Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping

Abstract

Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4–99; reverse transcriptase 38–247). The concordance among laboratories’ sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1–100%), compared to 15% (99.4%, 88.5–100%), 10% (99.2%, 87.4–100%), or 5% (98.5%, 86.4–100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping.

Are We Ready for NGS HIV Drug Resistance Testing? The Second “Winnipeg Consensus” Symposium

“…Compared to SS-based qualitative HIVDR tests, NGS-based quantitative HIVDR assays are far more complex. NGS consensus sequence-based EQA analysis by, direct application of strategies designed for SS methods, oversimplifies the intrinsic complexity of NGS HIVDR data output [ 52 , 53 ].…”

Technical Validation of a Hepatitis C Virus Whole Genome Sequencing Assay for Detection of Genotype and Antiviral Resistance in the Clinical Pathway

“…We surmise that the Sanger assay inaccurately detected this variant to be present at >20% as it estimates variant frequencies from the height of chromatogram peaks whereas the NGS method used by the WGS assay is based on clonal sequencing and quantifies variant frequencies from individual reads which is more accurate. A study comparing the accuracy of Sanger sequencing at 20% variant frequency threshold to NGS at 20% and 15% variant frequency thresholds showed a high level of agreement at 99.6% [96.1–100%; range] and 99.4% [88.5–100%; range], respectively ( Parkin et al, 2020 ). For the second discordant sample, the WGS assay detected the mixture M28MV in the NS5a gene whereas the Sanger assay detected the mixture M28AV.…”

Fact and Fiction about 1%: Next Generation Sequencing and the Detection of Minor Drug Resistant Variants in HIV-1 Populations with and without Unique Molecular Identifiers

“…If you wanted to improve the accuracy of estimating true abundance when trying to detect a variant at 1% then it is necessary to increase the number of observations by increasing the sample size; for example, sequencing 3000 genomes and detecting the variant 30 times allows you to report a variant at 1% abundance with a confidence interval of between 0.7% and 1.4%. We will remind you that every day, people are using NGS to make claims of 1% sensitivity [7,8] without any knowledge of the number of genomes sequenced.…”

“…In the recent WHO validation panel, the true abundance of minor variants was not known (since there is no validated method for their detection). Therefore to assess the various labs the "true" value was taken as the average value obtained by all of the labs [7]. Assuming there were differences in accuracy of the methods, such an approach would have the effect of making the less accurate results seem better and the more accurate results seem worse, probably not the ideal goal of a validation or testing panel.…”