Comparison of Four Different DNA Isolation Methods from MGIT Culture for Long-Read Whole Genome Sequencing of Mycobacterium tuberculosis

Background: Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapiddiagnosis. Next-generation sequencing (NGS), especially long-read whole genome sequencing (WGS), shows promise for rapidlydetecting TB and drug resistance, but it requires high-quality DNA, which is difficult to extract from Mycobacterium tuberculosisdue to its complex cell wall. Objectives: This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis, aiming tostandardize protocols for long-read WGS. Methods:Mycobacterium tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared asthe initial material for the DNA extraction protocol: Pellets from 1 mL McFarland 2 suspensions and all growing colonies fromtwo MGIT liquid cultures. Four DNA extraction methods were used: The cetyltrimethylammonium bromide (CTAB) method,GeneJET Genomic DNA Purification Kit, Quick-DNA Fecal/Soil Microbe Kit, and Genematrix Tissue/Bacterial DNA Purification Kit,with some modifications. DNA quality was assessed based on concentration, purity, and integrity. Results: Among the tested methods, the Quick-DNA Fecal/Soil Kit yielded approximately 85 ng/mL of DNA and a purity of 1.9 at260/280 nm from the colonial pellet of two MGIT tubes. However, lower intact DNA [DNA integrity number (DIN) similar to 6.8] wasobtained with this kit. The CTAB method provided the highest intact DNA (DIN similar to 9.5), although the purity of the DNA was notsufficient. Conclusions: Based on three repetitions of McF-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil Kit yielded thehighest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments.A pellet from two MGIT cultures (similar to 100 mu L) is suitable for long-read WGS with this kit. However, a larger sample size is required togeneralize these findings. For effective long-read sequencing of M. tuberculosis, DNA extraction protocols must be optimized tobalance yield, fragment size, and purity for accurate sequencing and drug resistance analysis.