In-house NGS-Based HIV Genotyping protocol v.01 v1
Year: 2024
Type: preprint
Abstract: This standard operating procedure (SOP) outlines the laboratory workflow for HIV drug resistance (HIVDR) genotyping assays performed on plasma samples. The assay utilizes next-generation sequencing (NGS) technology to identify mutations associated with antiretroviral treatment (ART) resistance and detect HIV-1 subtypes. Key Steps include; Sample Preparation and Processing: RNA Extraction: Viral RNA is extracted from plasma samples using a silica membrane spin column-based RNA extraction method. Amplicon Generation: Target regions of the HIV genome, including the protease, reverse transcriptase, and integrase genes, are amplified using RT-PCR. Library Preparation and Sequencing: Library Preparation: Amplified DNA fragments are prepared for sequencing using Illumina or Oxford Nanopore Technologies (ONT) library preparation kits. Sequencing: Prepared libraries are sequenced on Illumina MiSeq or ONT MinION Mk1C platforms, generating millions of short DNA sequences (FastQ files). Data Analysis and Interpretation: Bioinformatics Pipeline: Sequencing reads are analyzed using an in-house developed bioinformatics pipeline integrating Hydra, Sierra local, and the Stanford University HIV database. Web-based Analysis: Alternatively, sequencing reads can be directly analyzed using the Stanford University HIV database. Results Interpretation: Resistance mutations with ≥20% relative abundance are interpreted for predicted phenotypic resistance, and HIV-1 subtype is determined. Reporting: A comprehensive report summarizing resistance mutations, HIV-1 subtype, and potential treatment implications is generated. This SOP ensures accurate and efficient HIVDR genotyping, supporting informed ART optimization.
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W4404148890