Amplicon Sequencing using the Long-Read Sequencing Technologies

The World Health Organization (WHO) continues to emphasize the urgent need for a rapid, cost-effective, and user-friendly diagnostic method for tuberculosis (TB) and drug-resistant TB (DR-TB). Next-generation sequencing (NGS) technologies, endorsed by the WHO, have significantly improved the detection of DR-TB. Among these, targeted NGS (tNGS) enables focused detection of genetic mutations associated with drug resistance, eliminating the need for traditional culture-based diagnostics. One widely used tNGS assay provides rapid and comprehensive drug susceptibility testing but has been primarily optimized for sequencing platforms with high accuracy. However, the high cost of these sequencing systems has limited accessibility in low- and middle-income countries, particularly across Africa. Portable sequencing technologies present a promising alternative, offering flexibility and reduced infrastructure requirements. In this study, DNA was extracted from rifampicin-resistant TB (RR-TB) isolates, amplified using a tNGS assay, and sequenced on a portable sequencing platform. The same amplification products were also sequenced on a high-accuracy short-read sequencing platform to serve as a reference. Data from the portable sequencer were processed using a bioinformatics pipeline designed for long-read sequencing, while short-read sequencing data were analyzed using an established web-based application. The analysis showed that the long-read sequencing approach successfully identified high-frequency resistance-associated variants detected by short-read sequencing but exhibited limitations in detecting low-frequency variants.