Alisa Vershinina, PhD

Alisa Vershinina, PhD

Scientist (mostly) | artist (sometimes) | 'omics person

SNP calling from low-coverage ancient samples

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Published:

There are several potential problems if you call SNPs from low coverage ancient samples:

  • Reference bias

As has been stated here and elsewhere, samtools mpileup does quality recalibration by default. This recalibration does not work very well with aDNA data. To disable this, call snips using -B flag.

  • Not enough coverage (duh!)

If you ever need to recover snips from low-coverage genomes, you can use SequenceTools soft written by Stephan Schiffels.

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Creating consensus fasta using iupac codes

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Fasta files can be generated from vcf calls. There are two ways of doing that: (1) concatenate snips together (this can be done using either variants only or calling monomarphic (hom ref) variants as well and concatenating them too); (2) use reference genome as a backbone and incorporate variants into the reference. To incorporate information about heterozygotes, IUPAC substitution codes can be used. Here is a collection of scripts available:

Tools to deal with aDNA damage

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  • mapDamage –rescale option changes quality scores on bam files
  • PMDtools - filters out contaminant reads based on deamination profiles AND adjust scores too. Works on SAM files.
  • ATLAS toolset can do BQSR
  • AntCaller - genotype caller for ancient genomes. Works better than GATK according to the paper.
  • SpAl - estimate the proportion of spurious alignments in ancient DNA. Works with any species, as long as there is data on variation in the genome.

Should we BQSR ancient genomes?

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Published:

A regular protocol for variant discovery pipeline includes base call recalibration after an alignment to a reference genome. This is usually done by GATK. Recalibration accounts for the fact that quality scores sometimes can depend on a cluster density of the sequencing machine, length of the read, position of a bp in the read and other factors. When sequencing ancient DNA, all these parameters go out the window because of DNA post mortem damage (PMD). So should we recalibrate anyway?