At lunch today I sat beside someone who's been using Illumina sequencing to characterize the genetic and arrangement diversity that arises in a bacterial culture during growth from a single cell. He had lots of practical advice about our recombinome project. Most importantly, because diversity will have arisen in our recipient cells during the growth they do before we transform them, we need to make a DNA prep of a no-DNA added control part of the culture at the same time as we make a prep of the DNA-added transformant pool at the same time, and sequence this control DNA along with our transformed-cell DNA. And when we sequence our donor DNA, we should use the same DNA prep that we use for the transformation.
Other issues: Yeast extract contains yeast DNA that persists in culture media and can come through the DNA prep and contaminate the Illumina library. We grow our H. influenzae cells in 'brain heart infusion'. I think this contains yeast extract, but it may also contain DNA from the hearts and minds of cows. Illumina may have a protocol for removing DNA from culture media, or we could just DNase-treat it before we autoclave it (I wonder how much DNase, for how long...).
Concentration of DNA in the input sample is critical. There's a new way to use PCR to very accurately measure very low DNA concentrations, called 'Digital PCR'; it uses a version of limiting dilution where only some wells contain a DNA molecule.
We had a wonderful reception at the Monterey Bay Aquarium on Wednesday night. But we only got to see part of the aquarium, so now I'm going back to see the rest.
It's great that someone's looking into clonal variation. I'm sure that will be very cool work. We will at least get to address the problem with our controls.
ReplyDeleteI definitely agree that we want to transform with the same donor DNA that we sequence, along with sequencing recipient DNA from a no DNA control.
There's an additional control that may be interesting, but it's a bit pie-in-the-sky... Is there any reason to suspect that naturally competent cells could be in a hypermutable state? So, for the no DNA control, there could be two: (a) recipient competent cells, (b) recipient non-competent cells. They'd be taken from the same culture. If competence induced mutagenesis, there could be differences in the amount of clonal variation observed between the two different no DNA controls...
Another version of this I think we've talked about before: What would the "no DNA control" look like if we did or did not treat with DNase? DNA from dead cells in the recipient culture could be taken up by living competent cells and induce rearrangements, etc?
I hadn't thought about left-over DNA in the media... I'm not sure how terribly it would matter, except for reducing the number of mappable reads. The hearts and minds of cows are unlikely to contain DNA that would map to the Hin genome. Also, "contaminating" DNA would make an oddly useful control for uptake experiments, where we'd expect the hearts and minds of cows to be poorly taken up, relative to Hin DNA.
As a side-note, can we start calling BHI "heart and mind infusion"?
Anyways, say hi to the jellyfish for me...