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[DESCRIPTION]
<p>The intention of this protocol is to isolate high molecular weight DNA. This means you should avoid any pipetting without using a wide-bore or cut off pipette tip, vortexing, mixer shakers or anything else which generate a velocity gradient which may shear the DNA. In addition you should be very careful not to introduce nucleases by making up buffers with nuclease-free water. Avoid unnecessary heating and do not freeze, isolated DNA should be stored in the fridge, a good extraction will be stable for months. Currently tested on <em>E. coli </em>and human cell lines, however it is likely to work with many gram-negative bacteria and mammalian cells.</p>

[GUIDELINES]
<p>This protocol was d<span style="font-weight: 400;">eveloped by Josh Quick for the Nanopore WGS Consortium.</span></p>
<p> </p>
<p><strong>Protocol Reagents:</strong></p>
<p> </p>
<p>TLB:</p>
<p> </p>
<p>100 mM NaCl</p>
<p>10 mM Tris-Cl, pH 8.0</p>
<p>25 mM EDTA, pH 8.0</p>
<p>0.5% (w/v) SDS</p>
<p>20 µg/ml Qiagen RNase A (add fresh just before use)</p>
<p> </p>
<p>EB+Triton-X100:</p>
<p> </p>
<p>10 mM Tris-Cl pH 8.0, 0.02% Triton X-100</p>
<p> </p>
<p><strong>Expected Results for Human: </strong></p>
<p> </p>
<p>We have been able to generate 50-100,000 reads per flowcell (1-2 Gb) although when working with HMW DNA expect some variability. A read length N50 of 100 kb is a good result. The longest read sequenced with this protocol is 1.2 Mbp by Matt Loose.</p>
<p> </p>
<p> </p>
<p>Example run metrics:</p>
<p> </p>
<p>Total reads                   68169</p>
<p>Total base pairs           1731054841</p>
<p>Mean                           25393.58</p>
<p>Median                        2770</p>
<p>Min                              5</p>
<p>Max                             949165</p>
<p>N25                             219420</p>
<p>N50                             119444</p>
<p>N75                             53984</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rasc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/rasc2w6.png" data-ofn="expected_human.png" /></p>
<p> </p>
<p>Screen grab showing expected distribution from MinKNOW. A large amount of the data is off the scale which only extends to 72,000 events. Reads longer than this are not counted in the yield value which means the standard scaling factor of bases = events * 1.4 will not apply.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ratc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/ratc2w6.png" data-ofn="mion_results.png" /></p>
<p>Pore occupancy (strand / strand + single pore) of 0.8 should be achievable. Overall numbers in strand with RAD004 are often &gt;200 but fall quickly, regular restarts are needed to achieve the maximum yield.</p>
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<p> </p>
<p><span style="font-weight: 400;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rbkc2w6.png" data-src="https://s3.amazonaws.com/pr-journal/rbkc2w6.png" data-ofn="Screen Shot 2017-12-13 at 11.52.48 AM.png" /></span></p>
<p> </p>
<p>Pulsed-field gel, taken from <a href="http://biorxiv.org/content/biorxiv/early/2017/04/20/128835.full.pdf" target="_blank">http://biorxiv.org/content/biorxiv/early/2017/04/20/128835.full.pdf</a> showing DNA extracted using this method in lanes 8 and 9. Smear extends to about 350 kb but closely matches E. coli DNA extracted using the same method demonstrating it is the extraction method not the starting size of the chromosomes driving the size.</p>

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