Dry Panels Supporting External Quality Assessment Programs for Next Generation Sequencing-Based HIV Drug Resistance Testing

Abstract

External quality assessment (EQA) is a keystone element in the validation and implementation of next generation sequencing (NGS)-based HIV drug resistance testing (DRT). Software validation and evaluation is a critical element in NGS EQA programs. While the development, sharing, and adoption of wet lab protocols is coupled with the increasing access to NGS technology worldwide, rendering it easy to produce NGS data for HIV-DRT, bioinformatic data analysis remains a bottleneck for most of the diagnostic laboratories. Several computational tools have been made available, via free or commercial sources, to automate the conversion of raw NGS data into an actionable clinical report. Although different software platforms yield equivalent results when identical raw NGS datasets are analyzed for variations at higher abundance, discrepancies arise when variations at lower frequencies are considered. This implies that validation and performance assessment of the bioinformatics tools applied in NGS HIV-DRT is critical, and the origins of the observed discrepancies should be determined. Well-characterized reference NGS datasets with ground truth on the genotype composition at all examined loci and the exact frequencies of HIV variations they may harbor, so-called dry panels, would be essential in such cases. The strategic design and construction of such panels are challenging but imperative tasks in support of EQA programs for NGS-based HIV-DRT and the validation of relevant bioinformatics tools. Here, we present criteria that can guide the design of such dry panels, which were discussed in the Second International Winnipeg Symposium themed for EQA strategies for NGS HIVDR assays.

Establishment and application of a method of tagged-amplicon deep sequencing for low-abundance drug resistance in HIV-1

“…At present, the optimal threshold for NGS detection of clinical drug resistance mutations is still inconclusive. By strengthening the standardization of NGS operating procedures and data analysis in drug resistance detecting, NGS is expected to become a new standard method for HIV drug resistance genotyping ( Noguera-Julian et al, 2020 ).…”

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

“…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].…”

Microarrays and NGS for Drug Discovery

“…Leprohon et al have reviewed all critical information relevant to drug resistance and to resistance mechanism(s) in Leishmania infections generated from NGS analysis [211]. Furthermore, NGS has been successfully used for testing for HIV-1 drug resistance, although such studies are yet to be standardized [212][213][214][215][216]. Likewise, NGS and pyrosequencing have been used to investigate resistance of the Influenta A virus to baloxavir [217].…”