QIAseq SARS-CoV-2 kit, campaign visual for QIAseq DIRECT Library Kit, two scientists in a laboratory working on computer screens, (2nd version switched)

December 3, 2021 | Genomics

Certainty at a time of uncertainty: An accurate picture of circulating SARS-CoV-2 variants

Mutants, variants and vaccines: The twists and turns in the continually evolving COVID-19 pandemic

Since its emergence globally in early 2020, SARS-CoV-2 has continued to evolve and accumulate new mutations, some of which have conferred phenotypic advantages. The need for increased viral sequencing has exploded ever since the Alpha variant, otherwise known as B1.1.7, was first detected. The Alpha, Beta, Gamma, Delta, Mu and Omicron variants have presented with spike protein mutations that in some cases increase the transmissibility of the virus. The rise of these “Variants of Concern” (VOC)* and “Variants of Interest” (VOI)* has triggered a requirement for fast, accurate and scalable viral sequencing to match global demand.

Innovation at the heart of the public health response

From the start, public health labs were challenged with sequencing increasing numbers of samples and a growing need to ramp up genomic surveillance efforts. Determining the prevalence of circulating variants within the population was critical to enable the timely implementation of effective public health measures. With cases rising worldwide, highly transmissible, emerging variants were changing the trajectory of the pandemic. Faster, more accurate and high-throughput technologies were necessary to accelerate SARS-CoV-2 genomic surveillance efforts.

The hunt for potential new ‘vaccine escape’ mutants

Early reports of reinfection with SARS-CoV-2 meant greater scrutiny into circulating variants was necessary to determine whether specific mutations could enable the virus to evade the immune response (1). This meant a deeper look at variants responsible for ‘breakthrough infections.’ The hunt was on to track and trace potential new ‘vaccine escape’ mutants.

While technologies such as PCR are efficient at detecting existing variants, whole genome sequencing provides an unprecedented level of insight into the viral genome – and helps pinpoint new mutations as they arise. With the development of the QIAseq SARS-CoV-2 Primer Panel in June 2020, QIAGEN provided an optimized ARTIC-based NGS solution for targeted enrichment of the complete viral genome. Compared with other SARS-CoV-2 whole genome sequencing methods, the QIAseq SARS-CoV-2 Primer Panel enables preferential amplification of the SARS-CoV-2 genome by up to 100-fold and ensures high coverage, as shown in a published technology comparison study by Liu et al. (2).

COVID-19 in animals

There have been reports of cats, dogs and even zoo animals testing positive for – and indeed dying of – COVID-19. In June 2020, Danish mink farmers noticed that their animals were showing signs of respiratory illness. It was confirmed that mink had been infected with SARS-CoV-2 from humans. The virus then leaped from the mink back to humans. As a consequence of this compelling observation, the Danish government decided to sequence every positive COVID-19 case in Denmark.

The importance of sequencing all positive cases is that you really have a good picture of what's going on with the pandemic.

Dr. Mette Christiansen, head of the Diagnostic NGS Core Facility, Department of Molecular Medicine (MOMA), Aarhus University Hospital

“We went from having to sequence maybe 50 a week to having to sequence 1000 [samples]”, said Dr. Mette Christiansen, Head of the Diagnostic NGS Core Facility at the Department of Molecular Medicine (MOMA) Aarhus University Hospital. To meet elevated demand for high-throughput whole genome sequencing of COVID-19 positive samples, the NGS Core Facility at MOMA decided to deploy the QIAseq SARS-CoV-2 Primer Panel along with QIAseq FX DNA Library UDI Kits. “The reason for looking into different variants is the fear of vaccine escape mutants. We need to get high coverage during sequencing. One really important aspect is turnaround time – so getting the lab work finished as soon as possible. Another is to get as many positive sample sequences as possible with high enough quality”, she explained.

Genomics, QIAseq NGS Solutions, Dr. Mette Christiansen, Aarhus University hospital, COVID-1

Molecular biologist Mette Christiansen is the head of the Diagnostic NGS Core Facility at the Department of Molecular Medicine (MOMA) at Aarhus University Hospital, which handles NGS for all hospital departments and the wider region.

