Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of
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RESEARCH NOTE
Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis Wai Sing Chan1, Chun Hang Au1, Yvonne Chung1, Henry Chi Ming Leung2,3, Dona N. Ho1, Elaine Yue Ling Wong1, Tak Wah Lam2,3, Tsun Leung Chan1, Edmond Shiu Kwan Ma1 and Bone Siu Fai Tang1*
Abstract Objective: We designed and tested a Nanopore sequencing panel for direct tuberculosis drug resistance profiling. The panel targeted 10 resistance-associated loci. We assessed the feasibility of amplifying and sequencing these loci from 23 clinical specimens with low bacillary burden. Results: At least 8 loci were successfully amplified from the majority for predicting first- and second-line drug resistance (14/23, 60.87%), and the 12 specimens yielding all 10 targets were sequenced with Nanopore MinION and Illumina MiSeq. MinION sequencing data was corrected by Nanopolish and recurrent variants were filtered. A total of 67,082 bases across all consensus sequences were analyzed, with 67,019 bases called by both MinION and MiSeq as wildtype. For the 41 single nucleotide variants (SNVs) called by MiSeq with 100% variant allelic frequency (VAF), 39 (95.1%) were called by MinION. For the 22 mixed bases called by MiSeq, a SNV with the highest VAF (70%) was called by MinION. With short assay time, reasonable reagent cost as well as continuously improving sequencing chemistry and signal correction pipelines, this Nanopore method can be a viable option for direct tuberculosis drug resistance profiling in the near future. Keywords: Antibiotic resistance, Illumina MiSeq, MDR-TB, Nanopore MinION, NGS, Tuberculosis, XDR-TB, Xpert MTB/ RIF Introduction The increasing threat of tuberculosis (TB) drug resistance highlights the importance of prompt drug susceptibility test (DST) results for better patient care and infection control [1, 2]. Nevertheless, culture-dependent methods cannot provide a quick answer due to the fastidious nature of Mycobacterium tuberculosis (MTB). From literature, 24–61% of pulmonary TB cases were *Correspondence: [email protected] 1 Department of Pathology, Hong Kong Sanatorium & Hospital, Hong Kong, China Full list of author information is available at the end of the article
acid-fast bacilli (AFB) smear-negative [3, 4], with smearnegative, culture-positive TB accounting for 13% of TB transmission [5]. Novel diagnostic tools are needed for rapid detection of drug resistance from the technically demanding smear-negative specimens. Recent advent of next-generation sequencing (NGS) has facilitated comprehensive evaluation of MTB genome for drug resistance prediction [6]. Among various options in NGS market, Nanopore sequencers are ideal for infectious disease diagnosis, which requires short sampleto-answer time (Fig. 1). Despite its inferior sequencing accuracy [7], several groups utilized Nanopore MinION and successfully identified single nucleotide variants
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