Development and Comprehensive Evaluation of Culture-Independent, Long Amplicon-Based Targeted Next-Generation...

Area of Science:

• Genomics and Molecular Diagnostics
• Infectious Disease Research
• Antimicrobial Resistance (AMR) Studies

Background:

• Diverse antimicrobial resistance (AMR) profiles in tuberculosis (TB) necessitate advanced detection methods.
• Existing diagnostic methods may have limitations in comprehensively identifying AMR across multiple drugs.

Purpose of the Study:

• To develop and optimize a culture-independent, long amplicon-based targeted next-generation sequencing (tNGS) method.
• To predict AMR for 16 drugs within the Mycobacterium tuberculosis complex (MTBC).
• To evaluate the performance and clinical utility of the optimized tNGS method.

Main Methods:

• Utilized multiplex PCR to enrich 20 gene regions from MTBC.
• Sequencing performed on Oxford Nanopore Technologies (ONT) and Illumina platforms.
• Developed customized bioinformatics pipelines for data analysis and reporting; optimized ONT using Q20+ chemistry and R10.4.1 flow cell.

Main Results:

• The optimized ONT tNGS method requires only 15 high-quality reads per target gene for accurate variant identification.
• Achieved a rapid turnaround time of 4 hours and 50 minutes.
• Demonstrated 98.35% concordance with phenotypic drug susceptibility testing (pDST) and consistency with Xpert MTB/RIF assays; effectively identified Mycobacterium species with high resistance to clinical pathogen interference.

Conclusions:

• The optimized ONT tNGS method provides a rapid, accurate, and comprehensive approach for predicting AMR in TB.
• This culture-independent method simplifies primer design and is compatible with various sequencing platforms.
• The tNGS approach significantly improves upon previous methods in terms of speed, accuracy, and mutation coverage for AMR detection.