title: Colo829_1v1000_NovaSeq_Q5_Rep2
design: COLO829BL and COLO829 DNA was extracted using DNeasy Blood & Tissue Kit(Qiagen), and the mixture DNA samples was generated by spike-in 0.1% and 0.02% of COLO829 into COLO829BL. Primers for SNV targets were designed by Primer3 at size range of amplicons from 130bp to 170bp. PCR was performed by KAPA HiFi HotStart ReadyMix PCR Kit and NEBNext Q5 Hot Start HiFi PCR Master Mix, 10 M of each primer, 50 ng of COLO829BL, COLO829, two replicates of 0.1% mixture and two replicates of 0.02% mixture DNA for each target using the following PCR conditions: 95C for  5 min, 26 cycles of 98 C for 20 sec, 63 C for 15 sec, 72 C for 15 sec, and 72 C for 1 min, then storage at 4 C (Kapa HiFi HotStart); 98C for 30s,26 cycles of 98C for 10 sec, 65C for 15 sec, 72C for 20 sec, and 72C for 2min, then storage at 4C (NEBNext Q5). All amplicons were quality-checked on a 2% agarose E-gel (Invitrogen), then pooled in bins and purified by Agencourt Ampure XP Beads. 100ng of each pooled amplicon was end repaired, ligated adapter and enriched by 8 cycles of PCR using either KAPA HiFi HotStart ReadyMix PCR Kit or NEBNext Q5 Hot Start HiFi PCR Master Mix.