Comparison of Four Different DNA Isolation Methods from MGIT Culture for Long-Read Whole Genome Sequencing of <i>Mycobacterium tuberculosis</i>
| Author | Nazlı Arslan | en |
| Author | Ebru Demiray-Gurbuz | en |
| Author | Nuri Ozkutuk | en |
| Author | Nuran Esen | en |
| Author | Ayşe Aydan Özkütük | en |
| Orcid | Nazlı Arslan [0000-0002-5782-9288] | en |
| Orcid | Ebru Demiray-Gurbuz [0000-0003-2849-9029] | en |
| Orcid | Nuri Ozkutuk [0000-0002-2848-5914] | en |
| Orcid | Nuran Esen [0000-0001-9796-3003] | en |
| Orcid | Ayşe Aydan Özkütük [0000-0002-1710-2287] | en |
| Issued Date | 2024-09-30 | en |
| Abstract | Background: Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapid diagnosis. Next-generation sequencing (NGS), especially long-read whole genome sequencing (WGS), shows promise for rapidly detecting TB and drug resistance, but it requires high-quality DNA, which is difficult to extract from Mycobacterium tuberculosis due to its complex cell wall. Objectives: This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis, aiming to standardize protocols for long-read WGS. Methods: Mycobacterium tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared as the initial material for the DNA extraction protocol: Pellets from 1 mL McFarland 2 suspensions and all growing colonies from two 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 at 260/280 nm from the colonial pellet of two MGIT tubes. However, lower intact DNA [DNA integrity number (DIN) ~ 6.8] was obtained with this kit. The CTAB method provided the highest intact DNA (DIN ~ 9.5), although the purity of the DNA was not sufficient. Conclusions: Based on three repetitions of McF-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil Kit yielded the highest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments. A pellet from two MGIT cultures (~ 100 µL) is suitable for long-read WGS with this kit. However, a larger sample size is required to generalize these findings. For effective long-read sequencing of M. tuberculosis, DNA extraction protocols must be optimized to balance yield, fragment size, and purity for accurate sequencing and drug resistance analysis. | en |
| DOI | https://doi.org/10.5812/jjm-148070 | en |
| Keyword | <i>Mycobacterium tuberculosis</i> | en |
| Keyword | Whole Genome Sequencing | en |
| Keyword | Next Generation Sequencing | en |
| Keyword | Nanopore Sequencing | en |
| Keyword | DNA | en |
| Keyword | Isolation and Purification | en |
| Publisher | Brieflands | en |
| Title | Comparison of Four Different DNA Isolation Methods from MGIT Culture for Long-Read Whole Genome Sequencing of <i>Mycobacterium tuberculosis</i> | en |
| Type | Research Article | en |