On the basis of screening results of conventional blunt-end libraries, one individual from Kazburun 1 (kzb002) was identified as having exceptional preservation. To use the individual in a future high-coverage genome database, we commenced production of libraries with repaired damages using uracil-DNA-glycosylase (UDG) treatment to remove deaminated cytosine sites (28). Two new libraries were prepared using the standard blunt-end protocol listed above (27) albeit with an additional 3-hour incubation step at 37°C with 3 μl of Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs) instead of T4 DNA polymerase. After the incubation, 1 μl of T4 DNA Polymerase (Thermo Fisher Scientific) was added to the mix, and the mix was further incubated at 25°C for 15 min and at 12°C for 5 min. The libraries were tested using qPCR (as above) and subsequently amplified in five separate reactions of 25 μl with 1 μl of each 10 μM PCR primer (index primer P7 and IS4), 2.5 μl of an AccuPrime Pfx buffer, 0.5 μl of Herculase II Fusion DNA Polymerase (Agilent Technologies), 16 to 17 μl of ddH2O, and 4 μl of repaired DNA library. The thermocycling conditions consisted of an initial denaturation at 95°C for 2 min, 10 to 14 cycles of 95°C for 15 s, 60°C for 30 s, 68°C for 1 min, and a final extension at 68°C for 5 min. The libraries were pooled, cleaned with magnetic beads (Agencourt AMPure XP, Beckman Coulter), and quantified using the 2100 Bioanalyzer Instrument (Agilent Technologies).

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