Culture-free whole genome sequencing ofMycobacterium tuberculosisusing ligand-mediated bead enrichment method

Abstract

ABSTRACTBackgroundDirect whole genome sequencing (WGS) ofMycobacterium 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.MethodsWe 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 enrichMtbfrom clinical samples and performed WGS on Illumina platform.ResultsMtbwas 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 < 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 therpoBS441A 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 ofMtb.ConclusionsWe demonstrate the feasibility of magnetic bead-based enrichment for culture-free WGS ofMtbfrom clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection ofMtband drug resistance.