Time, Temperature, and Tiling Density When Capturing Ancient DNA

Enrichment of sequencing libraries through bait-target hybridization (a.k.a., "capture") is rapidly replacing PCR as the enrichment method of choice for ancient and degraded DNA analysis. Though successful in recent years, aDNA capture remains unpredictable and has much room for improvement. This is at least partially due to the fact that capture methods typically used were developed to target relatively long and abundant DNA fragments, which are qualities opposite those of typical ancient DNAs. In order to better tune capture to the unique properties of aDNA libraries, we performed a series of parameter sweeps to measure how hybridization time, temperature, and bait tiling density impact the specificity and sensitivity of capturing short, rare targets imbedded in complex environmental DNA backgrounds. Here we summarize the results of those experiments, and recommend specific experimental design considerations for improving degraded DNA capture.