Whole Genome Sequencing!

Model Organism: Worms Methods & Reagents
1

Hi all,

We are having problems with non-amplified genome preps for whole genome sequencing using illumina 50bp paired end sequencing. We have variable library quality and cluster density is unpredictable despite QC against known controls. We are using the gentra pure gene kit (now Qiagen) for the prep of the genomic DNA. We were wondering if it maybe down to contamination of some kind. We would appreciate any advice. Thanks!

2

I don’t know about the kit you’re using; I’ve had very good results making genomic DNA preps more or less according to Michael Koelle’s worm genomic DNA Southern blot protocol (agarose plates and sucrose flotation not necessary).

3

Are you using the nematode specific protocol? We’ve had great success with it and no contamination issues.

4

Looking over the protocol for the Gentra kit, and somewhat handicapped by the vagueness (there doesn’t seem to be a description of the Cell Lysis Solution, in particular), a couple of notes:

  1. The addition of a reducing agent (BME in the protocol I linked, possibly DTT) would make the digestion of the worm cuticles much, much more effective.
  2. The protocol says to spin down the precipitated DNA. In my experience (in at least one weird event, and with a different nematode), this is not your best option; spooling out genomic DNA can help to purify it away from other precipitates that will spin down with it.
  3. On the other hand, it claims to be much faster and maybe less hassle than the protocol I linked.
5

according to the safety sheet;

http://phoebe.colorado.edu/MSDS/Archive/Miscellaneous/MSDS-Puregene-Cell-Lysis-Gentra.pdf

nothing special, it’s an SDS-based lysis buffer.

For what it’s worth, home-grown protocols often seem to outperform commercial protocols (well I never :o) as this paper atests;

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011963

One of the methods (using CTAB) is also a tried and tested approach for worms (Schedl lab ex Fire protocol).

Steve

6

Thanks all. Lots of useful information here.

After receiving an email reply from the WGS facility we use it doesn’t look as if contamination is due to
foreign genomic DNA. Possibly carbohydrate contamination may be an issue here, though I’m always very careful to only use plates where the
worms have only just used up the food (or on the verge of…). I could have a go at the home made protocol but in answer to the question about the kit we currently use,
yes I do use the worm based protocol.
I used to use the CTAB method many years ago to prep bacterial gDNA, so I may just give a home grown method a go.

I look at all the other options you’ve suggested. Thanks again!