I am wondering how to extract the data of my NGS analysis. In my experience, I did site-directed saturation mutagenesis at 49 positions in my enzyme. So theoratically, i have all the possible mutations at each chosen positions. Then I did my own PCR fragment of 250 bp to avoid random sequences and barcode them depending of the YES/NO results of a screening. Finally I did the NGS illumina sequencing. The goal is to identify every mutations that i have for each positions in my pool. I am a beginner at bioinformatics and I am learning to use the tools in Galaxy. I mapped the sequences (12 millions) with my reference genome of 1400 bp and i can visualize them in IGV. But i would like an output file giving me the mutations obtained at specific positions.
I have looked in the litterature, but there is a lot of different technique for SNP, variant calling but often on human genome for cancer mutations which does not seem to fit my purpose.
Could somebody enlighten me on the tools to use and the easiest way to achieve my goal?