The advent of Next generation sequencing (NGS) has provided indisputable advancements in our knowledge and understanding of genomics and continues to equip biomedical science with critical insights and tools for the development of novel in-vitro diagnostics (IVD), vaccines, and drug and gene therapeutics. In addition, the role whole genome sequencing (WGS) plays in providing integral genomic data of infectious pathogens cannot be understated, especially throughout the many phases of the COVID-19 pandemic. However, having routinely used WGS in my lab for tracking SARS-CoV-2 variants of concern (VOC), I do feel it is necessary to consider the limitations and practicality of using WGS as the model screening method for high-throughput analysis.
During the COVID-19 pandemic, the Life Sciences Testing Center (LSTC) ran an average of ~5,500 PCR tests per day, with testing averages spiking up to ~8,000 due to holidays, high positivity rates, and emerging VOCs. Timely reporting of clinical PCR test results took an obvious precedence over sequencing endeavors, as should be the case. However, as positivity rates rose, so too did the pressure of WGS efforts to provide higher throughput SARS-CoV-2 variant analysis of positive specimens. Despite round-the-clock lab operations, we decided in March of 2021 that development of a more economic, sensitive, and higher throughput alternative to sequencing for viral surveillance was necessary.
For decades, TaqMan single nucleotide polymorphism (SNP) genotyping has been used as a scientifically well-established RT-PCR method for distinguishing allelic mutation status and copy number variations (CNV) across a host of diverse genomes (1-2). Recognizing these beneficial characteristics, we selected a series of TaqMan SARS-CoV-2 mutation panel (Thermo Fisher Scientific) SNP assays to construct adaptable “VOC SNP arrays” on 96-well optical plates. We established strict core criteria for variant identification to optimize number of samples per run, without impacting VOC discrimination. SNP arrays were designed using either an eight-assay or twelve-assay layout, with criteria for VOC confirmation requiring at least two defining and unique SNP profiles. To ensure assay layouts maintained their validity, we used data from our WGS runs in addition to open-source bioinformatics data to update SNP assays and VOC profiling.
To date, we have completed six comparability studies between TaqMan SNP genotyping and NGS for SARS-CoV-2 VOC surveillance and analyzed over 2,000 samples from March 2021 to August 2022. Each study sought to compare overall method performance (sensitivity and selectivity) in addition to specific analyses such as, cycle threshold (Ct) limit of detection (LoD) for VOC identification, economic cost-benefit analysis, and high-throughput turn-around-time (TAT). In our most recent study, experimental methodology was designed to compare real-time performance of three sequencing platforms against an eight-assay TaqMan SNP panel on thirty-four Omicron (B.1.1.529) samples to assess discrimination of sublineages harboring a high degree of sequence homology. In prior investigations, we have calculated our eight-assay TaqMan SARS-CoV-2 mutation panel layouts to be >5 to >11 times cheaper per reaction than WGS, regardless of the NGS platform used. In addition, our TaqMan genotyping approach has maintained a sensitivity of 93.33%-98.0%, and specificity of >97.0% with an observed cycle threshold LoD of ≥34, and an average turn-around-time to VOC result of ~8 hours for 96 samples, compared to >4 days for WGS.
On a final note, I would like to clarify/confirm the following: Whole genome sequencing is an absolutely critical technology for genomic surveillance of infectious disease, one which I will continue to utilize in my lab. Having said that, I do believe the allocation of time, funding and resources for alternative, population-scale biosurveillance programs should be diligently considered before deciding high-throughput “brute force” sequencing is the most sensible long-term option.
1. Cantsilieris S, Baird PN, White SJ. Molecular methods for genotyping complex copy number polymorphisms. Genomics. 2013 Feb;101(2):86-93. doi: 10.1016/j.ygeno.2012.10.004. Epub 2012 Oct 30. PMID: 23123317.
2. Ranade K, Chang MS, Ting CT, Pei D, Hsiao CF, Olivier M, Pesich R, Hebert J, Chen YD, Dzau VJ, Curb D, Olshen R, Risch N, Cox DR, Botstein D. High-throughput genotyping with single nucleotide polymorphisms. Genome Res. 2001 Jul;11(7):1262-8. doi: 10.1101/gr.157801. PMID: 11435409; PMCID: PMC311112.