The impact of next-gen sequencing on tracking SARS-CoV-2 variants

The evolution of SARS-CoV-2 has been unpredictable. Factors such as inequitable global vaccine distribution, long-haul COVID-19 among immunocompromised patients, and possible transmission between humans and other mammals, have contributed to the rapid increase in the number of mutations. The continued emergence and spread of new variants reinforce the critical role that genomic sequencing has in the enhanced surveillance of COVID-19.

To keep pace with an ever-evolving virus, efficient identification, and characterization in near real-time is critical. There are several ways to achieve this objective. PCR-based methods are efficient and sensitive but are limited in their ability to detect novel disease agents or aggressive mutations of known pathogens. Additionally, it only offers a binary, presence or absence result.

Next-generation sequencing (NGS) is a promising technology that overcomes these limitations. It is paramount to employ rapid and reliable genomic surveillance tools that track viral evolution. Through sequencing, specifically whole genome sequencing (WGS), researchers can determine the novel cause of infection and characterize the pathogen’s genetic material to better understand pathogenic virulence, transmission, susceptibility to antiviral agents, resistance to treatment, or vaccine targets aimed at curbing the pandemic.

Unfortunately, NGS methods have traditionally been labor-intensive, expensive, and time-consuming, with sequencers taking several days to generate data along with a significant amount of bioinformatics analysis to interpret meaningful results. This is far too long to have any meaningful impact beyond academic utilization and does not achieve the goal of reducing the immediate spread of infection. While these barriers dissuade many laboratories from adopting sequencing technology, there are examples of laboratories incorporating novel approaches to NGS-methods and adapting them for a rapidly changing landscape for infectious disease detection and public health intervention. The Nebraska State Public Health Laboratory (NPHL) is located on the University of Nebraska Medical Campus (UNMC) and is supported by both UNMC as well as the Nebraska Department of Health and Human Services. As the Assistant Director at NPHL, we knew early-on in the pandemic that, even though sequencing would be time-consuming, it would be integral to monitoring the virus.

At the start of the pandemic, NPHL was approached by a California based start-up, Clear Labs, that developed an automated solution for sequencing. NPHL piloted the system and was able to increase sequencing by three-fold to approximately 320 samples per week with minimal training, removing the requirement for staff to have expertise in sequencing. The Clear Dx WGS SARS-CoV-2 assay (RUO)* can process extracted RNA for up to 32 clinical samples in one run with a sample to result within 20 hours. The platform does this by automating PCR, library preparation, sequencing, and bioinformatic consensus genome creation (see Figure 1). The adoption of the Clear Labs Clear Dx SARS-CoV-2 WGS platform, and its turnkey approach, is helping NPHL better serve the residents of Nebraska and ensure that the Nebraska public health system has access to innovative technology that can help identify and manage future health events. Having an automated system builds laboratory capacity because staff and resources can be re-deployed to other testing needs to respond efficiently and effectively during the pandemic. The NPHL has been able to manage the sequencing workload of the entire State with 5 staff members due to the minimal hands-on time needed to set up a run (20-30 minutes) and the automated bioinformatics pipeline that allows staff to quickly analyze the data for a fast turn-around time for results

In addition to sequencing being able to provide information about disease transmission, it was important for public health officials to understand the distribution in the community. NPHL can track the rise and introduction of new variants such as Omicron through information gained from viral sequencing to implement appropriate public health countermeasures for an impacted area or population.

In the Fall of 2020, a novel mutation was detected and clustered to a group of elderly patients who lived in a nursing home. The patients had no symptoms, however, had high viral levels detected suggesting there may be an increase in transmissibility but decrease in virulence. NPHL researchers discovered that a specific mutation in the spike protein was impacting protein structure, conferring decreased virulence. Discoveries like this allow the scientific community to develop a more in-depth understanding of mutations that may have an impact on viral infectivity, pathogenicity, and transmission. Thus, NPHL continues to request and sequence samples across the state to actively monitor and track SARS-CoV-2 as part of pandemic preparedness.

