A mycobacterial DNA extraction protocol designed for resource limited settings generates high quality whole genome sequencing

Abstract

AbstractMycobacteria are major global human pathogens and includeMycobacterium tuberculosis, the causative agent of tuberculosis, andM. abscessus, an emerging multidrug resistant pathogen.M. abscessusaffects people with structural lung disease and those who are immunocompromised, most commonly causing pulmonary disease but also disseminated infections in the central nervous system and skin. High quality whole genome sequencing is essential to research mycobacterial epidemiology, pathogenesis and antimicrobial resistance. However, current DNA extraction protocols are time consuming, use toxic chemicals, require cold chain storage for certain reagents and can often result in poor quality, degraded DNA that directly impacts whole genome sequencing outputs. This is a particular challenge in low-income settings.Here, we report a novel optimised DNA extraction workflow forM. tuberculosisandM. abscessusthat invariably generates high quality Illumina short read sequencing data. We evaluated input culture CFU and physical cell disruption times. DNA quantity was determined using a Qubit fluorometer system with DNA integrity assessed using the Agilent TapeStation platform.We showed that this protocol facilitated complete genome assemblies ofM. abscessusandM. tuberculosisreference strains. There is no requirement for cold chain transport or storage of reagents, solvent extractions, or boiling to heat inactivate cultures, and the method does not require surfactant chemicals such as cetyltrimethylammonium bromide (CTAB).