I'm trying to develop a pipeline to determine mutations which are responsible for amino acid changes in genes associated with antibiotic resistance. I have roughly 300 bacrtial isolates.
My approach so far has not been fruitful, in short this is what i tried:
- Map all my fastq sequences individually to a gene using BWA.
- Converted output SAM to BAM, subsequently sorted all BAM files.
- Run FreeBayes in order to get a vcf file with SNP's
- Run VCF-annotator to get the amino acid change from the vcf file
My approach is probably flawed, but at this point I'm out of ideas. The results i get at the end are wrong.
Any help/input how I should approach this issue is greatly appriciated.