Experimental conditions improving in‐solution target enrichment for ancient <scp>DNA</scp>

<jats:title>Abstract</jats:title><jats:p>High‐throughput sequencing has dramatically fostered ancient <jats:styled-content style="fixed-case">DNA</jats:styled-content> research in recent years. Shotgun sequencing, however, does not necessarily appear as the best‐suited approach due to the extensive contamination of samples with exogenous environmental microbial <jats:styled-content style="fixed-case">DNA</jats:styled-content>. <jats:styled-content style="fixed-case">DNA</jats:styled-content> capture‐enrichment methods represent cost‐effective alternatives that increase the sequencing focus on the endogenous fraction, whether it is from mitochondrial or nuclear genomes, or parts thereof. Here, we explored experimental parameters that could impact the efficacy of <jats:styled-content style="fixed-case">MY</jats:styled-content>baits in‐solution capture assays of ~5000 nuclear loci or the whole genome. We found that varying quantities of the starting probes had only moderate effect on capture outcomes. Starting <jats:styled-content style="fixed-case">DNA</jats:styled-content>, probe tiling, the hybridization temperature and the proportion of endogenous <jats:styled-content style="fixed-case">DNA</jats:styled-content> all affected the assay, however. Additionally, probe features such as their <jats:styled-content style="fixed-case">GC</jats:styled-content> content, number of CpG dinucleotides, 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.</jats:p>