Targeted Sequencing with Synthetic Baits

The amplified libraries were pooled in equimolar ratios, totaling a final amount of 2 µg. An established protocol was followed (Maricic, Whitten & Pääbo, 2010 (link)) except that synthesized oligonucleotide baits were used instead of PCR products and the EB volume for final elution using Qiagen MinElute column was 20 µL instead of 15 µL. After 2 days of hybridization and subsequent elution steps, 1 µL of the final eluate was quantified by qPCR and 5 µL (in total 15 µL) was amplified in triplicate using the same kit and cycling conditions as described in the NGS library preparation for second round amplification of Illumina indexed libraries. The pooled PCR products were purified using the QIAquick PCR Purification Kit and was measured using the Tapestation 2200 (Agilent Technologies Catalog G2964AA). Hybridization capture libraries were sequenced at the National High-throughput DNA Sequencing Centre, Copenhagen, Denmark using Illumina MiSeq Reagent Kit v2 (300 cycles).