WGS of Thunnus thynnus: ancient sample
With the exception of the Norwegian samples, which were prepared as detailed in Eriksen et al. (2025), single-stranded libraries were built for both ancient and modern samples using the Santa Cruz Reaction (SCR) method (Kapp et al., 2021) from 1020 L of DNA, up to a maximum input of 150 ng. Prior to indexing, aDNA libraries were prepared under sterile conditions at the Ancient DNA Laboratory of the Department of Cultural Heritage, in facilities separate from those used for modern libraries. Library quality and the absence of amplification in negative controls were verified by qPCR following the SCR protocol; qPCR also informed the number of indexing cycles, and inhibited samples were discarded. Ligated DNA was dual-indexed with sample-specific 6-bp indices by PCR using 2X AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, USA) and 2550 M of each index primer for 1014 cycles (initial denaturation 95 C for 2 min; 1014 cycles of 95 C for 30 s, 60 C for 30 s, and 72 C for 70 s; final extension 72 C for 10 min). Amplified products were purified with AMPure XP beads (Agencourt) at a 1:1.2 ratio, eluted in 25 L EB buffer, and quantified on a Bioanalyzer 2100 (Agilent Technologies). All libraries were initially screened to assess DNA preservation and clonality by sequencing ~1 million reads per sample on Illumina HiSeq X, HiSeq 2500, or HiSeq 4000 instruments (100-bp paired-end). Libraries were then re-sequenced (150-bp paired-end) on HiSeq 4000 or NovaSeq S4 platforms at the Norwegian Sequencing Center (Oslo, Norway) and at Macrogen (Seoul, South Korea / Amsterdam, Netherlands) to the depths reported in Andrews et al. (2025). A second re-sequencing round was performed on the NovaSeq X Plus at Novogene UK (Cambridge) to achieve the final depths reported here. The same multiplexed libraries were submitted, with lane combinations chosen to balance sequencing output between modern and ancient samples.