Genome sequencing of CS7675 sample from Cova de la Sarsa (Valencia, Spain)
Sequencing libraries were constructed using the NEB’s NEBNext DNA Sample Prep Master Mix Set 2 (E6070) and Illumina-specific adapters, following established protocols33. Following the Bst fill-in step, the libraries were amplified and indexed in 50 µl PCR reactions containing 1X KAPA HiFi HotStart Uracil+ ReadyMix (KAPA Biosystems, Woburn, MA, USA) and 200 nM of each of Illumina’s Multiplexing PCR primer in PE1.0 (5’- AATGATACGGCGACCACCGAGATCTACACTCTTT CCCTACACGAC GCTCTT CCGATCT) and a custom-designed index primer with a six nucleotide index (5’- CAAGCAGAAGACGGCATAC GAGATNNNNNNGTGACTGGAGTTC). Thermocycling conditions were as follows: 1 min at 94°C, followed by 8-12 cycles of 15 sec at 94°C, 20 sec at 60°C, and 20 sec at 72°C, and a final extension step of 1 min at 72°C. The optimal number of cycles was determined by qPCR, following Meyer et al.33. The amplified libraries were then purified using Agencourt AMPure XP beads (Beckman Coulter, Krefeld, Germany) and quantified on an Agilent 2200 TapeStation (Agilent Technologies, Palo Alto, CA, USA).