Moin from Kiel,
we have an urgent question we currently analyze variants from exon based on amplicons. The short exons are covered by the end of the foreward and reverse read. Unfortunately the error-rate at the end of the reads is quite high. So the region with the high error-rate of both reads covers the exon region. Is reliable call still possible (excluding softclipped reads, reads with low quality,...) due to the high coverange of amplicon-data >20k reads per position. Attached an example from igv (softclipped reads shown as well).
I am currently working on a Exome sequencing projekt with older single-end SOLiD exomes and new paired-end exomes. In a first attempt (GATK 1.7 and best practices v3 back then) i tried calling and recalibrating all exomes together (at that time 120) without selecting for paired/single-end. As I already had validatet many variants I could check the quality of the calls and got very bad results, especially for InDels (previously called, true positive variants missing). My idee is that the UnifiedGenotyper has Problems mixing paired-end with single-end exomes.
Is there any official recommendation for this problem? My solution right now is to group the exomes in batches (40-50 Exomes) which ran on the same technology.
Also a second Problem/Question: For some individuals exomes where sequenced twice, and for some of these the first run was single-end and the second one was paired. The best practices mentions one should se all available reads for a individual for calling. Do you have any experience on how to handle these cases?
Any help is greatly appreciated!