Suggestions for WGS 5X Sequences
Hi Geraldine or Sheila,
I am in the process of customizing a GATK pipeline for processing aDNAA. I have processed a couple of 3000 year old WGS sequences so far using GATK best practices, and although the resulting VCFs have been ok, they are far from perfect, and I was looking for any suggestions you can offer on how to minimize reference bias both during alignment as well as variant calling. To give you an idea where things stand, I have organized a color coded spreadsheet summarizing CollectVariantCallingMetrics using various GATK tools. Here is a link to the sheet https://docs.google.com/spreadsheets/d/1iPw-afPV6Z4zGzqfOlna_UyYf87gBv1ghxzLzDkqfFw/edit?usp=sharing
The attached spreadsheet will show you the metrics with and without BQSR, and with or without VQSR.
I have also experimented with omitting some of the annotations you use in VQSR and have plotted everything in R, although the output with all annotations using -mG 4 was not bad.
So far my outputs have been using HaplotypeCaller in GVCF and joint genotyping using 2 or 3 samples, I plan to increase to 10 to see if that helps establish better evidence for variants at low depth sites
Any suggestions you can offer that I may try to reduce reference bias at low depth positions. Filtering out positions that passed VQSR but had a QUAL score <100 with vcftools helped some. Any suggestions with regards changing the prior likelihood of true and not true training sites and known sites during VQSR or to use different resources than the HapMap 1000G or Omni that you suggest.