Genome sequencing of Treponema denticola: wildtype and motility mutants
Briefly, 1 microgram of T. denticola genomic DNA was fragmented to ~400 bp using a Covaris M220 Focused-ultrasonicator (TrendBio, Australia). A 1 microlitre aliquot of sheared DNA was visualized using a LabChip GX Touch 24 Nucleic Acid Analyzer (PerkinElmer, USA) to ensure a peak fragment size of 400 bp. The DNA was end-repaired (Ion Xpress Plus Fragment Library Kit) and purified (Agencourt AMPure XP Kit, Beckman Coulter). Barcoded adaptors were ligated to the DNA and nick-repaired (Ion Xpress Plus Fragment Library Kit; Ion Xpress Barcode Adapters Kit) and then purified again (Agencourt AMPure XP Kit). The labelled library was then size-selected again using the Pippin Prep DNA Size Selection System (Sage Science), aiming for a target-peak size of ~480 bp collected over a specified range. Following sample purification (Agencourt AMPure XP Kit) the concentration of the unamplified library was determined using qPCR (Ion Library TaqMan Quantitation Kit); libraries were pooled in equimolar amounts of 26 pM.