Phylogenetic internal control for HIV-1 genotypic antiretroviral testing

Area of Science:

• Molecular Biology
• Virology
• Bioinformatics

Background:

• Genotypic testing for viral infections involves multiple critical steps requiring rigorous quality control.
• Established internal controls for RNA purification, RT-PCR, DNA sequencing, and analysis are essential.
• An additional phylogenetic quality control step can enhance the reliability of genotypic testing.

Purpose of the Study:

• To introduce and validate a phylogenetic quality control method for genotypic testing.
• To assess the utility of phylogenetic analysis in preventing sample mix-ups.
• To confirm the accurate assignment of viral sequences to individual patients, even with high divergence.

Main Methods:

• Routine comparison of new viral sequences with previous samples from the same patient using multiple alignment.
• Construction of neighbor-joining trees with the Kimura two-parameter method.
• Analysis of reverse transcriptase and protease gene sequences from 30 wild-type viral samples and longitudinal samples from 5 patients.

Main Results:

• Nucleotide sequence analysis demonstrated a mean inter-sample divergence of 2.9% in wild-type sequences.
• All sequence pairs from individual patients consistently clustered together in trees based on nucleotide sequences.
• Amino acid sequence-based trees did not always reliably cluster sequences from the same patient.

Conclusions:

• Phylogenetic analysis of nucleic acid sequences is a valuable tool for detecting sample mix-ups in genotypic testing.
• This method reliably confirms sequence origin from individual patients, irrespective of drug resistance mutations.
• Nucleotide sequence-based phylogenetic analysis offers superior accuracy for patient-specific sequence identification compared to amino acid analysis.