Sequencing Raw Data about Fidelity of RPA DNA Nucleic Acid Amplification

For the RPA reaction, following the procedure described earlier, incubation at 42℃ for 20 minutes was followed by the addition of an equal volume of DNA extraction reagent (Enol:Chloroform:Isoamylol=25:24:1, pH≥7.8) (Solarbio, China). After vortex mixing for 2 minutes, the mixture was centrifuged at 13,700 x g for 10 minutes, and the supernatant was collected for high-throughput sequencing.

PCR assays were conducted using a CFX96 system (Bio-Rad, Hercules, CA, USA) with the following program: initial denaturation at 95℃ for 5 minutes, followed by 30 cycles of denaturation at 95℃ for 15 seconds, annealing at 54℃ for 30 seconds, and extension at 72℃ for 15 seconds. The 50 µL PCR reaction mixture consisted of 25 µL of 2× Premix Taq (TaKaRa Taq version 2.0) or 2×PrimeSTAR Max DNA Polymerase (TaKaRa, Beijing, China), 1.25 µL each of 10 µM forward and reverse primers, 2 µL DNA template, and 20.5 µL ddH2O.