WGS of GIAB NA12878 amplified with EvaGreen on n6tec iconPCR
Human genomic DNA from GIAB reference material NA12878 (Coriell Institute) was enzymatically fragmented to ~150-200 bp using the NEB Ultra II FS DNA Library Prep kit (E7805). End repair, dA-tailing, and P5/P7 Illumina-compatible adapter ligation were performed per manufacturer protocol. Ligation products were purified with NEBNext Sample Purification Beads (0.8X ratio). Amplification was performed on the n6tec iconPCR instrument using NEB Q5 High-Fidelity DNA Polymerase in the presence of EvaGreen at 1X final concentration for 10 cycles (98C 30s; 10x [98C 10s, 65C 75s]; 65C 5min; hold 4C). Dye concentration and cycle count were set above standard iconPCR stop-point parameters to provide a stringent mutagenicity test. Amplified libraries were purified with NEBNext beads (0.8X). Libraries were quantified by Agilent Fragment Analyzer and qPCR, then sequenced PE 2x75 nt on an Element AVITI to ~100M reads per sample.