Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Genome sequence of a 45,000-year-old modern human from western Siberia. A high-coverage Neandertal genome from Chagyrskaya Cave. A high-coverage Neandertal genome from Vindija Cave in Croatia. The complete genome sequence of a Neanderthal from the Altai Mountains. Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA. A high-coverage genome sequence from an archaic Denisovan individual. After library preparation, molecules with uninformative short inserts (shorter than ~30−35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies).
The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 libraries. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3′ ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5′ adapter via blunt-end ligation. It has been shown that highly fragmented DNA is most efficiently converted into DNA libraries for sequencing if both strands of the DNA fragments are processed independently.
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