Maximizing Efficiency in SedaDNA Analysis: A Novel Extract Pooling Approach

In recent years, the field of ancient DNA (aDNA) has taken a new direction toward studying human population dynamics through sedimentary DNA (sedaDNA), enabling the study of past ecosystems. However, the screening of numerous sediment samples from archaeological sites remains a time-consuming and costly endeavor, particularly when targeting hominin DNA. Here, we present a novel high-throughput method that facilitates the fast and efficient analysis of sediment samples by applying a pooled testing method. Our approach involves combining multiple extracts, allowing users to parallelize laboratory procedures early in the sample preparation pipeline while effectively screening for the presence of aDNA. Pooled samples that exhibit aDNA signals can then undergo detailed analysis, while empty pools are discarded. We have successfully applied our extract pooling method to various sediment samples from Middle and Upper Paleolithic sites in Europe, Asia, and Africa. Notably, our results reveal that an aDNA signal remains discernible even when pooled with four negative samples. We also demonstrate that the DNA yield of double-stranded libraries increases significantly when reducing the extract input, potentially mitigating the effects of inhibition. By embracing this innovative approach, researchers can analyze large numbers of sediment samples for aDNA preservation, achieving significant cost reductions of up to 70% and reducing hands-on laboratory time to one-fifth.