WGS of Thunnus thynnus: ancient sample
With exception to the Norwegian samples which were prepared as detailed in (90), single-stranded libraries were built for both ancient and modern samples using the Santa Cruz Reaction (SCR) method (98) from 10-20 L of DNA, up to a maximum input of 150 ng. Prior to indexing, aDNA libraries were prepared in sterile conditions at the Ancient DNA Laboratory of the Department of Cultural Heritage, separate from those facilities used to prepare modern libraries. Library quality and the non-amplification of controls were confirmed using qPCR following the SCR method, which indicated the number of cycles required for indexing and inhibited samples to be discarded. Ligated DNA was double-indexed with sample-specific 6 bp indexes by amplification using 2X Amplitaq Gold 360 MM (Thermo Fisher Scientific, USA), and 25-50 M of each index for 10-14 cycles (2 min at 95C, cycles of 30 s at 95C, 30 s at 60C, and 70 s at 72C with a final extension of 10 min at 72C). Amplified products were cleaned by using AMPure XP beads (Agencourt) at a 1:1.2 ratio, eluted in 25 L in EB buffer and quantified using a Bioanalyzer 2100 (Agilent Technologies). All libraries were screened to explore DNA preservation and library clonality by sequencing ca. 1 million reads per sample on the Illumina HiSeq X, 2500 or 4000 platforms (100 bp paired-end). Libraries were then re-sequenced (150 bp paired-end) on the HiSeq 4000 or NovaSeq S4 Illumina platform, to the final sequencing depths reported. Sequencing and demultiplexing (allowing zero mismatches in the index tags) were performed at the Norwegian Sequencing Center (Oslo, Norway) and Macrogen facilities (Seoul, South Korea/Amsterdam, Netherlands) with care taken to avoid batch effects by pooling varied combinations of samples (Table S1).