Mitochondrial DNA Enrichment and Capture

Sequencing libraries were enriched for mitochondrial DNA using hybridisation capture enrichment15 (link). Two micrograms of each library were required for hybridisation capture. To obtain this amount of template library, four 1 μl aliquots of each stock library were PCR amplified in 100 μl reaction volume each with reactions again being terminated after reaching PCR amplification plateau. The four amplicons from each library were then pooled, purified using a Qiagen Minelute kit, and eluted in 20 μl of 0.1xTE. DNA extracted from modern Vicugna pacos (alpaca) samples was used to produce the capture bait as described in Maricic et al.15 (link). In brief, modern samples were extracted using a Qiagen DNeasy extraction kit, complete mitochondrial genomes were amplified by long range PCR as two separate fragments using primers based on the Vicugna pacos mitochondrial genome NCBI Reference Sequence: NC_002504.1 (Supplementary Table S2), purified using Qiagen QIAquick DNA purification kit and quantified using a thermo fisher scientific nanodrop 2000c spectrophotometer. Long range PCR products were pooled in 750ng equimolar amounts and fragmented through sonication. Biotinylated adapters were ligated to the sonicated mitochondrial genome fragments. Adapter ligated fragments were then bound to streptavidin coated beads to be used as bait in the hybridisation capture.