Genomic library preparation from highly degraded DNA is more efficient when library molecules are prepared separately from the complementary strands of DNA fragments. We describe a protocol in which libraries are constructed from single DNA strands in a three-step procedure: single-stranded ligation of the first adapter with T4 DNA ligase in the presence of a splinter oligonucleotide, copying of the DNA strand with a proofreading polymerase, and blunt-end ligation of the second double-stranded adapter with T4 DNA ligase.
Keywords: Ancient DNA; Library preparation; NGS; Single-stranded ligation; T4 DNA ligase.