The aim of this research project is to sequence the genomes of four Pseudomonas fluorescens isolates from two different cave environments. The resulting genome sequences will allow us to estimate of the selective pressure on the same species in two caves and then understand the overall selection pressures in nutrient starved caves. The additional P. fluorescens genomes already available for Pf-5, SBW25 and Pf0-1 will also allow comparative genomics for completely different environments and further analysis of the P. fluorescens pan-genome. I’ll outline our sequencing strategy below.
De Novo genome sequencing
If there are genomes already available for P. fluorescens strains then the obvious choice should be comparative based sequencing and assembly. However comparative analysis of three P. fluorescens species shows the genomes of P. fluorescens strains are much more dissimilar than might be expected for strains of the same species. Therefore it is likely the genomes our cave strains will be similarly unrelated to existing sequences.
Based on this we’re going to use 454 sequencing and perform de novo assembly of two P. fluorescens isolates; we’ll sequence a single isolate from two different cave sites. We would have preferred to have sequenced four genomes but this was ruled out by the low coverage per genome (~9X) and additional sample preparation costs which I’ve discussed previously. So instead we’ll sequence two genomes on a 454 plate split in half using the standard rubber gasket. Each sample will be prepared as a combination of paired-end and shotgun reads. This will provide uniform 35-40X coverage for each genome with the paired reads to improve assembly.
Comparative genome assembly
We ruled out comparative assembly using short but high read sequencing, such as Illumina or AB SOLiD because of the anticipated low sequence identity between P. fluorescens strains. Nevertheless once we have two de novo assembled cave genomes hopefully we can do comparative assembly of other cave isolates using these as scaffolds. Previous P. fluorescens genomes show a low sequence identity but we hope that isolates of the same species from the same site will have enough genome similarity to allow one genome to act as a scaffold for the comparative assembly of a second.
Therefore we will take a two further isolates of the same species from each site and send them for AB SOLiD sequencing. This will provide 90X coverage for each genome at a much cheaper price compared with 454 sequencing. Hopefully the combination of 454 and AB SOLiD will produce large amounts of data to compare variability between strains and sites.