Experimental conditions improving in solution target enrichment for ancient DNA

High throughput sequencing has dramatically fostered ancient DNA research in recent years. Shotgun sequencing, however, does not necessarily represent the best-suited approach for this field due to the extensive contamination of samples with exogenous environmental microbial DNA templates. DNA capture-enrichment methods instead represent cost-effective alternatives that increase the sequencing focus on the endogenous fraction, whether it be mitochondrial or nuclear genomes, or parts thereof. Here we explored experimental parameters that could impact the efficacy of MYbaits in-solution capture assays of ~5,000 nuclear loci or the whole genome. We found that varying quantities of the starting DNA template or probes had little effect on the capture results. The tiling of the probes, the hybridisation temperature, and the proportion of endogenous DNA all affected the assay, however. In addition, probe molecular features such as the GC content, the number of CpG dinucleotides, the sequence complexity and entropy, and self-annealing properties need to be carefully addressed during the design stage of the capture assay. The experimental conditions and probe molecular features identified in this study will improve the recovery of genetic information extracted from degraded and ancient remains.