Whence Gibbs?

I successfully worked out how to command the Gibbs Motif Sampler to analyze the new genome sequences. I've only done it for two of them, because a better option has appeared.

A new version of the Gibbs motif software is available. It gives the option of using a 'centroid' sampling method that combines the best sites found in different runs (runs initiated with different random-number seeds), rather than simply taking all the sites identified in the run that had the best score. This has the big advantage of eliminating most of the weakly-matched 'false positive' sites.

It took me a few days to work out how to get it running on the computer cluster (the helpful administrator reset some permissions for me). The new release includes a version that runs in the Mac terminal, and I now have that working too. But it didn't take long to discover that it runs about 100-fold (no, I'm not exaggerating) slower than the usual (non-centroid) version. This means that a good run analyzing a whole genome would take several weeks (or more?); getting rid of the false positives isn't worth that big an investment.

But the very helpful Gibbs expert has again offered to help - he says the centroid version shouldn't be slower at all. So I've sent him the test file I've been using (2% of the genome) plus examples of the output I get. He's going to see if he can find the problem and fix it.

Lots of Gibbs search progress

Yesterday I worked out a way to nudge the Gibbs motif sampler into finding the Neisseria meningitidis DUS (their term for their uptake signal sequence). Even though the DUS is present in Neisserial genomes even more frequently than the H. influenzae USS is in its genome, the sampler couldn't find it without prompting. This may be because it's much shorter than the USS (only 12 contiguous bp vs 22 bp spread over 29 positions), or for some other reason I don't understand.

I didn't want to give the sampler a prior file specifying the pattern to look for, so instead I added two lines of fake sequence with a very high frequency of the DUS to the start of the genome file. This 'seed' was enough to get the sampler started on the right motif. Once it's started it has no trouble finding the DUS, and I can later delete the seeded DUSs from the list it generates.

This morning I obtained the A. pleuropneumoniae genome sequence my collaborators have been working with, split it into pieces, and generated reverse complements of both it and the N. meningitidis genome, and combined each genome's forward and reverse-complement sequences into single 'F+RC' files for searching. I did this because I need to have the sampler search both strands, and (I think) I have better control if I tell it to search just the sequence I've given it. The A. pleuropneumoniae USS is very similar to but even longer than the H. influenzae USS, so I did test runs with the 'prior' masking file I'd used for H. influenzae to make sure everything worked.

I did this and all my other tests using only 10% of the genome and only one orientation, because I wanted them to run very fast and because the guys who manage the computer cluster want all long runs to be entered through their 'Fair Share' queueing system. And now I've successfully queue'd requests for full-genome searches. I don't expect to get the results until tonight or tomorrow.

I also emailed my collaborators to let them know I'm finally back working on this project. The PI is on vacation, but the bioinformatician has been taking advantage of his absence to work full time on it! She's going to send me her new data and rewrite in a few days, so I'm not going to do any work on the manuscript until then. I could go ahead and do Gibbs analysis of all the genomes we might want to consider, but I think I should wait to see how the three main foci of our work (H. influenzae, A. pleuropneumoniae and N. meningitidis) fit into the manuscript.