title: Ancient Plasmodium vivax mtDNA from 1942-1944, Spain.
design: Double stranded libraries were built using the NEBNext DNA Sample Prep Master Mix Set 2 (E6070, New England Biolabs) and Illumina specific adapters from, following manufacturer’s instructions with minor modifications to the protocol, as described below. Given the fragmented nature of aDNA, the initial nebulization step was skipped. The first module of the protocol, corresponding to the end-repair reaction, was performed in 50 μl reactions using 30 μl of aDNA extract, 5 μl of NEBNext 10X end repair buffer, 2.5 μl of end repair enzyme mix and 12.5 μl of molecular grade water. The reactions were incubated for 20 min at 12°C and 15 min at 37°C, and purified using MinElute spin columns (Qiagen) using 10X PB buffer, and eluted in 30 μl of buffer EB after a 10 minute incubation at 37ºC. The second module, the adapter ligation step, was performed in 50 μl reactions using 30 μl of end-repaired DNA, 5 μl of Illumina-specific adapters from (24) with a final concentration of 0.25 μM, 10 μl of 5X Quick Ligation Buffer and 5 μl of Quick T4 Ligase. The reactions were incubated for 15 min at 20°C and purified using QiaQuick spin columns (Qiagen) and 5X PB, before being eluted in 30 μl of buffer EB after a 10 minute incubation at 37ºC. The third module, the adaptor fill-in step, was performed in a 50 μl reaction with 30 μl of adapter-ligated DNA, 5 μl of adapter fill-in Reaction Buffer, 3 μl of Bst Polymerase, and 12 μl of molecular grade water. The reaction was incubated for 20 min at 37°C followed by 20 min at 80°C to inactivate the Bst polymerase.