Targeted-Sequencing Workflows for Comprehensive Drug Resistance Profiling of Mycobacterium tuberculosis Cultures Using Two Commercial Sequencing Platforms: Comparison of Analytical and Diagnostic Performance, Turnaround Time, and Cost

Clin Chem. 2020 Jun 1;66(6):809-820. doi: 10.1093/clinchem/hvaa092.

Abstract

Background: The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics.

Methods: A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports.

Results: Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities.

Conclusion: Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis.

Keywords: Mycobacterium tuberculosis; Illumina MiSeq; Nanopore MinION; drug-resistant TB; next-generation sequencing; targeted sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Barcoding, Taxonomic
  • Drug Resistance / genetics*
  • High-Throughput Nucleotide Sequencing / economics
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Multiplex Polymerase Chain Reaction
  • Mycobacterium tuberculosis / classification*
  • Mycobacterium tuberculosis / genetics
  • Sequence Analysis, DNA / economics
  • Sequence Analysis, DNA / methods*
  • Workflow*