Can Gatk Base Quality Recalibration Be Used With Amplicon Sequencing?

Question

[Casbon]

GATK can recalibrate the quality scores in a sequencing run. However, would amplicon sequencing produce a rich enough data set to train the recalibrater.

For example, pretend I have one amplicon in my sequencing run with a novel SNP at a given position. This position would presumably get marked as low quality (since all the reads disagree with the reference). This means that trying to call SNPs from recalibrated scores would produce incorrect qualities, especially at variant sites?

Answer 1

[Casbon]

RTFM:

A critical determinant of the quality of the recalibation is the number of observed bases and mismatches in each bin. The system will not work well on a small number of aligned reads. We usually expect well in excess of 100M bases from a next-generation DNA sequencer per read group. 1B bases yields significantly better results.

Looks like recalibrating amplicon sequencing would be wrong, especially if using low numbers of pooled amplicons.

[Istvan Albert]

Thanks for following up on this once you figured it out.

[Casbon]

It may not be as bad as it seems if you have a few amplicons you could turn off the DiNucleotide covariates.