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

Abstract

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% (..