Culture-Free Whole Genome Sequencing of Mycobacterium tuberculosis Using Ligand-Mediated Bead Enrichment Method

• Published in: Open forum infectious diseases 2024-07, Vol.11 (7), p.ofae320
• Main Authors: Vasanthaiah, Shruthi, Verma, Renu, Kumar, Ajay, Bandari, Aravind K, George, John, Rastogi, Mona, Manjunath, Gowrang Kasaba, Sharma, Jyoti, Kumar, Abhishek, Subramani, Janavi, Chawla, Kiran, Pandey, Akhilesh
• Format: Article
• Language: English
• Subjects: Diagnostic Methods and Tools
• ISSN: 2328-8957; 2328-8957
• DOI: 10.1093/ofid/ofae320

Abstract Background Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background. Methods We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform. Results Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%–28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%–94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF. Conclusions We demonstrate the feasibility of magnetic bead–based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance. Graphical Abstract Graphical Abstract This graphical abstract is also available at Tidbit: https://tidbitapp.io/tidbits/culture-free-whole-genome-sequencing-of-mycobacterium-tuberculosis-using-ligand-mediated-bead-enrichment-method