Modified cetyltrimethylammonium bromide DNA extraction for Mycobacterium tuberculosis whole genome sequencing

Efficient extraction of high-quality genomic deoxyribonucleic acid (DNA) from Mycobacterium tuberculosis (MTB) is a critical step for whole genome sequencing (WGS) and downstream molecular applications. However, the lipid-rich MTB cell wall continues to limit DNA recovery, and existing protocols do not consistently address this challenge. This study aimed to refine the cetyltrimethylammonium bromide (CTAB)-based DNA extraction protocol to achieve improved DNA yield and purity for downstream WGS applications. We conducted a cross-sectional laboratory-based protocol refinement study nested within an ongoing MTBC genetic diversity and drug resistance study in western Kenya, using archived Mycobacterium Growth Indicator Tube (MGIT) cultured isolates. The entire MGIT broth from the MTBC-positive isolates was centrifuged before extraction to maximize bacterial biomass recovery, followed by an extended lysozyme incubation to promote enzymatic cell wall disruption. The process combined enzymatic lysis, mechanical disruption, CTAB-based purification, and isopropanol precipitation. Deoxyribonucleic acid concentration and purity were determined by NanoDrop spectrophotometry. A total of 325 MTB isolates were processed. Deoxyribonucleic acid (DNA) concentrations ranged from 5.41 to 2,052.60 ng/μL (median: 65.21 ng/μL), with A260/280 purity ratios between 1.36 and 2.08. 60% of samples achieved DNA concentrations ≥50 ng/μL, and overall, 89% (290/325) of the samples successfully passed sequencing quality control. The majority of the samples were successfully sequenced. The refined CTAB protocol effectively overcomes the challenge posed by the MTB cell wall, yielding high-quality DNA suitable for WGS. It provides an accessible and scalable approach for laboratories in high TB burden and resource-limited settings.