Let’s Tweap again: Economic and SNP retrieval optimisation for target enrichment of ancient DNA

Abstract Advancements in ancient DNA (aDNA) research have enabled the recovery of individual human genomes through target enrichment approaches. Despite the decreasing cost of next-generation sequencing, enrichment methods targeting single-nucleotide polymorphisms (SNPs) compiled in RNA/DNA hybrid panels remain costly and require further authentic optimisations. Here, we present the Tweap ( Tw ist ch eap ) protocol as a cost-effective alternative to the commercialised Twist protocol for target enrichment via in-solution hybridisation of ancient human DNA by replacing the streptavidin-coated superparamagnetic beads for binding the hybridised targeted DNA. We evaluated the protocol on 14 double-stranded DNA libraries from ancient human individuals originating from a hot and humid climate zone known to accelerate DNA degradation. The selected individuals reflected similar preservation levels and a broad range of endogenous human DNA content (0.79 – 41.69 %). We optimised and simplified both the Twist and Tweap protocols for target enrichment, particularly for libraries with ultra-low library complexity, making it reproducible in any life sciences laboratory. The modified Tweap protocol not only reduced overall reagent costs and consumption, contributing to more climate-conscious research, but also recovered up to 7% more unique SNPs when applying multi-pooling of libraries from the same individual. Our findings demonstrate that the Tweap protocol is a viable, efficient, and scalable alternative for SNP capture in ancient DNA (aDNA) studies, supporting broader accessibility and sustainability in ancient DNA research.