Genome sequencing of Porphyromonas gingivalis W50 PG1879 mutant
Briefly, 1 microgram of P. gingivalis genomic DNA was fragmented to ~400 bp using a Covaris M220 Focused-ultrasonicator (TrendBio, Australia). A 1 microlitre aliquot of sheared DNA was visualised using a LabChip GXII 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), purified (Agencourt AMPure XP Kit, Beckman Coulter), barcoded adaptors ligated to the DNA then nick-repaired (Ion Xpress Plus Fragment Library Kit; Ion Xpress Barcode Adapters Kit) and purified again (Agencourt AMPure XP Kit, Beckman Coulter). The labelled library was then size-selected using the Pippin Prep DNA Size Selection System (Sage Science), aiming for a target-peak size of ~480 bp collected over the specified range of 407 to 543 bp. Following sample purification (Agencourt AMPure XP Kit, Beckman Coulter) the concentration of the unamplified library was determined using qPCR (Ion Library TaqMan Quantitation Kit); 26 pM of library was sequenced.