In the race against the virus, hours and minutes matter

A vaccine escape mutant remains the biggest threat to combating the COVID-19 pandemic. Detection of new variants and cataloging existing variants within populations requires robust whole genome sequencing technologies that provide high uniformity and coverage. With increased demand, labs worldwide are under intense pressure to sequence greater numbers of samples as quickly as possible. However, many of the current NGS workflows are complex and multi-step, are difficult to automate and allow limited sample multiplexing.

Also affecting the speed of variant analyses are global supply chain issues. Scientific laboratories rely heavily upon the use of sterile disposable plasticware in molecular biology applications. Labs continue to face critical shortages of plasticware such as filter tips as well as reagents, which means their processing capabilities – and consequently time-to-result – are severely impacted.

These limitations highlighted a clear need for a dramatically faster, high-throughput NGS solution that requires less plasticware. In response to this challenge, in early 2021, QIAGEN developed the QIAseq DIRECT SARS-CoV-2 Kit – a rapid, high-throughput NGS solution to accelerate SARS-CoV-2 genomic surveillance.

Compared to current ARTIC-based workflows, the QIAseq DIRECT SARS-CoV-2 Kit is 50% faster and involves a streamlined 4-hour enrichment and library prep workflow, which can also be readily automated. For improved scalability and efficiency, up to 768 samples can be multiplexed per lane or flow cell, allowing labs to ramp up sequencing efforts by processing >6000 samples in parallel**.

Designed for short-read sequencers, the QIAseq DIRECT SARS-CoV-2 Kit generates ~250 bp amplicons for improved uniformity and better coverage across the SARS-CoV-2 genome compared with the most widely-used amplicon-based options. There’s also a 50% reduction in plasticware use with the QIAseq DIRECT SARS-CoV-2 Kit. With its ultrafast workflow and superior NGS performance metrics, this solution is a game-changer for labs short on time and resources.

The data analysis bottleneck: Simplifying SARS-CoV-2 sequence data analysis

An additional pressure point for labs is analyzing and interpreting the large and rapidly collected viral datasets. Researchers need to track and visualize phylogenetic divergence. The QIAseq DIRECT SARS-CoV-2 Kit is supported by the QIAGEN Digital Insights CoV-2 Insights Service, an integrated, cloud-based solution tailored for viral genome analysis, characterization and reporting of SARS-CoV-2 variants and lineages. This means researchers can seamlessly go from generating SARS-CoV-2 NGS data to developing deeper insights with ease.

SARS-CoV-2 and wastewater testing

Wastewater-based epidemiology (WBE) is gaining momentum globally as a key approach to predicting and tracking the spread of COVID-19. NGS can provide additional insight in this field compared to PCR approaches as its possible to sequence the entire viral genome at depth with optimized workflows, from sample prep to data analysis. This means that NGS can be used to alert authorities of potentially new variants and strains in a population, even before these variants are detected in clinical cases (3).

Dr. Davida Smyth is Associate Professor of Microbiology at Texas A&M University San Antonio, Texas. Prior to this role, she was Associate Professor of Biology at Eugene Lang College of Liberal Arts at The New School, New York.

"Whole genome sequencing gives us a snapshot of the entire genome. We want to see if there are additional locations across the [SARS-CoV-2] genome that are under selection or are behaving differently with respect to the vaccine interventions."

Dr. Davida Smyth, Associate Professor of Microbiology at Texas A&M University San Antonio, Texas.

Dr. Davida Smyth, Associate Professor of Microbiology at Texas A&M University San Antonio, Texas, and her collaborators have been tracking and monitoring SARS-CoV-2 from New York City wastewater samples (4). “If you are doing wastewater epidemiology for surveillance, you need to be able to compare across samples,” she stated. Every wastewater sample is different, with varying levels of RNA and inhibitors. “Each RNA sample that we get is different, and we only have a small volume of it. You’re getting a mixture of genetic material, and you're getting viruses and bacteria as well, you're getting the genomic signature from the hosts that have also contributed to the water“, she explained. With resources stretched and ongoing shortages with plasticware such as filter tips, processing large numbers of samples was increasingly becoming a challenge. The lab needed a fast, reliable NGS solution that reduced its dependency on plasticware. High genome coverage and uniformity were also critical parameters for variant-calling in wastewater samples.

