Hi. I am doing a matched Normal-Tumor mutation detection work.
When evaluating my pipelines, I noticed that some somatic mutations are not showing if I follow the best practice for somatic detection by Broad institute.
After I checked the bam files aligned by different pipelines with IGV, I found that the difference was made in the IndelRealigner process.
My question is, I have an impression that it is better to align the normal / tumor reads as input together, and then produce separated output through the --nWayout option in GATK IndelRealigner, but how would it be better to do so than aligning the normal / tumor sample separately? Comparing the separately-aligned result (1way) and aligned-together result (2way), I found a mutation showing in 1way output (with allele frequency 10/168), while it is almost gone in 2way output (1/170). I checked the alignment with IGV, and I think the mutation might be resulted by artifact, which means the the IndelRealigner made the right decision. However, I am still not sure if it is always better to trust the 2way result. I am worried about missing some somatic mutations and not knowing about it, since somatic mutation usually has lower allele frequency.
Does anyone know how to interpret the benefit of using 2way output over 1way?
Any reply would be greatly appreciated.