Culture-free whole genome sequencing of Mycobacterium tuberculosis 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 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 < 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement RV is supported by Ramalingaswami Fellowship, Department of Biotechnology (DBT) Govt. of India (ID No. BT/HRD/35/02/2006) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the institutional review board of Kasturba medical college, Manipal Academy of Higher Education (IEC:526/2018). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data supporting the findings of this manuscript have been submitted to NCBI and can be accessed through the SRA submission portal with accession number PRJNA1052384 (https://submit.ncbi.nlm.nih.gov/subs/sra/SUB14001592/overview).