The tiling amplicon design of the QIAseq DIRECT SARS-CoV-2 Kit generates high-yield libraries with superior sequence uniformity and depth of coverage. The accelerated workflow, increased throughput and reduced need for plasticware make the QIAseq DIRECT SARS-CoV-2 Kit highly suited for wastewater monitoring.

Davida and her team decided to complement their targeted sequencing approach for wastewater samples with whole genome sequencing using the QIAseq DIRECT SARS-CoV-2 Kit, as well as other similar solutions. “Whole genome sequencing gives us a snapshot of the entire genome. We want to see if there are additional locations across the [SARS-CoV-2] genome that are under selection or are behaving differently with respect to the vaccine interventions and so on. “Out of the whole genome sequencing kits we worked with, the coverage with the QIAseq DIRECT SARS-CoV-2 Kit was far better”, she emphasized.

Unlock the potential of NGS for wastewater-based SARS-CoV-2 epidemiology

Explore how Davida and her colleagues navigated the challenges of varying viral concentrations and viral genome fragmentation to conduct SARS-CoV-2 NGS from wastewater samples.

Watch the webinar

Altered miRNA expression in COVID-19 patients

Determining the host response to SARS-CoV-2 infection can provide invaluable insights into viral pathogenesis and COVID-19 progression. miRNAs are promising biomarkers of infectious disease. Their altered expression in COVID-19 patients can help predict SARS-CoV-2 infection with high accuracy. In a recent study, Dr. Ryan Farr and his team from the Australian Centre for Disease Preparedness (ACDP), CSIRO, investigated circulating miRNA profiles in the plasma of ten COVID-19 patients and ten age- and gender-matched healthy donors. Using the QIAseq miRNA Library Kit and QIAseq miRNA NGS 48 Index IL, they determined that among patient samples collected during early-stage disease, COVID-19 induced differential expression of 55 host-encoded miRNAs, with miR-31, -4742 and -3125 strongly up-regulated and miR-1275, -3617 and -500b down-regulated. The study revealed that three miRNAs (miR-423-5p, miR-23a-3p and miR-195-5p) could identify early-stage COVID-19 with 99.9% accuracy. This three-miRNA “plasma signature” returned to that of healthy controls as patients recovered from disease (5).

Dr. Ryan Farr is a Research Scientist at the Australian Centre for Disease Preparedness (ACDP), CSIRO.

Accurate prediction of SARS-CoV-2 infection using miRNA profiling in COVID-19 patients

We examined this fascinating topic in a webinar where Dr. Ryan Farr from CSIRO discussed how an AI multivariate machine-learning approach led to the development of a robust miRNA biomarker signature of COVID-19.

Watch the webinar

Pandemic preparedness and future outlooks

As one of the most influential global events in the last 100 years, it is an understatement to say that the COVID-19 pandemic has turned the world upside down. This public health emergency has had a devastating impact on many lives and economies and has plunged the world into a ‘new normal’ where we’ve had to adapt our lives dramatically. It has indisputably highlighted the need for pandemic preparedness moving forward. There is a clear requirement for robust, reliable technologies for the detection, testing and surveillance of infectious agents to enable an effective and timely public health response.

Innovation and collaboration are fundamental to any long-term strategy for addressing the ongoing pandemic and mitigating future pandemics. QIAGEN is at the forefront of these efforts, combining its knowledge and expertise with innovation. Together, we can help shape the outcome of the ongoing pandemic.

* In September 2021, the US government reclassified several Variants of Concern to “Variants Being Monitored” (VBM), and further reclassifications may occur through the course of the COVID-19 pandemic. See here for details.

** Over 6000 samples may be multiplexed on a single Illumina NovaSeq dual S4 flow cell.