TITLE: No Title
AUTHORS: 

[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><strong>Acknowledgements:</strong></p>
<p> </p>
<p>This protocol was d<span style="font-weight: 400;">eveloped by Josh Quick for the Nanopore WGS Consortium (https://github.com/nanopore-wgs-consortium). Updating this protocol to keep it working with each new version of the rapid kit would not be possible without the contributions of Nick Loman, Matt Loose and John Tyson. We would also like to thank David Stoddart, Simon Mayes and Daniel Turner at Oxford Nanopore for their support. Please follow on Twitter for latest updates and results:</span></p>
<p><span style="font-weight: 400;"> </span></p>
<p><span style="font-weight: 400;">@Scalene</span></p>
<p><span style="font-weight: 400;">@pathogenomenick</span></p>
<p><span style="font-weight: 400;">@mattloose</span></p>
<p><span style="font-weight: 400;">@DrT1973</span></p>
<p> </p>
<p> </p>
<p><strong>Citation:</strong></p>
<p> </p>
<p><a href="https://doi.org/10.1101/128835" target="_blank">https://doi.org/10.1101/128835</a>.</p>
<p> </p>
<p> </p>
<p><strong>Protocol Reagents:</strong></p>
<p> </p>
<p>TLB:</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>10 mM Tris-Cl pH 8.0, 0.02% Triton X-100</p>
<p> </p>
<p> </p>
<p><strong>Expected Results for Human: </strong></p>
<p> </p>
<p>The NA12878 data in rel4 was generated using RAD002 kits. We put a lot of effort into reproducing these run metrics using the updated RAD003 kits and although we discovered many optimisations to the standard protocol, read lengths in particular fell short of what we had previously generated. The RAD004 kit featured changes to the tethering chemistry and we have observed a significant improvement in the performance of the kit. The yield for ultra-long read flowcells is expected to be lower than those with standard libraries, we attribute this to membrane damaged caused by the very high concentrations of DNA. We have been able to generate 50-100,000 reads per flowcell (1-2 Gb) although when working with HMW DNA we expect some variability due to DNA heterogeneity. A read length N50 of 100 Kb is considered an excellent result. The <a href="https://twitter.com/mattloose/status/954147458778587136" target="_blank">longest read</a> sequenced with this protocol is 1.2 Mbp by Matt Loose.</p>
<p> </p>
<p> </p>
<p><strong>Example run metrics for RAD004:</strong></p>
<p> </p>
<p> </p>
<p>General summary:</p>
<p><br />Number of reads:       33,907<br />Total bases:                1,324,105,498<br />Median read length:   17,472<br />Mean read length:      39,051.1<br />Read length N50:       90,923<br />Active channels:         492</p>
<p> </p>
<p>Top 5 longest reads and their mean basecall quality score:</p>
<p><br />1:   975,623 (10.26)<br />2:   862,356 (10.28)<br />3:   722,772 (7.62)<br />4:   709,230 (10.19)</p>
<p>5:   699,282 (9.88)</p>
<p> </p>
<p>Run metrics from NanoStats.txt in Guppy output.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/sjbia8e.png" data-src="https://s3.amazonaws.com/pr-journal/sjbia8e.png" data-ofn="image (1).png" /></p>
<p>Read length histogram view from MinKNOW 1.23 in playback mode after one hour. A lot 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/si8ia8e.png" data-src="https://s3.amazonaws.com/pr-journal/si8ia8e.png" data-ofn="image.png" /></p>
<p>Channel states view from MinKNOW 1.23 in playback mode from the start of the run. Pore occupancy (strand / strand + single pore) of 0.75 should be achievable with this method. Overall numbers in strand with RAD004 are often &gt;200 but fall quickly, regular restarts are needed to achieve the maximum yield.</p>
<p> </p>
<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 different extractions, this method is 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>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.