With the onset of the SARS-CoV-2 pandemic, Clear Labs quickly added clinical sequencing to expand beyond its core business of food safety testing to help with the fight against the COVID-19 pandemic. It used its automated platform as the framework for development of two NGS assays, a SARS-CoV-2 diagnostic/strain surveillance assay, and a SARS-CoV-2 whole genome sequencing (WGS) assay. Currently, more than 50% of state public health laboratories (PHLs) use the Clear Dx platform for WGS. The assays have significantly increased the PHLs SARS-CoV-2 sequencing efforts and have aided in the identification and surveillance of existing and emerging strains, such as the Omicron sub lineage, BA.2. Both assays are fully automated and use integrated bioinformatic pipelines to generate high quality consensus genomes.

The Clear Dx platform is custom-built with a PCR instrument and sequencers housed within a robot that automates pipetting, minimizing the requirement for manual pipetting by staff. Once sequences are generated, the data is analyzed through a bioinformatics pipeline that produces raw sequencing data (FASTQ) and high quality assemblies (FASTA) that can be exported for further analysis or to public databases such as the Global Initiative for Sharing Avian Influenza Data (GISAID) and National Center for Biotechnology Information (NCBI), allowing for data sharing with the national and international scientific community in near real time.

There are numerous benefits associated with adopting an automated system:

  1. Minimal personnel time required for sequencing set up and analysis, freeing up staff to perform other required tasks in the laboratory
  2. Ease in set up and training so that laboratory staff with varying expertise and training have the ability to run the instrument, resulting in minimal disruption in laboratory operations and testing
  3. Fast turn-around time (<20 hours) from starting the instrument to obtaining a result, resulting in a faster response to outbreak investigations, variant tracking, and surveillance
  4. Increasing the testing volume to meet the demands of the pandemic
  5. Reduction in user errors to ensure accurate and consistent results

The clinical community has embraced automation to enhance laboratory capacity and workflow efficiencies. Automation of sequencing will allow for all laboratories to contribute to generating data with regards to pathogens that are seen regularly in the clinical laboratory, such as multi-drug resistant organisms, organisms associated with Hospital Acquired Infections (HAI), and many more. Utilization of NGS on a routine basis will enable collection of data that can help to expand development of more precise clinical diagnostics, new antibiotic formulations, vaccines, etc. to tackle these organisms that continue to affect the health of our communities.

As we are envisioning a future with increased availability of automated solutions, the complement to automating sequencing will be to automate technology-enhanced analytics that have the capability to analyze data in a standardized and accessible fashion. Automation of genomic data analysis can remove the burden of manual analysis to enable faster identification of outbreak clusters; clearer understanding of the course of spread of a pathogen through a hospital or community; predicting the evolution of the pathogen as a form of risk modeling for public health policy decision-making; and many more benefits. In addition, cloud-connected systems can enable near real-time data extraction and insights, minimizing the lag time that currently exists for data consolidation across states. To have an effective national and international pathogen surveillance system, it is important to recognize and overcome the persisting challenges with multi-jurisdictional data stewardship, data privacy protection, and data sharing..

Academic, clinical, and public health laboratories have been integral in the pandemic response through their sequencing efforts. Whole genome sequencing has been an important tool for obtaining insight about SARS-CoV-2 that assisted public health officials design targeted strategies to help minimize the spread and curb the reach of the virus across all communities. The utilization of NGS for SARS-CoV-2 surveillance has been a great asset for the public health and scientific communities. Prior to COVID-19, sequencing was conducted for special projects, and in a limited capacity, due to cost and the extensive time requirement for set up. The pandemic has introduced the necessity for performing sequencing more frequently and for more pathogens. Democratizing genomics through lowering costs, automation, integrated bioinformatics, and a curated public database, paves the way for the establishment of a critical genomic infrastructure that can accelerate rapid data exchange and deliver near real-time pathogen insights to better prepare for future pandemic that may arise. 

Ramin Khaksar, Chief Scientific Officer of Clear Labs, a supplier of fully automated, next-generation sequencing (NGS) for genomic surveillance and diagnostics

Baha Abdalhamid, MD, PhD, D (ABMM), Assistant Professor, Department of Pathology and Microbiology at the University of Nebraska Medical Center