<p>Hi all,</p>

<p>Our lab has sequenced 20 different strains of the same bacterial species.  I now want to analyse these data, i.e., link genomic data to virulence (some of the strains are virulent, others aren't) and other phenotype differences.</p>

<p>I started with de novo assemblies and mapping to an annotated reference genome.  However, it is unclear to me how to proceed now in a good way.  Most of the information for comparative genomics I find on the web applies only to the human genome or one-to-one comparisons of bacterial genomes.</p>

<p>Is there a standard workflow for this kind of analysis?  Does anybody know of any good tutorials/references/articles?  How would you proceed?</p>

I don't think there is a single best standard approach to this, but [Bakker et al.][1] did a comparative genomics approach on Listeria with the goal of identifying virulence genes. You could simply try to replicate their methods on your data.

The main focus seems to be on the gene level, identifying a subset of virulence related candidate genes, and then look for whole gene deletions. Thereby I would also include biological knowledge on bacteria in general and knowledge about this specific species, as to what are good candidate sets of genes (e.g. Type III secretion system, etc.). The aim is to identify if there are differences in the inventory of genes.

In addition and in case, the whole gene deletion/insertion approach does not yield good candidate genes, I would run the whole raw data through a variant calling pipeline and look at smaller variation like small insertions or deletions, frame-shifts, mutations in CDS and promoter sequences. To accomplish this a standard pipeline (aka. [BWA][2] into [samtools][3]) could  be used. You can then check variants common for certain phenotypes and check their effect on the protein level. Further, looking for non-coding elements might be interesting too.

 [1]: http://www.biomedcentral.com/1471-2164/11/688/
 [2]: http://bio-bwa.sourceforge.net/
 [3]: http://samtools.sourceforge.net/