Abstract
Next generation sequencing (NGS) is a trending new standard for genotypic HIV-1 drug resistance (HIVDR) testing. Many NGS HIVDR data analysis pipelines have been independently developed, each with variable outputs and data management protocols. Standardization of such analytical methods and comparison of available pipelines are lacking, yet may impact subsequent HIVDR interpretation and other downstream applications. Here we compared the performance of five NGS HIVDR pipelines using proficiency panel samples from NIAID Virology Quality Assurance (VQA) program. Ten VQA panel specimens were genotyped by each of six international laboratories using their own in-house NGS assays. Raw NGS data were then processed using each of the five different pipelines including HyDRA, MiCall, PASeq, Hivmmer and DEEPGEN. All pipelines detected amino acid variants (AAVs) at full range of frequencies (1~100%) and demonstrated good linearity as compared to the reference frequency values. While the sensitivity in detecting low abundance AAVs, with frequencies between 1~20%, is less a concern for all pipelines, their specificity dramatically decreased at AAV frequencies <2%, suggesting that 2% threshold may be a more reliable reporting threshold for ensured specificity in AAV calling and reporting. More variations were observed among the pipelines when low abundance AAVs are concerned, likely due to differences in their NGS read quality control strategies. Findings from this study highlight the need for standardized strategies for NGS HIVDR data analysis, especially for the detection of minority HIVDR variants.
Highlights
Generation sequencing (NGS) is a trending new standard for genotypic HIV-1 drug resistance (HIVDR) testing
The six clinical laboratories that participated in this study included the National HIV and Retrovirology Laboratory (NHRL) at JC Wilt Infectious Disease Center, Winnipeg, Canada; BC Center for Excellence in HIV/AIDS (BC-CfE), Vancouver, Canada; Division of Infectious Diseases, Brown University (BU), Alpert Medical School, Providence, USA; IrsiCaixa AIDS Research Institute, Badalona, Spain; Center for Research in Infectious Diseases (CIENI), National Institute of Respiratory Diseases, Mexico City, Mexico; and Departments of Pathology and Medicine, Case Western Reserve University (CWRU), Cleveland, USA
The assessment of all amino acid variants (AAVs) versus HIV drug resistant mutations (DRMs) allowed for a much larger data set, and better coverage of variations at various frequency ranges with different genetic context
Summary
Generation sequencing (NGS) is a trending new standard for genotypic HIV-1 drug resistance (HIVDR) testing. Several recommendations emerged from this meeting covering standards and best practices for (1) NGS read quality control (QC)/quality assurance (QA); (2) NGS read alignment and reference mapping; (3) HIV variant calling and variant QC; (4) NGS HIVDR interpretation and reporting; and (5) general analysis data management[40] Such recommendations remain to be fully implemented in the currently available pipelines and those to be developed. To determine whether the NGS-based HIVDR data analysis pipelines are concordant, we compared the performance of five commonly-applied NGS HIVDR pipelines including, HyDRA25, MiCall[38], PASeq.[36], Hivmmer[39] and DEEPGEN37 (see Methods) for HIV amino acid variant (AAV) detection and quantification. All five pipelines successfully processed NGS data; differences in reporting AAV frequencies, especially when they occur at low frequencies support the need to standardize the processing steps in the pipelines, in the area of quality control criteria
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