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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
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HIV-1 genotypic resistance testing using single molecule real-time sequencing.

Stéphanie Raymond1, Nicolas Jeanne2, Camille Vellas1

  • 1INSERM UMR1291 - CNRS UMR 5051 - Université Toulouse III, Toulouse Institute for Infectious and Inflammatory Diseases, Toulouse, France; CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, France.

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology
|July 28, 2024
PubMed
Summary

Long-Read Single Molecule Real-time (SMRT) sequencing shows promise for HIV-1 resistance testing, accurately detecting mutations in both RNA and DNA samples. This advanced method aids in characterizing genotypic resistance and haplotyping for improved patient management.

Keywords:
HIV-1 polymeraseHaplotypesLong readsNext-generation sequencing

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Area of Science:

  • Virology
  • Genomics
  • Molecular Diagnostics

Background:

  • HIV-1 resistance testing is crucial for effective clinical management.
  • Next-generation sequencing (NGS) methods are increasingly available in clinical virology laboratories.

Purpose of the Study:

  • To evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing for HIV-1 polymerase genotyping.
  • To assess SMRT sequencing's utility in characterizing antiretroviral resistance.

Main Methods:

  • Analyzed 111 prospective clinical samples (plasma and leukocyte-enriched blood) using Sanger sequencing, Vela NGS, and SMRT sequencing.
  • Developed a SMRT sequencing protocol and bioinformatics pipeline for resistance inference.
  • Employed both haplotype and variant calling approaches for data analysis.

Main Results:

  • Successful sequencing of HIV-1 polymerase achieved in 98% of plasma RNA samples (viral load >4 log copies/mL).
  • SMRT sequencing detected 98% of resistance-associated mutations (RAMs) identified by Sanger sequencing.
  • Quantification of RAMs by SMRT and Vela NGS showed strong correlation (Spearman's ρ = 0.82).

Conclusions:

  • SMRT sequencing is a performant method for HIV-1 genotypic resistance characterization in both RNA and DNA clinical samples.
  • Long-read sequencing offers a novel tool for HIV-1 mutation haplotyping and resistance analysis.