A method for reconstructing the structure of double-stranded DNA fragments on a single molecule level and its application to Neanderthal DNA

Despite the large number of molecules that can be sequenced in parallel with current DNA sequencing technologies, the extensive manipulations involved in the preparation of DNA samples for sequencing have hitherto made it impossible to accurately determine the structure of double-stranded DNA fragments being sequenced. We here present a method, MatchSeq, which combines single-stranded DNA library preparation with computational sequence matching, allowing double-stranded DNA fragments to be reconstructed on a single molecule level. The application of MatchSeq to Neanderthal DNA reveals that 5' overhangs are slightly more abundant in ancient DNA than 3' overhangs, that single-stranded parts in the interior and at the termini of DNA fragments are enriched in pyrimidines, and that cytosine deamination occurs in both single- and double-stranded context close to molecule ends. MatchSeq can be combined with routine sequencing of fragmented double-stranded DNA isolated from any biological source and opens the possibility to study DNA fragmentation in living systems or post-mortem at an unprecedented level of resolution.