title: Removing the bottleneck in whole genome sequencing of Mycobacterium tuberculosis for rapid drug resistance analysis: a call to action.
abstract: Whole genome sequencing (WGS) can provide a comprehensive analysis of Mycobacterium tuberculosis mutations that cause resistance to anti-tuberculosis drugs. With the deployment of bench-top sequencers and rapid analytical software, WGS is poised to become a useful tool to guide treatment. However, direct sequencing from clinical specimens to provide a full drug resistance profile remains a serious challenge. This article reviews current practices for extracting M. tuberculosis DNA and possible solutions for sampling sputum. Techniques under consideration include enzymatic digestion, physical disruption, chemical degradation, detergent solubilization, solvent extraction, ligand-coated magnetic beads, silica columns, and oligonucleotide pull-down baits. Selective amplification of genomic bacterial DNA in sputum prior to WGS may provide a solution, and differential lysis to reduce the levels of contaminating human DNA is also being explored. To remove this bottleneck and accelerate access to WGS for patients with suspected drug-resistant tuberculosis, it is suggested that a coordinated and collaborative approach be taken to more rapidly optimize, compare, and validate methodologies for sequencing from patient samples.
authors.affiliation: Lung Infection and Immunity Unit, Department of Medicine, Division of Pulmonology and UCT Lung Institute, University of Cape Town, Old Main Building, Groote Schuur Hospital, Observatory, Cape Town, South Africa. Electronic address: Ruth.Mcnerney@uct.ac.za.
authors.forename: Ruth
authors.initials: R
authors.lastname: McNerney
authors.affiliation: Faculty of Infectious and Tropical Diseases and Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, UK.
authors.forename: Taane G
authors.initials: TG
authors.lastname: Clark
authors.affiliation: Faculty of Infectious and Tropical Diseases and Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, UK.
authors.forename: Susana
authors.initials: S
authors.lastname: Campino
authors.affiliation: Department of Microbiology, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India.
authors.forename: Camilla
authors.initials: C
authors.lastname: Rodrigues
authors.affiliation: FIND, Campus Biotech, Geneva, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Dolinger
authors.affiliation: Lung Infection and Immunity Unit, Department of Medicine, Division of Pulmonology and UCT Lung Institute, University of Cape Town, Old Main Building, Groote Schuur Hospital, Observatory, Cape Town, South Africa.
authors.forename: Liezel
authors.initials: L
authors.lastname: Smith
authors.affiliation: Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Cabibbe
authors.affiliation: Lung Infection and Immunity Unit, Department of Medicine, Division of Pulmonology and UCT Lung Institute, University of Cape Town, Old Main Building, Groote Schuur Hospital, Observatory, Cape Town, South Africa.
authors.forename: Keertan
authors.initials: K
authors.lastname: Dheda
authors.affiliation: Critical Path Institute, Tucson, Arizona, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Schito
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D060005:Genotyping Techniques; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods; D013183:Sputum / Q000382:microbiology; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000188:drug therapy / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial
keywords: Clinical isolate; Drug resistance; Next-generation sequencing; Sputum; Tuberculosis