Extraction of high‐molecular‐weight DNA from Streptococcus spp. for nanopore sequencing in resource‐limited settings

Abstract

The long‐read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource‐limited settings, such as field conditions or low‐ and middle‐income countries. For this purpose, protocols for extracting high‐molecular‐weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram‐negative bacteria. However, we found that without modification, these protocols are unsuitable for gram‐positive Streptococcus spp., a major threat to fish farming and food security in low‐ and middle‐income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high‐molecular‐weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish‐pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high‐molecular‐weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low‐copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource‐limited settings‐adapted protocol for high‐molecular‐weight DNA extraction could be implemented successfully for similarly recalcitrant‐to‐lysis gram‐positive bacteria, and it represents a method of choice for MinION‐based disease diagnostics in low‐ and middle‐income countries.