Wednesday, March 16, 2011

Phase II

The Human Microbiome meeting was awesome...

Here's a 16S phylogeny of bacteria that live in the human airway:
here's some other places on the body

Anyways, cool stuff.

So my ears are itching, because yesterday the local genome centre was doing quality control on our libraries of Haemophilus influenzae recombinants (and controls)… If all goes well, we'll get scads of data in a couple weeks!

But I also need to get the work of our "Phase II" plan for Genome BC off the ground quickly, so we'll have samples to submit within a couple months of getting this incoming "Phase I" data.

The goal is to make maps of transformation frequency across the H.flu genome, when our standard lab strain Rd is transformed by DNA from clinical isolates with varying distributions of genetic variation. We hope to be able to investigate the sequence factors controlling the chance of homologous recombination, not only the effects of divergence, but also the role of chromosomal position, for example proximity to transformation hotspots.

There are two parts to making these transformation maps, the experiment itself and the sequence analysis. For the latter, we have worked out a strategy, but I'll just give a brief synopsis here: The challenge is basically to be able to make frequency estimates that are nearly as low as the sequencing error rate. We plan to handle this by using the very high co-transformation rate of adjacent SNPs and the high density of SNPs between H.flu isolates. Thus we will use information within reads and between paired reads to distinguish true transformation from spurious errors.

The basic experiment is really straightforward: (1) Incubate donor DNA with competent cells, allowing for uptake and recombination. (2) Purify the recipient chromosomes. (3) Sequence these to extremely high genomic coverage and measure the donor-specific allele frequency at each polymorphic site.

While that sounds simple, there will be a bunch of challenges ahead in getting this done properly (and on-time). We've got a pretty got a pretty good plan for handling the issues, so I think we'll be able to make appropriate samples for sequencing:
  1. We need as highly competent of cultures as possible. Our sensitivity will depend on sequencing chromosomal DNA fragments from as few non-competent cells as possible.
  2. We need very high complexity libraries, since we will be sequencing to several thousand fold genomic coverage of each library.
  3. We need replication.
  4. We need more than one clinical isolate to donate its DNA (so that we can actually make this into a real experiment).
  5. We need controls.
  6. We need to show that we don't have to worry about donor DNA contamination (i.e. donor sequences that weren't recombined into the chromosome).
So the first thing to do is make the competent-est competent cultures I can. Our sensitivity will depend on sequencing DNA fragments from as few non-competent cells as possible.

This week, I'm going to test MIV transformation of wild-type Rd and a couple of our hypercompetent mutants (murE and sxy-1) with/without cyclic AMP to see which gives the highest transformation frequencies (and least congression). (I'll start just with our regular old MAP7 DNA). We reason that this will not alter the transformation frequency map in an important way, because the hypercompetent mutants and addition of cAMP affect the regulation of competence, rather than its consequences.

I am tempted to try transforming H.flu circa 1961 and make the crazy blood brain heart media and do an anoxic transformation. Their transformation frequencies were so high in the sixties! It might be worth doing, though the difference could perhaps be something other than the culture conditions (perhaps our strain has changed?)… It would also require, ahem, some fairly odious preparation...

Anyways, I'll try and keep the blog updated on this experiment's progress (and later the sequence analysis) as things move along…

Tuesday, March 1, 2011

Uptake specificity as a tool for microbiome research

image credit

Sorry for neglecting you, blog.

So Rosie and I are headed to the Human Microbiome Meeting here in Vancouver in just over a week. This means there's two posters to make! AAccK! We'll be a little out-of-place at the meeting, since our work is mostly in the lab, but we're hoping to learn more about the normal environment of H. influenzae and maybe even find people with access to mucus interested in bacterial genetic exchange.

So our posters won't be directly about the microbiome, but instead one will be about our genomics of natural transformation experiment, and the other will be about the uptake specificity measurements using deep sequencing. As I was thinking about the future of the uptake specificity work, it occurred to me that we could use H. influenzae as a way to filter Haemophilus and other Pasterellaceaen chromosomal fragments from a lung mucus DNA sample, for example. This would at least be a good way to screen out the gobs and gobs of human DNA likely there (at least most of it) and maybe focus a microbiome project down to a narrowish group... save a lot of money that way...