Culture-free whole genome sequencing of <i>Mycobacterium tuberculosis</i> using ligand-mediated bead enrichment method

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 from clinical samples due to low number of bacilli against a high background. Methods We prospectively collected 34 samples (sputum, n=17; bronchoalveolar lavage, BAL, 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% (IQR = 7.9-39.3). There was a positive correlation between load of bacilli on smears and genome coverage (p-value &lt; 0.001). We detected 58 genes listed in the WHO 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 5/34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin which is not covered by Xpert MTB/RIF. This approach also allowed us to identify mixed infections in eight samples (BAL=4/8, pus=2/3 and sputum= 2/10) including samples that were infected with three or more different strains of Mtb . 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.