I am working on 10 bacterial genomes (1 reference and 9 mutant) sequenced by Illumina technology. My main aim is to find SNPs that are common in 9 genomes but absent in reference genomes. In last, I would like to do the automatic annotation of those SNPs. Until now, I have done the following steps and wondering if I am on the right path.
First: Extracted the common SNPs in 9 mutant genomes
vcf-isec -n +9 -f 1.vcf.gz 2.vcf.gz 3.vcf.gz 4.vcf.gz 5.vcf.gz 6.vcf.gz 7.vcf.gz 8.vcf.gz 9.vcf.gz | bgzip -c > isec1.vcf.gz
Second: tab index
tabix -p vcf isec1.vcf.gz
Third: Extracted SNps that are present in isec1.vcf.gz but absent in reference strain
vcf-isec -c -f isec1.vcf.gz reference.vcf.gz > isec2.vcf
Four: Automatic annotation of isec2.vcf
java -jar snpEff.jar eff -no-downstream -no-upstream -no-utr -no-intergenic -v database isec2.vcf
Most of the SNPs were observed in intergenic region. Should I include these intergenic SNPs or not? Any other suggestions of selecting SNPs.