Question: Can Gatk Base Quality Recalibration Be Used With Amplicon Sequencing?
2
gravatar for Casbon
9.9 years ago by
Casbon3.2k
Casbon3.2k wrote:

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?

gatk snp • 3.6k views
ADD COMMENTlink modified 11 months ago by Biostar ♦♦ 20 • written 9.9 years ago by Casbon3.2k
3
gravatar for Casbon
9.9 years ago by
Casbon3.2k
Casbon3.2k wrote:

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.

ADD COMMENTlink modified 21 months ago by RamRS27k • written 9.9 years ago by Casbon3.2k

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

ADD REPLYlink written 9.9 years ago by Istvan Albert ♦♦ 84k

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

ADD REPLYlink written 9.9 years ago by Casbon3.2k
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