Powder

Fifteen DNA extracts (L1–L15) were prepared using 100–200 mg of bone powder from two ~30 000-year-old Neandertal bones (Vi33.25 and Vi33.26 from Vindija Cave, Croatia) following the protocol of Rohland et al. (22 (link)). Two negative controls (L16 and L17) were carried through the extraction process. Sequencing libraries were prepared from the extracts using a previously published protocol (9 ) with the following modifications: (i) All SPRI purification steps were substituted by spin column purification (MinElute PCR purification kit, Qiagen). (ii) For L11-L15 and L17, USER enzyme mix (New England Biolabs) was added to the blunt-end repair reaction to remove uracils (23 ). (iii) Adapter concentration in the ligation reaction was reduced to 0.25 µM of each adapter. (iv) No purification step was performed after adapter fill-in with Bst polymerase. Instead, the enzyme was heat inactivated at 80°C for 20 min. The reaction mix was then used directly as template for PCR.
All libraries were amplified twice by PCR, using a polymerase that is capable of copying across deoxyuracils for the first, and a proof-reading polymerase for the second amplification. Using 5′-tailed primers (‘indexing primers’; see Supplementary Table S1 for all primer sequences), indexes were added to both ends of the library molecules during the first amplification. The entire library volumes were used as templates in 100 µl PCR reactions containing 1× Thermopol buffer (NEB), 5 U AmpliTaq Gold (Applied Biosystems), 250 μM each dNTP and 400 nM each indexing primer. Cycling conditions were comprised of an activation step lasting 12 min at 95°C, followed by 10 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 30 s and elongation at 72°C for 40 s, with a final extension step at 72°C for 5 min. The index combinations used for each library are listed in Supplementary Table S2. PCR products were purified using the MinElute PCR purification kit and eluted in 20 µl EB. An amount of 5 µl of the eluates were used as template for the second round of amplification, which was performed in 100 µl reactions containing 1× Phusion High Fidelity Mastermix (NEB) and the primers IS5 and IS6 (9 ) at a concentration of 400 nM each. Cycling conditions were comprised of an activation step lasting 30 s at 98°C, followed by 10 cycles of denaturation at 98°C for 20 s, annealing at 60°C for 30 s and elongation at 72°C for 40 s, with a final extension step at 72°C for 5 min. PCR products were purified using the MinElute PCR purification kit and eluted in 10 µl EB. The concentrations of all libraries were determined on a Bioanalyzer 2100 (Agilent) using DNA 1000 chips.
Libraries were either directly pooled and sequenced (no-CAP experiment) or enriched for mitochondrial DNA. Enrichment was performed either individually (experiment SP-CAP) or in bulk (experiment MP-CAP) using a protocol detailed in Maricic et al. (21 (link)). After enrichment, the libraries in the SP-CAP and MP-CAP experiments were amplified for 24 cycles using Phusion polymerase under the conditions described above. Libraries were purified using the MinElute PCR purification kit, quantified on a Bioanalyzer 2100 and pooled in equimolar ratios.