Fig 1. Workflow of ATAE coupled with PSGS.

Dark-field microscopy, MLST, and determination of TPA DNA copies were performed on the UZ1974 clinical sample taken from a syphilis positive patient. TPA cells were concentrated in the sample using polyclonal antibodies conjugated with biotin, which were bound to streptavidin covered beads. Prior to NGS, whole genome amplification (WGA) was carried out using multiple displacement amplification using phi 29 polymerase; WGA products were purified using QIAEX II beads. The number of TPA DNA copies was monitored using the nested PCR protocol for polA detection [20]. Using the BWA MEM algorithm, the whole set of obtained reads from NGS (Illumina NextSeq 500) was mapped to the human genome reference (hg38), removed, and the rest of the reads were mapped to the TPA reference genome (GenBank Acc. No. CP004011.1).