFi HotStart ReadyMix (KAPABIOSYSTEMS, Wilmington, MA, USA)</p>
<p>Phusion High-Fidelity DNA Polymerase (New England Biolabs)</p>
<p>Dimethylsulfoxyde 100% (Thermo Fisher scientific)</p>
<p>50 µM adapters mix primers (see table 1) (Eurofins, Vergèze, France) HPLC purification (see recipes)</p>
<p>10X Oligo Hybridation Buffer (see recipes)</p>
<table>
<tbody>
<tr>
<td width="122">
<p><strong>Names</strong></p>
</td>
<td width="356">
<p><strong>Sequences</strong></p>
</td>
</tr>
<tr>
<td width="122">
<p><strong>IS1_adapter P5.F</strong></p>
</td>
<td width="356">
<p>A*C*A*C*TCTTTCCCTACACGACGCTCTTCCG*A*T*C*T</p>
</td>
</tr>
<tr>
<td width="122">
<p><strong>IS2_adapter P7.F</strong></p>
</td>
<td width="356">
<p>G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCG*A*T*C*T</p>
</td>
</tr>
<tr>
<td width="122">
<p><strong>IS3_adapter P5+P7.R</strong></p>
</td>
<td width="356">
<p>A*G*A*T*CGGAA*G*A*G*C</p>
</td>
</tr>
<tr>
<td width="122">
<p><strong>IS4_ind PCR P5</strong></p>
</td>
<td width="356">
<p>AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT</p>
</td>
</tr>
<tr>
<td width="122">
<p><strong>I1_ind PCR P7</strong></p>
</td>
<td width="356">
<p>CAAGCAGAAGACGGCATACGAGATcctgcgaGTGACTGGAGTTCAGACGTGT</p>
</td>
</tr>
</tbody>
</table>
<p>* PTO = phosphothioate oligonucleotide bond</p>
<p> </p>
<p>Recipes:</p>
<table>
<tbody>
<tr>
<td width="264">
<p><strong>Oligo hybridization buffer (10X)</strong></p>
</td>
</tr>
<tr>
<td width="264">
<p><strong>500 mM NaCl</strong></p>
</td>
</tr>
<tr>
<td width="264">
<p><strong>10 mM Tris-Cl, pH 8.0</strong></p>
</td>
</tr>
<tr>
<td width="264">
<p><strong>1 mM EDTA, pH 8.0</strong></p>
</td>
</tr>
</tbody>
</table>
<p>Adapter mix ready to use.</p>
<table>
<tbody>
<tr>
<td width="262">
<p><strong> </strong></p>
<p><strong>Reagent</strong></p>
</td>
<td width="66">
<p><strong>Volume (μL)</strong></p>
</td>
<td width="79">
<p><strong>[Final]</strong></p>
</td>
<td width="97">
<p><strong>[Combined]</strong></p>
</td>
</tr>
<tr>
<td width="262">
<p><strong>IS1_adapter_P7.F (500 μM) </strong></p>
</td>
<td width="66">
<p>20</p>
</td>
<td width="79">
<p>200 μM</p>
</td>
<td width="97">
<p><strong>100uM each</strong></p>
</td>
</tr>
<tr>
<td width="262">
<p><strong>IS3_adapter_P5+P7.R (500 μM) </strong></p>
</td>
<td width="66">
<p>20</p>
</td>
<td width="79">
<p>200 μM</p>
</td>
<td width="97">
<p><strong>100uM each</strong></p>
</td>
</tr>
<tr>
<td width="262">
<p><strong>Oligo hybridization buffer (10X)</strong></p>
</td>
<td width="66">
<p>5</p>
</td>
<td width="79">
<p>1X</p>
</td>
<td width="97">
<p>1X</p>
</td>
</tr>
<tr>
<td width="262">
<p><strong>H2O </strong></p>
</td>
<td width="66">
<p>5</p>
</td>
<td width="79">
<p>-</p>
</td>
<td width="97">
<p>-</p>
</td>
</tr>
</tbody>
</table>
<p>Mix and incubate each reaction in a thermal cycler for 10 s at 95°C, followed by ramp from 95°C to 12°C decreasing of -0,1°C/s. Pool both reactions to obtain adapters mix ready to use.</p>

[STEPS]
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