Sequencing of Mycoplasma genitalium genomes using a tiling amplicon method on the Nanopore MinION

Mycoplasma genitalium is challenging to work with and new methods are needed to study this bacterium directly in clinical samples. This study designed and validated a proof-of-concept polymerase chain reaction (PCR)-based 'tiling' methodology to sequence M. genitalium genomes. Primers were designed to produce 2.5 kb amplicons covering the 580 kb genome with a minimum overlap of 100 bp. Analysis was performed using the laboratory strain G37 and a clinical isolate. Amplicons were sequenced on the Oxford Nanopore MinION using ligation sequencing. Reads were mapped to a reference to produce a consensus genome. A total of 262 primer pairs were designed and amplification was successful for 99.5% (261/262) of 2.5 kb amplicons, with G37 genome coverage of 99.5% (mean read depth, 1973X). Using larger 5kb amplicons, amplification was successful for 92.4% (121/131) of primer pairs, with a coverage of 92.2% (mean read depth, 223X). When validated on a clinical isolate, 98.3% coverage was achieved (read depth, 443X). In conclusion, this study developed a PCR-based tiling approach to whole genome sequencing of M. genitalium by designing and validating a set of 262 primer pairs.