WGA sequencing of Treponema pallidum subsp. pallidum from isolate UZ1974 using Anti-Treponemal Antibodies Enrichment
TPA cells in the swab extract (PBS) were concentrated in the sample using polyclonal antibodies conjugated with biotin (Pierce Treponema pallidum Antibody PA1-73103; ThermoFisher Scientific, Waltham, MA, USA). Antibodies were bound to streptavidin coated magnetic beads (Dynabeads, CELLLection Biotin Binder Kit, ThermoFisher Scientific, Waltham, MA, USA). TPA cells were separated in the following steps: i) biotin-streptavidin binding (260 l of beads, 240 l of PBS, and 2 l of anti-treponemal antibodies were mixed together and incubated at 4 C overnight using end-over-end rotation); ii) removal of unbound antibodies (the mixture was twice washed with 500 l of PBS); iii) incubation with swab extracts for one hour at room temperature (200 l); iv) washing with PBS, and v) DNA isolation (QIAamp DNA Blood mini-kit, Qiagen, Hilden, Germany). Whole genome amplification (WGA) was carried out using Multiple Displacement Amplification with phi 29 polymerase (REPLI-g Single Cell Kit, Qiagen, Hilden, Germany). The WGA products were purified using QIAEX II beads according to the manufacturers recommendations (Qiagen, Hilden, Germany).