long-read sequencing of the tprK gene
The DNA extracting from 14 primary syphilis samples and 14 secondary syphilis samples was directly used for PCR amplification of tprK. PCR was conducted with tprK-specific primers appended to 16-bp PacBio barcodes under the following conditions: 98C for 2 minutes followed by 35 cycles of 98C for 10 seconds, 62C for 15 seconds and 68C for 2 minutes with a final elongation at 68C for 5 minutes. The resulting tprK amplicon was purified using 0.6x volumes of AMPure XP beads. barcodes (Table S1) under the following conditions: 98C for 2 minutes followed by 35 cycles of 98C for 10 seconds, 62C for 15 seconds and 68C for 2 minutes with a final elongation at 68C for 5 minutes. The resulting tprK amplicon was purified using 0.23x volumes of AMPure XP beads ((Beckman Coulter, Inc., CA, USA).