HIV-1 Drug Resistance Assay Using Ion Torrent Next Generation Sequencing and On-Instrument End-to-End Analysis...

Area of Science:

• Medical Diagnostics
• Molecular Biology
• Virology

Background:

• Antiretroviral therapy management for HIV-1 relies on sequencing the pol gene.
• Sanger sequencing, the current standard, has limitations in detecting minor resistance variants.
• Next-generation sequencing (NGS) offers enhanced sensitivity for detecting low-frequency variants, enabling earlier intervention.

Purpose of the Study:

• To develop and validate a comprehensive NGS protocol and analysis pipeline for HIV-1 drug resistance testing.
• To simplify the implementation of NGS in clinical laboratories by addressing assay design and data analysis challenges.
• To compare the performance of the developed NGS assay against established Sanger sequencing methods.

Main Methods:

• Development of an end-to-end NGS protocol using AmpliSeq multiplex PCR and Ion Torrent S5 XL sequencing.
• Integration of Stanford's HIVdb resistance algorithm into a Torrent Suite software plugin for automated analysis.
• Validation using archived samples, comparing NGS results with ViroSeq Sanger sequencing data.

Main Results:

• The NGS assay demonstrated high sensitivity (98.2%) and specificity (99.0%) with an optimal variant frequency threshold of 10%.
• 91% of drug resistance mutations were detected by both NGS and Sanger sequencing, with NGS identifying additional variants.
• The assay showed high reproducibility, no cross-reactivity with other viruses, and a limit of detection of 500 copies/mL.

Conclusions:

• The developed NGS assay provides a sensitive, specific, and reproducible method for HIV-1 drug resistance testing.
• The integrated analysis pipeline simplifies implementation in clinical settings, requiring no additional bioinformatics infrastructure.
• This approach facilitates earlier detection of resistance and informed modification of HIV-1 antiretroviral therapy.