identifying SNPs from short read sequencing data

Question

[reaset41]

I would like to mutagenize a 1kb bacterial gene. Then I would subject the strains to fitness filter and sequence the resulting pool of genes. Is it possible to detect SNPs from short read sequencing data? Any links or tutorials would be appreciated

[GenoMax]

Short answer is yes. Are you planning to sequence the entire genome after mutagenesis or only the selected region? In any case the analysis will consist of aligning the short reads to genome or the 1kb region (depending on how you sequence) and then call SNP's. Since this is a bacterial gene/genome you could use callvariants.sh from BBMap suite.

[reaset41]

Thank you--this is helpful. I was planning to only sequence the selected mutagenized region (~1kb). If there are multiple SNPs within the 1kb region, I imagine this tool will not be able to evaluate co-occurring SNPs because it is not assembling the length of the gene?

[GenoMax]

You will first align the reads to the region of interest and then call variants.

I was planning to only sequence the selected mutagenized region

Are you planning to do this by amplifying the region?

[reaset41]

Yes I will planning to amplify the region prior to sequencing. Thank you, this is very helpful