TITLE: No Title
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[DESCRIPTION]
The following protocol is used to construct shotgun libraries from RNA virus assemblages. This method is designed to detect all RNA viruses, regardless of their genome orientation, and therefore provides a broader assessment of RNA virus diversity compared to the single-gene–based method described in "<a href="https://www.protocols.io/view/A-degenerate-primer-reverse-transcriptionpolymeras-dse6bd?guidelines" target="_blank">A degenerate primer reverse transcriptionpolymerase chain reaction–based protocol to determine the diversity of picorna-like viruses</a>."

[GUIDELINES]
<strong>Equipment</strong>—Thermocycler, heating block, incubator, gel electrophoresis unit, gel documentation system <br /><br /><strong>Supplies</strong>—1.7 mL sterile, nucleic acid–free microcentrifuge tubes; 0.2 mL sterile, nucleic acid-free PCR tubes; sterile razor blades; QIAamp Viral RNA Mini kit (Qiagen); MinElute Gel Extraction kit (Qiagen); MinElute PCR Purification kit (Qiagen); MinElute Reaction Cleanup kit (Qiagen); PCRTerminator End Repair kit (Lucigen); CloneSmart HCKan Blunt Cloning kit with Ecloni Supreme cells (Lucigen); Turbo DNA-free Kit (Applied Biosystems); electroporation cuvettes <br /><br /><strong>Solutions, reagents, and media</strong>—Superscript III Reverse Transcriptase and buffers (Invitrogen), 10 µM dNTP mix, RNase Out (Invitrogen), Klenow Fragment, 3′-5′ exo – (New England Biolabs); primers (Table 1): 0.5 × TBE (45 mM Tris-borate, 1 mM EDTA [pH 8.0]) electrophoresis buffer; nucleic acid-free, sterile water; Ampligold Taq Polymerase and buffers (Applied Biosystems); 0.02-filtered SM Buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris pH 7.5)<br /><br /><strong>Steps:</strong><br /><br /><em>Sample collection, purification, and extraction</em>—The ideal starting amount for this method is on the order of nanograms or greater of purified viral RNA, however the sensitivity of RTPCR suggests that sub-nanogram starting template will be successful. Because of how little RNA is present in a viral genome, the virus community in tens to thousands of liters of seawater must be isolated and concentrated before one can proceed with shotgun library construction. Other chapters in this book (Wommack et al. 2010, this volume; Steward and Culley 2010, this volume) discuss approaches to concentrating viral communities from seawater. <br /><br />RP-SISPA will not discriminate between viral and non-viral nucleic acids, and it is therefore critical that only pure viral RNA is present as template. We have found that purification through two sequential cesium chloride gradients (Protocol B in Lawrence and Steward 2010, this volume) is effective for removing contaminating cellular and exogenous nucleic acids prior to library construction.<br /><br /><strong>Table 1: RT and PCR primer details</strong><br /><br />
<table>
<tbody>
<tr>
<td>Name</td>
<td>Sequence (5'-3')</td>
<td>°C annealing</td>
<td>~Product (bp)</td>
</tr>
<tr>
<td>Mpl.sc1F</td>
<td>TIGCIGGWGAYTWYARM</td>
<td>50</td>
<td>500</td>
</tr>
<tr>
<td>Mpl.sc1R</td>
<td>YTCCTTWTCRGSCATKGTA</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>Mpl.sc2F</td>
<td>ITWGCIGGIGATTWCA</td>
<td>43</td>
<td>500</td>
</tr>
<tr>
<td>Mpl.sc2R</td>
<td>CKYTTCARRAAWTCAGCATC</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>Mpl.sc3F</td>
<td>TIATIGMKGGIGAYTA</td>
<td>49</td>
<td>500</td>
</tr>
<tr>
<td>Mpl.sc3R</td>
<td>TTMARGAAIKMAGCATCTT</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>Mpl.cdhF</td>
<td>GMIGGTGAYTAYAGCGCTTWYGAY</td>
<td>44</td>
<td>500</td>
</tr>
<tr>
<td>Mpl.cdhR</td>
<td>ATACCCAATGCCTYTTIARRAA</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>RdRp1</td>
<td>GGRGAYTACASCIRWTTTGAT</td>
<td>50</td>
<td>450</td>
</tr>
<tr>
<td>RdRp2</td>
<td>MACCCAACKMCKCTTSARRAA</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>SL1</td>
<td>CAGTCCAGTTACGCTGGAGTC</td>
<td>50</td>
<td>NA</td>
</tr>
<tr>
<td>SR2</td>
<td>GGTCAGGTATGATTTAAATGGTCAGT</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>FR26RV-N</td>
<td>GCCGGAGCTCTGCAGATATCNNNNNN</td>
<td>NA</td>
<td>NA</td>
</tr>
<tr>
<td>FR40RV-T</td>
<td>GCCGGAGCTCTGCAGATATC(T)20</td>
<td>NA</td>
<td>NA</td>
</tr>
<tr>
<td>FR20RV</td>
<td>GCCGGAGCTCTGCAGATATC</td>
<td>65</td>
<td>NA</td>
</tr>
<tr>
<td colspan="4">NA, not applicable</td>
</tr>
</tbody>
</table>
<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bbfc2w6.jpg" alt="" data-src="https://s3.amazonaws.com/pr-journal/bbec2w6.jpg" data-ofn="fig1.jpg" /><br /><br /><strong>Figure 1.</strong> RP-SISPA Schematic. This figure is based on Djikeng et al. (2008). Figure 1A shows the three primers used in RP-SISPA. FR26RV-N is composed of the 20 bp 5′ primer sequence, FR20RV (black bar), and a random hexamer at the 3′ end (light blue bar). Primer FR40RV-T is a composite of the FR20RV primer (black bar) and a 20 bp poly T tail (red bar). FR20RV (black bar) is used in PCR. The reverse complement of FR26RV-N (green and orange bar) and FR40RV-T (green and yellow bar) are shown. In Figure 1B, cDNA is synthesized from viral RNA (gray segmented line) with reverse transcriptase primed with FR26RV-N and FR40RV-T. To synthesize the second strand, FR26RV-N is annealed to the newly synthesized cDNA strand where it primes Klenow exo – DNA polymerase “gap filling” activity, resulting in an FR20RV site on both 5′ and 3′ ends. The DNA templates, which represent random stretches of the initial viral RNA genomes, are then amplified with the FR20RV primer to generate more material for cloning and sequencing.

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