Development and verification of a novel tiling PCR method for long-range HIV-1 sequencing in a diagnostic setting

Area of Science:

• Virology
• Genetics
• Molecular Biology

Background:

• Genotyping of new Human Immunodeficiency Virus type 1 (HIV-1) infections is crucial for effective antiretroviral therapy.
• Traditional sequencing methods focus on the pol region, limiting comprehensive genomic analysis.
• Next-generation sequencing (NGS) adoption, spurred by the SARS-CoV-2 pandemic, highlights the need for optimized HIV-1 diagnostic sequencing.

Purpose of the Study:

• To design and validate a novel tiling PCR method for routine, robust NGS-based sequencing of longer HIV-1 genome regions.
• To adapt successful tiling PCR strategies from other viral diagnostics for HIV-1 sequencing.
• To improve the efficiency and comprehensiveness of HIV-1 genotyping for clinical diagnostics.

Main Methods:

• Development of a novel set of tiling PCR primers to amplify the 5' half of the HIV-1 genome in six overlapping 1,000 bp segments.
• Optimization of the assay for rapid turnaround from sample to sequencer in under 24 hours.
• Validation using a comparison panel of patient samples to assess sequence generation and amplification of key drug resistance regions.

Main Results:

• The tiling PCR method successfully generated HIV-1 sequences from 100% of samples in the comparison panel.
• Complete protease, reverse transcriptase, and integrase regions were amplified in over 90% of samples with viral loads >5000 copies/mL.
• The novel method identified seven additional drug resistance mutations compared to historical methods, enhancing diagnostic accuracy.

Conclusions:

• The developed designer tiling PCR is a promising and efficient method for routine NGS-based diagnostic sequencing of HIV-1.
• This approach facilitates comprehensive genotyping, aiding in the selection of effective antiretroviral treatments.
• The method's speed and accuracy represent a significant advancement in HIV-1 molecular diagnostics.