Figure 1

mtDNA hybridisation enrichment protocol. mtDNA baits were prepared using 2 × 8 kb mtDNA long-range PCR products (spanning 16,569 base pairs), generated using a DNA extract from a present-day sample (of a known haplotype) and the Roche LR Expand PCR kit. The products were fragmented by physical shearing to create 200 bp to 600 bp fragments prior to end labelling with biotin. The DNA library was prepared as follows. Damaged DNA leaves 5′ and 3′ overhangs. T4 DNA polymerase was used to polish the DNA by creating blunt ends and T4 PNK phosphorylated 5′ ends, which is required for adaptor ligation. T4 ligase attached universal hybridisation adaptors (Uni-hyb A and Uni-hyb B) to the phosphorylated ends. Klenow polymerase filled in the short-arm adaptor ligation to create double-stranded adaptors (through the use of deoxyribonucleotide triphosphates - dNTPs). Adaptor complementary primers and Taq polymerase amplified the entire library to immortalise the sample. Single-stranded probe DNA was mixed with single-stranded library DNA and left to hybridise overnight (in the presence of blocking oligos). Biotinylated probe and bound library DNA were fixed to streptavidin beads on a magnetic rack, and non-specific or weakly bound library DNA was washed away through a series of three stringency washes (by increasing temperature and decreasing salt concentration) from the library–probe–streptavidin interaction. The single-stranded library DNA was converted to double stranded DNA and eluted from the probe–streptavidin interaction using the Bst strand-displacing enzyme (in the presence of dNTPs). Bst recognises nicks in the template and displaces library DNA into solution. Probe DNA remained bound to the magnet. Eluted library DNA was enriched through low cycle PCR, using adaptor complementary primers. Library DNA was then prepared for next-generation sequencing. mtDNA, mitochondrial DNA; PCR, polymerase chain reaction.