Extending the spectrum of DNA sequences retrieved from ancient bones and teeth.

The number of DNA fragments surviving in ancient bones and teeth is known to decrease with fragment length. Recent genetic analyses of Middle Pleistocene remains have shown that the recovery of extremely short fragments can prove critical for successful retrieval of sequence information from particularly degraded ancient biological material. Current sample preparation techniques, however, are not optimized to recover DNA sequences from fragments shorter than ∼35 base pairs (bp). Here, we show that much shorter DNA fragments are present in ancient skeletal remains but lost during DNA extraction. We present a refined silica-based DNA extraction method that not only enables efficient recovery of molecules as short as 25 bp but also doubles the yield of sequences from longer fragments due to improved recovery of molecules with single-strand breaks. Furthermore, we present strategies for monitoring inefficiencies in library preparation that may result from co-extraction of inhibitory substances during DNA extraction. The combination of DNA extraction and library preparation techniques described here substantially increases the yield of DNA sequences from ancient remains and provides access to a yet unexploited source of highly degraded DNA fragments. Our work may thus open the door for genetic analyses on even older material.

Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing.

DNA preserved in ancient bones, teeth and sediments is typically highly fragmented and present only in minute amounts. Here, we provide a highly versatile silica-based DNA extraction protocol that enables the retrieval of short (≥35 bp) or even ultrashort (≥25 bp) DNA fragments from such material with minimal carryover of substances that inhibit library preparation for high-throughput sequencing. DNA extraction can be performed with either silica spin columns, which offer the most convenient choice for manual DNA extraction, or silica-coated magnetic particles. The latter allow a substantial cost reduction as well as automation on liquid-handling systems. This protocol update replaces a now-outdated version that was published 11 years ago, before high-throughput sequencing technologies became widely available. It has been thoroughly optimized to provide the highest DNA yields from highly degraded samples, as well as fast and easy handling, requiring not more than ~15 min of hands-on time per sample.