Sampling, DNA preparation and sequencing

Stockholm

Samples LOW002, LOW003, LOW006, LOW007, LOW008 and PON012 were processed at the Archaeological Research Laboratory at Stockholm University, Sweden, following methods previously described⁸. In brief, this involved extracting DNA by incubating the bone powder for 24 h at 37 °C in 1.5 ml of digestion buffer (0.45 M EDTA (pH 8.0) and 0.25 mg ml^–1 proteinase K), concentrating supernatant on Amicon Ultra-4 (30-kDa molecular weight cut-off (MWCO)) filter columns (MerckMillipore) and purifying on Qiagen MinElute columns. Double-stranded Illumina libraries were prepared using the protocol outlined in ref. ⁴⁸, with the inclusion of USER enzyme and the modifications described in ref. ⁴⁹.

Samples 367, PDM100, Taimyr-1 and Yana-1 were processed at the Swedish Museum of Natural History in Stockholm, Sweden, following previously described methods⁸. In brief, this involved extracting DNA using a silica-based method with concentration on Vivaspin filters (Sartorius), according to a protocol optimized for recovery of ancient DNA⁵⁰. Double-stranded Illumina libraries were prepared using the protocol outlined in ref. ⁴⁸, with the inclusion of USER enzyme.

Samples ALAS_024, VAL_033, ALAS_016, VAL_008, HMNH_007, HMNH_011, VAL_050, VAL_005, DS04, VAL_037, VAL_012, VAL_011, VAL_18A, IN18_016 and IN18_005 were processed at the Swedish Museum of Natural History in Stockholm, Sweden, following previously described methods for permafrost bone and tooth samples⁵¹. In brief, this involved DNA extraction using the methodology of ref. ⁵² and double-stranded Illumina library preparation as described in ref. ⁴⁸, with dual unique indexes and the inclusion of USER enzyme. Between eight and ten separate PCR reactions with unique indexes were carried out for each sample to maximize library complexity. The libraries were sequenced alongside samples HOV4, AL2242, AL2370, AL2893, AL3272 and AL3284 across three Illumina NovaSeq 6000 lanes with an S4 100-bp paired-end set-up at SciLifeLab in Stockholm.

Potsdam

Samples JAL48, JAL65, JAL69, JAL358, AH574, AH575 and AH577 were processed at the University of Potsdam. Pre-amplification steps (DNA extraction and library preparation) were conducted in separated laboratory rooms specially equipped for the processing of ancient DNA. Amplification and post-amplification steps were performed in different laboratory rooms. DNA was extracted from bone powder (29–54 mg) following a protocol…