Confirming Multiplex RT-qPCR Use in COVID-19 with Next-Generation Sequencing: Strategies for Epidemiological Advantage

Abstract

Rapid identification and tracking of emerging SARS-CoV-2 variants are critical for understanding the transmission dynamics and developing strategies for interrupting the transmission chain. Next-Generation Sequencing (NGS) is an exceptional tool for whole-genome analysis and deciphering new mutations. The technique has been instrumental in identifying the variants of concern (VOC) and tracking this pandemic. However, NGS is complex and expensive for large-scale adoption, and epidemiological monitoring with NGS alone could be unattainable in limited-resource settings. In this study, we explored the application of RT-qPCR-based detection of the variant identified by NGS. We analyzed a total of 78 deidentified samples that screened positive for SARS-CoV-2 from two timeframes, August 2020 and July 2021. All 78 samples were classified into WHO lineages by whole-genome sequencing and then compared with two commercially available RT-qPCR assays for spike protein mutation(s). The data showed good concordance between RT-qPCR and NGS analysis for specific SARS-CoV-2 lineages and characteristic mutations. RT-qPCR assays are quick and cost-effective and thus can be implemented in synergy with NGS for screening NGS-identified mutations of SARS-CoV-2 for clinical and epidemiological interest. Strategic use of NGS and RT-qPCR can offer several COVID-19 epidemiological advantages.

Optimization of the Illumina COVIDSeq™ protocol for decentralized, cost-effective genomic surveillance

“…The lowest sequencing depth of >200x and 90% genome coverage were found adequate for accurate variant detection while validating this assay for clinical testing according to the CAP (College of American Pathologists) guidelines for NGS-based Laboratory Developed Test (LDT) [ 6 ]. Importantly, we sequenced 153 samples using this modified approach, and 152/153 (99.34%) samples (PCR Ct < 30) resulted in the correct variant using DRAGEN COVID Lineage (v3.5.4), and 142/153 (92.81%) samples achieved 90% genome coverage with an average depth of >200x ( Supplementary Table-1 ).…”

“…We have adopted this modified protocol for sequencing 153 genomes from East Texas, USA, and compared the results with PCR-based variant detection [ 8 ]. High accuracy and reproducibility of this approach have been demonstrated in validating the COVIDSeq™ RUO assay for clinical application according to Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) guidelines [ 6 ]. We have only scrutinized this procedure for accurate detection of circulating variants rather than detecting new mutations and variant frequency in the mixed variant population.…”

Association of Diabetes with Meningitis Infection Risks: A Systematic Review and Meta-Analysis

“…Infectious diseases remain a signifcant threat to the wellbeing of people and the economies of countries worldwide [7,[50][51][52][53][54][55]. Our study included 16847 meningitis cases with 7 prospective and 9 retrospective study designs.…”

Deciphering microbiota of acute upper respiratory infections: a comparative analysis of PCR and mNGS methods for lower respiratory trafficking potential

“…Yet from the clinical point of view, recognition of potential pathogenicity is crucial to estimate etiological relevance, to optimize treatment, and understand outbreak conditions. Similarly, the recognition of broader pathogenic microorganisms can be pivotable to draw on for decentralized epidemiological layouts [29]. Assuredly, underdiagnosed upper respiratory (co)infections have potential for trafficking to the lower respiratory region and trigger adverse harm, especially in immunosuppressed hosts [30][31][32][33].…”