TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes a generalizable, long-read, low-input metagenomic sequencing approach ('VirION') for the survey of viral communities. A significant obstacle in adopting long-read technology for viral metagenomics lies in obtaining the amount of DNA required; e.g. viral DNA extraction from 20 L of seawater yields far less than the micrograms of DNA recommended for efficient long-read sequencing. To overcome this limitation, we developed a Long-Read Linker-Amplified Shotgun Library approach for long-read viral metagenomics. The VirION method has been demonstrated to be as relatively quantitative as short-read methods, and analyses that combined VirION long-read data with Illumina, short-read data, captured many abundant and ubiquitous viral genomes that were missed by short-read assemblies. This approach was also shown to have overcome issues of microdiversity, and to have captured more genomic islands than short-read assemblies. Thus, VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches.</p>

[BEFORE_START]
<p><strong>Sample requirements</strong></p>
<p> </p>
<p>&lt;1 μg of viral metagenomic DNA in 47 μl of elution buffer (EB) (e.g. 10 mM Tris-Cl, pH 8.5) or nuclease free water (NFW)</p>
<p> </p>
<p>Important note: EDTA compromises flow cell pores, so do not use TE to re-suspend/elute viral DNA; If viruses were obtained by chemical flocculation and resuspension, further cleaning (post DNA extraction with Wizard DNA Clean-Up System) may be required (e.g. using DNeasy PowerClean Pro Kit (Cat No./ID: 12997-50).</p>
<p> </p>
<p><strong>Stopping stages</strong></p>
<p> </p>
<p>If pausing (e.g. overnight) either after PCR adapter ligation and clean-up (Stage ‘C’) or after amplification and clean-up (Stage ‘D’), some reagents will not be required immediately: check protocol to ensure reagents are not defrosted unnecessarily. Replace reagents taken from freezer to -20°C when they have been used and are no longer required (check later stages).</p>

[GUIDELINES]
<p><strong>Method contents</strong></p>
<p>A: DNA fragmentation</p>
<p>B: End-prep and clean-up</p>
<p>C: PCR adapter ligation and clean-up <strong>(possible stop stage)</strong></p>
<p>D: Amplification and clean-up <strong>(possible stop stage)</strong></p>
<p>E: 2<sup>nd</sup> End prep and clean-up</p>
<p>F: Adapter ligation</p>
<p>G: Adapter bead binding</p>
<p>H: Elution of library</p>
<p>I: Preparation of host computer and performing Flow cell QC</p>
<p>J: Priming the flow cell</p>
<p>K: Loading the library</p>
<p>L: Completing the experiment</p>
<p> </p>
<p>Note: Process sheared, diluted commercial lambda DNA as a postivie control, through stages A-E, in order to verify success of amplification stages.</p>
<p> </p>
<p><strong>Equipment and consumables</strong></p>
<ul>
<li>Vortex mixer</li>
<li>Pipettes P1000, P200, P100, P20, P10 and P2</li>
<li>Tips: 1000 μl, 200 μl, 10 μl</li>
<li>Magnet for bead separation (a decent make, e.g. DynaMag™ Spin Magnet)</li>
<li>Micro centrifuge *(model: Eppendorf 5424, if possible)</li>
<li>Covaris g-TUBE (1 per sample, plus one for a positive control)</li>
<li>1.5 ml Eppendorf LoBind centrifuge tubes (Important: some plastics inhibit library prep.) </li>
<li>2 ml thin-walled PCR tubes</li>
<li>Thermal cycler set for: 
<ul>
<li>Program 1: 5 min at 20°C; 5 min at 65°C; 1 min 20°C; hold at 20°C</li>
<li>Program 2: 3 mins at 95°C; 15 cycles of: 15 secs at 95°C, 15 secs at 62°C, 5 min at 72°C, followed by one cycle of 5 min at 72°C; and hold at 4°C</li>
</ul>
</li>
<li>Qubit fluorimeter</li>
<li>Qubit reaction tubes and Qubit dsDNA HS and BR Assay Kits</li>
<li>Heat block set to 55°C</li>
<li>Timer</li>
<li>Filled Ice box</li>
<li>Bioanalyzer/TapeStation</li>
<li>Hula mixer (rotator: optional)</li>
<li>Desktop/laptop:
<ul>
<li>Min. available memory: 50 GB</li>
<li>Sleeptimer and update turned off</li>
<li>For latest minimum computer specs: https://community.nanoporetech.com</li>
</ul>
</li>
<li>MinION Mk 1B</li>
</ul>
<p><strong>Reagents</strong></p>
<ul>
<li>AMPure XP beads resuspended and at RT</li>
<li>SQK-LSK108 sequencing kit (Oxford Nanopore)</li>
</ul>
<p>Defrost following reagents on ice:</p>
<ul>
<li>‘PCR Adapters’ (PCA),</li>
<li>‘Primers’ (PRM),</li>
<li>‘Running Buffer with Fuel Mix’ (RBF),</li>
<li>'Library Loading Beads' (LLB)‘</li>
<li>Adapter Mix’ (AMX 1D) Note: leave in freezer until 10 min before needed</li>
</ul>
<p>Defrost following reagents at RT:</p>
<ul>
<li style="list-style-type: none;">
<ul>
<li>‘Adapter Bead Binding’ (ABB)</li>
<li>‘Elusion Buffer’ (ELB)</li>
</ul>
</li>
</ul>
<ul>
<li>NEB Next Ultra II End-repair / dA-tailing Module (E7546S):
<ul>
<li>‘Ultra II End-Prep Buffer’ - defrost at RT; check for precipitate; vortex well to dissolve if necessary</li>
<li>‘Ultra II End-Prep Enzyme Mix’ - defrost on ice</li>
</ul>
</li>
<li>NEB Blunt / TA Ligase Master Mix (M0367S) – check for precipitate; take out of freezer 10 min before use and keep on ice</li>
<li>NEB Next High-Fidelity 2X PCR Master Mix (Product number: M0541), take out of freezer ~15 min before use and put on ice</li>
<li>Nuclease free water (NFW)</li>
<li>Freshly prepared 80% EtOH (with NFW)</li>
<li>Commercial lambda DNA sheared as detailed in stage ‘A’, and diluted to match sample concentration</li>
</ul>
<p> </p>

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