WGS sequencing of Treponema pallidum subsp. pallidum (TPA) from isolate CW29 using DpnI enrichment approach.
Isolation of DNA: Swab extracts (prepared by submersion of swabs into 1.5 ml of PBS and agitation for 5 min at room temperature) and tissue sample (25 mg) were used for isolation of DNA using a QIAamp DNA Blood Mini kit and a DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to manufacturer's recommendations. DNA binding enrichment using restriction endonuclease DpnI: Clinical DNA samples (10-40 l) were added to DpnI coated beads in 1.7 ml Eppendorf tubes in a final volume of 50 l, as described previously (Barnes et al, 2014, Liu et al, 2016). The beads were mixed by end-over-end rotation for 30 minutes and the DpnI-magnetic beads were separated using a magnetic stand. The beads were washed once with Wash Buffer (10 mM Tris pH 7.9, 500 mM NaCl, 10 mM CaCl2, 0.1% Tween 20) followed by a single Binding Buffer wash (10 mM Tris pH 7.9, 50 mM NaCl, 10 mM CaCl2, 0.01% Tween 20). DNA was eluted from beads by incubation with 20 l of 5 M guanidinium thiocyanate at room temperature for 5 minutes and subsequently desalted via dialysis for 45 minutes using 20,000 MWCO Slide-A-Lyzer MINI dialysis cups (Thermo Scientific, Waltham, MA). Library generation and next-generation sequencing: The Nextera XT DNA library Preparation Kit (Illumina, San Diego, CA) was used to produce barcoded libraries for all DpnI enriched fractions. Library products were amplified using 19 cycles and DNA was purified with AMPure XP beads (New England BioLabs, Ipswich, MA, USA) with an elution volume of 20 l. Library quality and size distributions were determined with the Lab Chip GX Touch-HT (Perkin Elmer, Waltham, MA, USA) and High Sensitivity DNA Analysis Kit (Perkin Elmer, Waltham, MA, USA). Libraries were diluted for sequencing and pooled as appropriate for the targeted sequencing depth on a single flow cell. NextSeq runs were prepared using NextSeq 500/550 300 Cycle High Output v2 (FC-404-2004), loaded at approximately 3.9 pM. All runs were configured to obtain 149 nucleotide paired-end read lengths.