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[DESCRIPTION]
<p><span style="font-weight: 400;">Nanopore sequencing  (Oxford Nanopore Technologies MinION instrument) requires high quality, high molecular weight DNA (HMW) to produce long sequence reads. Extracting high purity, HMW DNA is a difficult challenge, especially for a biotrophic rust fungus. This is because the biomass for DNA extraction is restricted to spores which are physically tough and rich in complex polysaccharides and lipids. When DNA extracted from spores precipitated with ethanol or isopropanol, many other impurities like polysaccharides and lipids get coprecipitated along with DNA which renders DNA purity unsuitable for Nanopore sequencing. These impurities cannot be removed with any subsequent DNA purification with either paramagnetic beads (SPRI) or re-precipitation with ethanol and isopropanol. These impurities always co-precipitated with the DNA which absorbs light at 230 nm. In an attempt to improve purity, I tried precipitating DNA with cationic detergent CTAB (Hexadecyl-Trimethyl-Ammonium Bromide) which has been used to precipitate DNA in the past but not employed extensively. CTAB is a cationic surfactant which selectively complex with DNA and precipitates it out of the solution in the presence low salt concentration (0.4M NaCl) while leaving other impurities. </span></p>
<p><span style="font-weight: 400;">Unlike isopropanol or ethanol precipitated DNA which looks highly viscous, the CTAB precipitated DNA lack similar viscosity and therefore indicative less or no impurities. Moreover, with CTAB precipitation I get ~ 20-25 ug crude DNA per 100 mg of spores which is almost double the amount I get with isopropanol and ethanol precipitation. The crude DNA once washed with 1 V homemade SPRI beads solution, gives a 260/280 ratio ~1.8-2 and 260/230 2.0-2.2.</span></p>

[GUIDELINES]
<p><span style="font-weight: 400;">I adopted this protocol from “Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues” by Jean‐René Arseneau, Royce Steeves, and Mark Laflamme.</span></p>
<p><span style="font-weight: 400;">https://onlinelibrary.wiley.com/doi/full/10.1111/1755-0998.12616</span></p>
<p><span style="font-weight: 400;">https://doi.org/10.1111/1755-0998.12616</span></p>
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<p><span style="font-weight: 400;"><strong>Note:</strong> We started DNA extraction and optimization with the aim to get pure DNA from the rust spores which meet all the purity standard needed for Nanopore sequencing. This protocol gives the DNA that meets all the purity standards like the 260/280 and 260/230 ratios and the Nanodrop and Qubit concentration 1:1, </span><strong>despite this we could not get a good nanopore run out of this DNA</strong><span style="font-weight: 400;">. </span></p>
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<p><span style="font-weight: 400;">This could be due to some contaminants are getting copurified with the DNA which interferes with the nanopore sequencing. These contaminants are invisible to the Nanodrop light spectrum.</span></p>
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<p><span style="font-weight: 400;">This probably might just be an issue with DNA extracted from rust spores and for other species, the protocol might just work fine.</span></p>
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<p><span style="font-weight: 400;">We are planning to purify this DNA with low melting point (LMP) agarose plugs to test if the sequencing run improves.</span></p>
<p><span style="font-weight: 400;">We would update the protocol as and when we succeed in getting an awesome run!</span></p>

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