Abstract
Ancient DNA (aDNA) analysis remains challenging due to low endogenous DNA content of degraded samples. Hybridisation-based in-solution enrichment has emerged as an effective tool for targeting genomic regions, enhancing endogenous DNA yield while minimising overall sequencing effort. Despite their widespread use, the performance of different probe kits in capture efficiency remains insufficiently understood, particularly in nonhuman model organisms. In this study, we examined the performance of two commercially available custom probe systems, the RNA-based myBaits and DNA-based Twist, in enriching endogenous aDNA (0.9%-90.1%) extracted from crow bones collected from the early to late Holocene (100-14,000 years ago). The target regions included a panel of 104 K genome-wide single nucleotide polymorphisms (SNPs) identified from modern populations of the Corvus corone species complex. Both custom probe kits substantially improved fold enrichment and target site detection rates compared with shotgun sequencing. Between the two kits, myBaits consistently achieved higher capture efficiency. In contrast, Twist retained a greater proportion of endogenous DNA, but most of this originated from off-target regions, resulting in lower target efficiency under our experimental conditions. Twist demonstrated higher coverage in regions with extreme GC content, highlighting its utility for applications targeting GC-rich genomic regions. These findings provide insights into the performance of commercially available DNA enrichment methods and help guide study design.