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Amplicon Sequencing using the Long-Read Sequencing Technologies
Published on: August 29, 2025
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HIV-1 gag genotypic resistance testing using short-read and long-read next-generation sequencing technologies.
Camille Vellas1, Lila Poiteau2, Stéphanie Raymond1
1CHU de Toulouse, Laboratoire de Virologie, Toulouse, France; INSERM UMR1291-CNRS UMR5051-Université Toulouse III, Toulouse Institute for Infectious and Inflammatory Diseases, France.
Summary
Next-generation sequencing methods effectively detect HIV-1 gag mutations for monitoring lenacapavir resistance. Both short-read and long-read sequencing show high concordance in people living with HIV-1.
Area of Science:
- Virology
- Genomics
- Antiretroviral Therapy
Background:
- Lenacapavir is a novel HIV-1 capsid inhibitor approved for multidrug-resistant infections and pre-exposure prophylaxis.
- Accurate sequencing of HIV-1 gag sequences and resistance-associated mutations (RAMs) is crucial for managing lenacapavir treatment in people living with HIV-1 (PLWH).
Purpose of the Study:
- To compare short-read and long-read next-generation sequencing (NGS) methods for HIV-1 gag genotyping.
- To assess the efficacy of these methods in detecting RAMs relevant to lenacapavir resistance.
Main Methods:
- Comparison of DeepChek® gag HIV-1 (short-read) and PacBio gag SMRT (long-read) NGS platforms.
- Analysis of HIV-1 gag sequences from blood samples (plasma HIV-1 RNA and cell HIV-1 DNA) of 47 PLWH.
Main Results:
- Both NGS methods demonstrated robust performance for sequencing plasma HIV-1 RNA.
- Low viral load in cell HIV-1 DNA samples posed a sequencing challenge for both approaches.
- High nucleotide concordance (99.2%–100%) was observed between the methods, with concordant interpretation of lenacapavir resistance mutations in most samples.
Conclusions:
- Both short-read and long-read NGS are effective for HIV-1 gag genotyping in clinical settings.
- These methods support monitoring resistance to HIV-1 capsid inhibitors like lenacapavir.
- Minor differences in consensus sequences were noted between the sequencing platforms.
