Whole Genome Sequencing of ‘Treponema vincentii’ using the MinION

Abstract

Objectives: To sequence a clinical isolate of ‘Treponema vincentii’ using the recently-introduced Oxford Nanopore Single Molecule Real Time (SMRT) sequencing MinION platform. Methods: Axenic culture of an oral spirochete species ‘Treponema vincentii’ (OMZ 861; originally isolated from the human oral cavity by Dr. Chris Wyss), was maintained anaerobically in TYGVS medium. Genomic DNA was extracted using Promega Wizard genomic DNA purification kit. Whole genome sequencing library was prepared using the Nanopore sequencing kit (Oxford Nanopore Technologies) and sequenced in a minION Mk1B device on a R9 flowcell (MIN104) for 48 hours. The reads were basecalled with Metrichor 2D Basecalling RNN. Fastq extraction was done with poretools-0.5.1 and de novo assembled using Canu-1.3. The draft genome was annotated using RASTv2. Results: The sequencing generated a total of 207,172 reads and 776 Million Bases (MB). High quality reads account for a total yield of 321 MB (>100 x coverage) with an average length of 3,800 bases and the longest read of 40,980 bases. Most of the quality reads were generated within the first 24 hours indicating future run for a shorter period of time would provide sufficient coverage. Assembly in Canu resulted in one single contig (2.58 MB) with GC content at 45.73%, which matches those of the reference strains T. vincentii ATCC 35580 (2.51 MB and 45.69%) and F0403 (2.69 MB and 45.5%). Preliminary comparison of the functional systems among all 3 strains revealed high congruence indicating a consistent metabolic functionality of this species. Notably OMZ 861 contained Aminoglycoside N6'-acetyltransferase (EC 2.3.1.82) that was absent in the other two reference strains that may contribute to antibiotic resistance. Conclusions: Nanopore minION sequencing technology was demonstrated quick and powerful approach for whole genome sequencing of oral bacterial isolates. Simple and streamlined sample preparation and short turnaround time ensure contiguous genome assembly. The resultant single contig genome facilitates the characterization and understanding of this species.