TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol optimized from Oxford Nanopore Technology 1D native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) for sequencing amplicons of fungal marker genes (ITS, EF1a) using PCR.</p>
<p> </p>
<p>Thanks to Dr. Benjamin Schwesinger for his suggestions about the protocol.</p>
<p>Thanks to Dr. Leon Smith (ANU, Linde lab), Prof. Wieland Meyer (USydney) and Dr. Laszlo Irinyi for their contribution of fungal samples.</p>

[BEFORE_START]
<p>prepare PCR reaction for sequencing. I tried different DNA polymerases including the NEB LongAmp suggested by ONT, NEB Q5 and Promega GoTaq. I found Q5 always produce the best amount of products consistently. LongAmp is with good quality but the yield is too low. GoTaq produces as good amount as Q5 but Q5 is with higher accuracy based on previous experiences.</p>
<p> </p>
<p>With Q5 polymerase, 30 cycles of 100ul reaction will provide you more than enough PCR products (following manufactures protocol). I used 1 volume of magnetic beads (AMPure, Beckman) to clean up the PCR reaction. but for short fragments, I think column based should also work fine. Also, I tested some homemade megnetic beads for DNA purification and they yield the same DNA compared to AMPure Beads. I have not got the protocol for making our beads out yet, but Protocol.io must have similar ones which is worth to test using your sample.</p>
<p> </p>
<p>I included the beads clean up step for PCR reaction in this protocol.</p>

[GUIDELINES]
<p>This is the protocol to extend experimental details of the official MinION sequencing protocol using the native barcoding kit (EXP-NBD103) and 1D ligation kit (SQK-LSK108). I adapted this kit for amplicon sequencing of fungal marker genes (ribosomal RNA and Elongation factor 1a) of individual fungal species.</p>
<p> </p>
<p>By now I sequenced 45 known fungal species to set up this workflow and 11 field samples containing disease infected wheat. The primer I used is listed here and I also attached the brief summary in the excel files:</p>
<p> </p>
<table style="width: 879.417px; border-color: black;">
<tbody>
<tr style="height: 20px;">
<td style="width: 62px; text-align: center; height: 20px;">name</td>
<td style="width: 39px; height: 20px;">
<p style="text-align: center;">orientation</p>
</td>
<td style="width: 242px; height: 20px;">
<p style="text-align: center;">sequence</p>
</td>
<td style="width: 501px; height: 20px;">
<p style="text-align: center;">description</p>
</td>
<td style="width: 495.417px; height: 20px;">
<p style="text-align: center;">reference</p>
</td>
</tr>
<tr>
<td style="width: 62px;">NS3</td>
<td style="width: 39px;">
<p>FWD</p>
</td>
<td style="width: 242px;">
<p>GCAAGTCTGGTGCCAGCAGCC</p>
</td>
<td style="width: 501px;" rowspan="2">
<p>used to amplify fungal rRNA gene region for species identification, bind with small subunit</p>
</td>
<td style="width: 495.417px;" rowspan="6">
<p>Raja, H. A., Miller, A. N., Pearce, C. J., &amp; Oberlies, N. H. (2017). Fungal identification using molecular tools: a primer for the natural products research community. Journal of Natural Products, 80(3), 756-770.</p>
</td>
</tr>
<tr style="height: 20px;">
<td style="width: 62px; height: 20px;">LR6</td>
<td style="width: 39px; height: 20px;">REV</td>
<td style="width: 242px; height: 20px;">CGCCAGTTCTGCTTACC</td>
</tr>
<tr style="height: 20px;">
<td style="width: 62px; height: 20px;">EF1-983F</td>
<td style="width: 39px; height: 20px;">FWD</td>
<td style="width: 242px; height: 20px;">GCYCCYGGHCAYCGTGAYTTYAT</td>
<td style="width: 501px; height: 20px;" rowspan="2">used to amplify fungal Elongation Factor 1 gene region for species identification</td>
</tr>
<tr style="height: 20px;">
<td style="width: 62px; height: 20px;">EF1-2218R</td>
<td style="width: 39px; height: 20px;">REV</td>
<td style="width: 242px; height: 20px;">ATGACACCRACRGCRACRGTYTG</td>
</tr>
<tr style="height: 20px;">
<td style="width: 62px; height: 20px;">RPB2-5f</td>
<td style="width: 39px; height: 20px;">FWD</td>
<td style="width: 242px; height: 20px;">GAYGAYMGWGATCAYTTYGG</td>
<td style="width: 501px; height: 20px;" rowspan="2">used to amplify fungal RNA polymerase II gene region for species identification</td>
</tr>
<tr style="height: 20px;">
<td style="width: 62px; height: 20px;">RPB2-7CR</td>
<td style="width: 39px; height: 20px;">REV</td>
<td style="width: 242px; height: 20px;">CCCATRGCTTGYTTRCCCAT</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p>The original protocol is here: <strong>https://community.nanoporetech.com/protocols/1d-native-barcoding-genomi/v/nbe_9006_v103_revo_21dec2016 </strong>(May need an account of ONT).</p>
<p> </p>
<p>In general, the majority of steps are the same but I reasonably scaled down a little for some steps to save some reagents (optimized for using 12 barcodes, so can be scaled up if using fewer barcodes). Also, I attached an Excel file with calculations that will help proceed the corresponding steps.</p>
<p> </p>
<p>Please let me know your feedback and suggestions. I will super appreciate your help! my email address is: yiheng.hu@anu.edu.au</p>

[STEPS]
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