Thursday, October 23, 2008

The Hassles of High Throughput Sequencing #1

As my lab strives to adopt increasingly high throughput molecular practices, we've hit a few bumps along the way. One involves the first step in the process of obtaining DNA sequence data: extraction. As a molecular biologist, I've grown up in the era of kits: I've always done genomic extractions by pulling a Qiagen/Promega/Viogene kit off the shelf, dropping a chunk of tissue into a tube and following instructions. This simple and convenient approach, however, is challenged by the needs of the very high throughput facility we're working with. They're asking for genomic DNA with a 260/280 ratio (a measure of a sample's quality that reflects the ratio of DNA to undesirable proteins) between 1.8 and 2.0. Essentially, they're asking for genomic DNA that is completely free of protein contaminants. Unfortunately, kits that rely on a silica filters tend to give us ratios in the 1.2-1.3 range. After a week of experimentation, we've been able to get samples with ratios in the 1.6-1.7 range, but only via phenol-chloroform extraction. Although I've spent my career avoiding a method that has always been described as "unpleasant", several gel-based methods make the phenol-chloroform process relatively painless (check out Phase Lock Gel from Eppendorf [apparently discontinued] and MaXtract from Qiagen). I'm sure we're not the first to encounter this problem, but hopefully this post will help others avoid the trouble of extracting dozens of samples only to be told later that the resulting samples weren't good enough!


Fatty said...

I still use CTAB extraction for plants, which is approximately as fast as any kit, but often with better results (more DNA). I almost never quantify 260/280, though. (I did the first few times). I find it unreliable, and doing it right seems to be a labor-intensive waste of time:

fatty said...

P.S. The date on that link is telling about the last time I did the 260/280 spec. Also, if you begin to select the link, it will highlight the whole thing, so you can paste it. Or try this.

Todd Oakley said...

We're in the middle of prepping cDNA for 454 EST sequencing. The first step, we use Trizol, a phenol based reagent, which gives us good yield of RNA.
Then, we need to clean up the DNA after an RT reaction and amplification.

We found that doing phe:Chl extraction led to huge losses in DNA. We lost far less with a Qiagen PCR spin clean up kit. The OD ratios we are getting are a little low.

To quantify, we are using a nanodrop spec, which seems to have been inconsistent lately. In my experience, as fatty said, I find OD very unreliable.

Kevin Zelnio said...

Todd, we gave up on quantifying with the spec. I never seemed to correlate with band strength of quality.

We are doing all our sequencing ourselves. For microsat development the silica filter extraction has been working great. The only problem is our centrifuge's only hold 24 tubes at a time. I use Chelex on the tissue that I will test the microsats on (doing my first run today or tomorrow). We did have problems getting DNA from a deep sea snail using the silica filter though and had success with getting DNA, and following through with getting clones, extracting with Chelex.

The real problem as I see it is having the ABI sequencer run on the windows platform! Our sequencer just got knocked out yesterday because of a virus on the server. :(

Glor said...

If you're getting results you like with silica, but want to speed things up for a large number of samples you should try Promega's 96-well plate system. Although the digestion phase can be a bitch (due to the difficulty of sealing a plate that's going to sit on a hot block for 12+ hours) the rest of the process is a breeze. Normally you'd need to invest in a vacuum system as well, but it looks like they're currently offering one for free as part of a promotion.