We are re-sequencing several strains of our bacteria (Rhodobacter sphaeroides strains) to identify mutations and differences from the reference R. sphaeroides genome we have. I've used breseq
and GATK
to identify SNPs and indels with success in the past. However, we are sequencing older strains in which we have replaced a gene of interest with an antibiotic gene. When I process these samples with breseq
or GATK
it does not list the gene of interest as being mutated, but does identify other SNPs and indels.
I have verified that the gene of interest lacks coverage by plotting the BAM files, but this can be laborious for many strains.
Is there a good tool that can identify gene replacement instances such as this? We are likely going to be sequencing many, many more strains in the future and I would like to have a way to detect these without having to manually look at the coverage for the region in the genome.
Thanks! That's a really clever way to go about it and detect these kind of knock-ins/knock-outs.
Not quite as elegant as just using PCR (which is also performed); but the genome is being sequenced to identify off-target effects, anyway; so these ratios are used as a secondary confirmation.