Three Assays for in-Solution Enrichment of Ancient Human DNA at More than a Million SNPs

In-solution enrichment for hundreds of thousands of single nucleotide polymorphisms (SNPs) has been the source of >70% of all genome-scale ancient human DNA data published to date, making it economical to study ancient samples with low proportion­­­s of human DNA, and increasing the rate of conversion of sampled remains into interpretable genetic data. So far, nearly all such data has been generated using a set of bait sequences targeting about 1.24 million SNPs (the '1240k reagent'), but synthesis of the reagent has been cost-effective for only a few laboratories. In 2021, two companies, Daicel Arbor and Twist BioSciences, made available assays that target the same core set of SNPs along with supplementary content. We test all three assays on a common set of 27 ancient DNA libraries, and show that all three are effective at enriching many hundreds of thousands of SNPs. For all assays, one round of enrichment produces data that is as useful as two. In our testing, the “Twist Ancient DNA” assay produces the highest coverages, greatest uniformity on targeted positions, and almost no bias toward enriching one allele more than another relative to shotgun sequencing. A caveat is that the 1240k and Twist Ancient DNA results were based on protocols we optimized in our lab, whereas for Daicel Arbor we used the manufacturer's recommended protocol at the time of our testing, and thus our results for Daicel Arbor should be interpreted as a lower bound on performance that leaves substantial room for further optimization. We also identify hundreds of thousands of targeted SNPs for which there is minimal allelic bias when comparing 1240k data to either shotgun or Twist data. This makes it straightforward to co-analyze the large datasets that have been generated using 1240k and Twist capture, and shotgun sequencing approaches.