Sequencing Raw Data about Fidelity of RPA DNA Nucleic Acid Amplification

The high-throughput sequencing samples were prepared by both RPA and PCR amplification targeting the CHS1 gene with the same primers. 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. Both RPA and PCR products were amplified with adapters and barcodes suitable for Illumina NovaSeq sequencing. Raw reads were obtained from Tsingke Biotechnology (Beijing, China) and analyzed using Python scripts (Python 3.11.8), filtering reads with an average Phred quality (Q score) of at least 25.