Qiamp dna mini prep kit

DNA was extracted from treponeme cultures and other bacterial cultures using the QIAmp DNA mini-prep kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions or by heat lysis. The heat lysis protocol for DNA extraction was performed as follows: one ml of culture was centrifuged for 5 min at 9,300 x g and the supernatant was discarded. The remaining pellet or a colony was resuspended in 50 μl of nuclease free water and vortexed vigorously. The suspension was incubated on a dry heat block for 20 minutes at 100°C, and then centrifuged for 10 min at 20,000 x g. The resulting supernatant (DNA template) was transferred to a labeled microcentrifuge tube and stored at -20°C.

Genomic DNA was extracted using a Qiagen QIAmp DNA mini prep kit (Qiagen) with lysis step. DNA was quantified, and quality assessed using the Nanodrop 2000 (ThermoFisher Scientific) Nextera XT libraries were prepared for sequencing using the benchtop Illumina MiSeq sequencer. Paired-end reads (2x300 bp) were generated using the v3-600 reagent cartridge. Using the online bioinformatics platform EDGE (v1.5.1), powered by MRC CLIMB¹ (Connor et al., 2016 (link); Li et al., 2017 (link)), sequenced reads were trimmed (unpaired reads smaller than 50 bp or with less than 5 times coverage were discarded) and assembled using SPAdes (Bankevich et al., 2012 (link)) (v3.9.1). Coding sequences were annotated with prokka (v1.11) and ShortBRED (Kaminski et al., 2015 (link)) was used to search assembled genomes for Antibiotic Resistance genes (CARD database) (Jia et al., 2017 (link)) and for virulence genes from (VFDB) (Chen et al., 2016 (link)).