Extraction of high-molecular-weight DNA from Streptococcus spp. for long-read sequencing in resource-limited settings

MinION is an affordable and portable device suitable for whole genome sequencing in resource-limited settings (RLS), which is ideal for diagnostics and control of S. iniae and S. agalactiae disease outbreaks in low-middle income countries (LMIC) finfish aquaculture. However, this requires a DNA extraction method which can recover high-molecular weight (HMW) DNA from species of gram-positive bacteria that are recalcitrant to lysis and is appropriate for use in RLS/LMIC, i.e. uses non-hazardous, cheap reagents and equipment. Here we established an efficient and simple protocol that satisfies these criteria and verified its suitability for MinION sequencing of diverse fish-pathogenic streptococcal strains with the rapid barcoding library. The value of HMW DNA extraction coupled with long read sequencing was demonstrated by the recovery of a novel plasmid in one of the S. iniae genome assemblies, which we named pSI1 to reflect that it is the first published report of natural plasmids in this species.