Culture-Free Whole Genome Sequencing of <i>Mycobacterium tuberculosis</i> Using Ligand-Mediated Bead Enrichment Method

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 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.