I wrote some time ago about the protocol I used to prepare HMW DNA for the new HA412 assembly. The advantage of that protocol is that it doesn’t need much tissue to start with, it’s quick and can work quite well. However, it is also quite unreliable, and will sometimes fail miserably.

To prepare HMW DNA for H. anomalus I tried a different protocol, suggested by Allen Van  Deynze at UC Davis. They used it on pepper to prepare HMW DNA for 10X linked reads (the same application I had in mind), and obtained fragments of average size ~150-200 Kb. The resulting 10X assembly was quite spectacular (N50 = 3.69 Mbp for a 3.21 Gbp genome) and was recently published.

The Van Deynze protocol (basically a scaled-up CTAB) worked much better than the Mayjonade one in my hands. However, it is a longer protocol and needs 2 grams of very young leaves as starting material (about the amount of young leaves you can collect in a 50 ml tube). Getting that many leaves form a single plant takes some patience, especially if, like me, you are trying to get etiolated tissue to minimize chloroplast contamination (I covered the tips of individual branches for 4-5 days with dark cloth).

On a pulse field gel (PFGE) my DNA had fragments of average size ~100-150Kb, which is pretty good. [Unfortunately, the actual weighed mean size of single-stranded used for 10X library construction (as estimated by 10X’s Chromium Controller) was ~17 Kb, despite the fact that Genome Quebec performed a size-selection >50 Kb. The difference with the PFGE is not unexpected and is probably due to single-strand breaks, but still sucks a bit.] It actually turns out that the problem probably was with the BluePippin size-selection before the 10X prep done at the sequencing center (they loaded too much DNA and smaller fragments were carried through). They have re-done the 10X library and GregO is re-attempting an anomalus assembly – hopefully this time it will work better.

Going forward, I was hoping to switch to extracting nuclei and then use a Qiagen MagAttract kit. The grinding needed to break the cell wall is probably what causes most of the DNA shearing (getting large DNA fragment from animal cells is generally much easier than from plants). Isolating nuclei should bypass that problem, while at the same time getting rid of chloroplasts. I still haven’t found a good occasion to waste my time on trying to make that work on an amount of starting material small enough to be obtained from a single plant; however, I already have all the reagents and a possible protocol, if anyone wants to try.

Van Deynze CTAB HMW DNA extraction