Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing

Abstract

The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) established a robust proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. While many of the lessons learned during the development of such programs may also apply to next generation sequencing (NGS)-based HIVDR assays, challenges remain for the ongoing evaluation of NGS-based testing. These challenges include a proper assessment of assay accuracy and the reproducibility of low abundance variant detection, intra- and inter-assay performance comparisons among laboratories using lab-defined tests, and different data analysis pipelines designed for NGS. In collaboration with the World Health Organization (WHO) Global HIVDR Laboratory Network and the Public Health Agency of Canada, the Rush VQA program distributed archived proficiency testing panels to ten laboratories to evaluate internally developed NGS assays. Consensus FASTA files were submitted using 5%, 10%, and 20% variant detection thresholds, and scored based on the same criteria used for SS. This small study showed that the SS External Quality Assurance (EQA) approach can be used as a transitional strategy for using NGS to generate SS-like data and for ongoing performance while using NGS data from the same quality control materials to further evaluate NGS assay performance.

Technologies for HIV-1 drug resistance testing: inventory and needs

“…]. As a transition strategy for the external quality assessment of NGS-based HIV DR assays, it may be useful to use external quality assessment strategies originally implemented for the external quality assessment of HIV DR assays based on Sanger population sequencing [43].…”

“…The external quality assessment of NGS-based HIV DR testing is very demanding and not all requirements have been conclusively defined yet [19 ▪▪ ,33 ▪▪ ,34 ▪▪ ,40,41 ▪▪ ,42 ▪▪ ]. As a transition strategy for the external quality assessment of NGS-based HIV DR assays, it may be useful to use external quality assessment strategies originally implemented for the external quality assessment of HIV DR assays based on Sanger population sequencing [43].…”

Next Generation Sequencing for HIV-1 Drug Resistance Testing—A Special Issue Walkthrough

“…The next group of manuscripts present lessons from the past and how they might impact our way forward when considering NGS for HIV-1 drug resistance testing. Jennings et al [21] discuss challenges in applying experience earned from a Sanger-based EQA strategy within the National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) program towards NGS HIV-1 drug resistance assays, including already started initiatives. Lee et al [22] then present unique requirements and challenges in conducting EQA for NGS-based HIV-1 drug resistance testing, and consider the differences such a program might mandate as compared to the conventional Sanger sequencing approach.…”