My PI has assigned me a task to gather evidence to determine whether or not a list of particular species of Campylobacter and Helicobacter code for a list of several proteins. To put it simply I’ve been asked, “Do species X, Y, Z code for proteins A, B, C?” The genomes of these bacterial species have been fully sequenced and I am able to retrieve the FASTA files for each of them. I am having trouble deciding on the proper method to screen these genomes for the genes. I have been given the names of the proteins, i.e. VgrG and HCP, but the amino acids sequences listed on databases are annotated to different species of the bacteria. I seem to be stuck in a grey area and am having trouble connecting the overwhelming amount of resources available on the NCBI website. I have not found a specific guide to perform this task - Could you please provide me with a solution to my problems?
First of all, don't reinvent the wheel, search for annotated homologs first. Assuming you don't find them, the next step is:
Collect the sequences of your query proteins (not genes) from one species in a multifasta file.
Run a tBLASTn with those sequences against the genomes of all the other species of interest.
Analyze. Look for HSPs with a certain level of sequence identity. The details of what threshold you choose will depend on your data, I can't help you there.
While the tBLASTn approach is going to be more sensitive, an easier method that can find gene structures and not just HSPs is to use
exonerate -m p2g proteins.fa genome.fa
Finally, for a more sensitive search than either of the above, useful for finding more distant homologs, run a Psi-tBLASTn of your proteins against the genomes of interest.
In all of these approaches, the tricky bit is to choose the right threshold. Unfortunately, there is no golden rule here. It all depends on how closely related your species are and how similar the homologs. You will have to try a few different ones before you can choose the right one.
Do multiple sequence alignment of your protein sequences. This will give you evolutionary relationship between your sequences.