Hi all,
We are using amplicon based method to enrich the target region of given SNPs. And GATK UnifiedGenotyper is applied to call the given/specific variants.
In order to test the robust the our panel, we replicate the same samples, and compared the calling results between two runs.
Over 2000 variants we are looking for, we found that some of them showed different genotypes.
We defined ABRatio as the percentage of reads stand for alt allele.
Similar depth was found between two run, however, slight ABRation change was found.
In the first run, ABRatio=0.06, wildtye genotyes 0/0 was assigned for the sample. However, 0.07 was found for this same sample in the second run. Heterozygous genotype 0/1 was assigned.
We are wondering if there is any method that can diminish this disconcordance? From calling parameter or just experiments?
Best,
Junfeng
If I got you correctly, what you are describing sounds like a normal variation in NGS experiments that you observe even if sequencing the exact same sample twice. Check the overall reproducibitity/correlation of your experiment. If there is no systematic bias, then there is not much you can do about it. High-throughput comes at the price that a single event is measured with less precision that in low-throughput experiments. Correct me if I got your question wrong.
Yes, the overall reproducibility is quite good. Only 1-8 variants showed disconcordance against 2000, which might come from the enrichment bias.
However, what I am talking is the bioinformatics or calling issue. GATK consider 0.06 as wildtype but heterozygous for 0.07.
The base quality is quite good. I am wondering why the slight change of alt allele percentage can lead the different calling result, and what I can do to get the same genotypes.