title: Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA.
abstract: This protocol describes a method for converting short single-stranded and double-stranded DNA into libraries compatible with high-throughput sequencing using Illumina technology. This method has primarily been developed to improve sequence retrieval from ancient DNA, but it is also applicable to the sequencing of short or degraded DNA from other sources, and it can also be used for sequencing oligonucleotides. Single-stranded library preparation is performed by ligating a biotinylated adapter oligonucleotide to the 3' ends of heat-denatured DNA. The resulting strands are then immobilized on streptavidin-coated beads and copied with a polymerase. A second adapter is attached by blunt-end ligation, and library preparation is completed by PCR amplification. We estimate that intact DNA strands are recovered in the library with ∼50% efficiency. Libraries can be generated from up to 12 DNA or oligonucleotide samples in parallel within 2 d.
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. marie_gansauge@eva.mpg.de
authors.forename: Marie-Theres
authors.initials: MT
authors.lastname: Gansauge
authors.forename: Matthias
authors.initials: M
authors.lastname: Meyer
mesh_terms: D019921:Biotinylation; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000737:chemistry*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded