Abstract

Abstract In recent years, massive parallel sequencing has revolutionised the study of degraded DNA , thus enabling the field of ancient DNA to evolve into that of paleogenomics. Despite these advances, the recovery and sequencing of degraded DNA remains challenging due to limitations in the manipulation of chemically damaged and highly fragmented DNA molecules. In particular, the enzymatic reactions and DNA purification steps during library preparation can result in DNA template loss and sequencing biases, affecting downstream analyses. The development of library preparation methods that circumvent these obstacles and enable higher throughput are therefore of interest to researchers working with degraded DNA . In this study, we compare four Illumina library preparation protocols, including two “single‐tube” methods developed for this study with the explicit aim of improving data quality and reducing preparation time and expenses. The methods are tested on grey wolf ( Canis lupus ) museum specimens. We found single‐tube protocols increase library complexity, yield more reads that map uniquely to the reference genome, reduce processing time, and may decrease laboratory costs by 90%. Given the advantages of single‐tube library preparations, we anticipate these methods will be of considerable interest to the growing field of paleogenomics and other applications investigating degraded DNA .