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NGS During the COVID-19 Pandemic and Beyond

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Next-generation sequencing (NGS) has been a fundamental tool used throughout the COVID-19 pandemic, helping scientists to study the SARS-CoV-2 viral genome and identify mutations. The need for improvements in speed, simplicity and cost effectiveness are driving developments in sequencing methods such as the Midnight Panel – an enrichment method that can reduce sequencing time by more than half.

Technology Networks recently spoke with Dr. Nick Downey, NGS collaborations lead at IDT, to learn more about the ways that NGS is being used as part of the response to the pandemic and gain insights into how and why this varies globally. In this interview, Nick also tells us more about the Midnight Panel and shares his views on how the pandemic has influenced the future use of sequencing programs in public health.

Anna MacDonald (AM): Can you describe the roles that NGS has played during the COVID-19 pandemic? Why is it so important to study the SARS-CoV-2 viral genome?

Nick Downey (ND):
In the early days of the pandemic, one of the first steps was to characterize the virus and understand how quickly it was spreading. NGS was used to decode the first SARS-CoV-2 sequence very early in the initial outbreak, and this sequence was the springboard for many tools to slow the pandemic. First, the initial sequence provided information to do basic research on effective assays to detect the virus. Perhaps most importantly, the early sequence of SARS-CoV-2 allowed researchers to begin building reagents for development of the eventual vaccines. The ability to begin this process so early and in a directed manner almost certainly allowed vaccine development to begin much earlier than disease outbreaks of the past.

Soon, NGS enrichment protocols were being used to reduce the amount of sequencing required and allowed more research samples to be tested simultaneously. Getting this data has identified the emergence of new strains such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), and most recently B.1.617.2 (delta). Early identification of a new strain allows researchers to initiate studies that provide data for Public Health assessment sooner. We saw this with the cases around the mutations mentioned. Identification of the variants triggered research studies into their infectivity, ability to evade immune responses, and geographical spread.

With the rollout of vaccinations, there is heightened concern around a new strain that might avoid the immune response, and this can be evaluated based on the genome data. When done on a large scale, researchers can use this information to study vaccine effectiveness based on circulating variants and provide important data to evaluate whether vaccine boosters may be required in the future.

AM: In what ways do NGS strategies vary by country? What are the reasons for these differences?

The use of NGS as part of the SARS-CoV-2 response has been quite different globally. I do not think there is a single reason for this, and most countries have acted within their situation. An earlier leader in sequencing was the UK, and its government committed early in the pandemic to using NGS to track viral sequences with a goal of looking at 10% of samples that test positive. Other countries have taken a more decentralized approach and may not have prioritized sequencing as a go-to method. This has evolved during the pandemic as needs and resources change from country to country.

Setting up national sequencing surveillance is not trivial. One difficulty is that this requires close coordination to share data, and so countries with more distributed organizations face more difficulty than a country with a single organization. For some countries, there were low caseloads at the beginning of the pandemic, so the perceived need or utility of sequencing may have been deprioritized in the context of other Public Health initiatives. And of course, accessibility of the specialized equipment and trained personnel are always an issue. Personnel burdened with detecting SARS-CoV-2 at the peak of the pandemic did not have the ability to perform sequencing experiments in tandem.

AM: The WHO urged countries to prioritize increasing sequencing capacity. What challenges must be overcome to improve sequencing speed and throughput? 

The challenges will be different depending on the region or country involved. As mentioned, coordination across groups is needed for data sharing and standardization of data format. Logistics around getting positive samples to a sequencing facility, accounting for different sample preparation/treatment at those different locations, and accounting for orders of magnitude differences in the amount of viral material in the sample all add up to challenges for streamlining and speeding up the process of getting material onto a sequencer. And then finally, as you increase the data generated, you need systems that can handle the volume and connections to move the data to more centralized repositories.

A fortunate challenge some countries are now facing is a drastic drop in positive samples. When caseloads are high, positive samples are abundant and it’s easy to choose low Ct samples to sequence. The drop in high titer samples influences the prioritization of higher sensitivity methods over high throughout methods.

While it’s often assumed increasing throughput requires large capacity sequencers, it’s actually technically challenging to fill these machines. Each sample must be tagged with an index (used to identify data from different samples), and there are limited numbers of indexes available. Generating thousands of samples with unique tags takes time and effort. In addition, large capacity sequencers tend to run for longer periods to generate the data. So there is a balance of speed and cost, between trying to load more samples on a single machine or distributing the samples across several smaller machines to save a few hours (the latter choice also reduces the need for those indexes). 

AM: Can you tell us more about Midnight Panel? What sets this method apart?

The Midnight Panel* was designed by Drs Nikki Freed and Olin Salinder at Massey University in New Zealand and is designated for research use only. The original method is intended for use with the Oxford Nanopore sequencing platform. The panel consists of multiplex PCR primer sets that copy a series of 1200 bp amplicons. The primers are split into two pools to create SARS-CoV-2 genome fragments with an overlapping design. This leads to fewer amplicons required to cover the genome. That, in time, results in more even coverage. In addition, the fewer primer binding sites mean there is a lower probability for a new variant to inhibit primer binding resulting in amplicon dropout.

While longer amplicons might be even more attractive, there is a balance with the functionality of the reverse transcriptase enzyme (needed to copy the viral RNA into DNA) to copy large enough regions to allow the subsequent amplification. The Midnight Panel balances this amplicon length and reverse transcriptase function challenge. The longer amplicons have been used on the Pacific Biosciences sequencing machines. Additionally, the amplicon sequencing library is easily converted to Illumina compatible sequencing libraries via fragmentation (enzymatic or sonication).

AM: How do you think the pandemic has influenced the future use of sequencing programs in public health?

In retrospect, we’ve come to realize that SARS-CoV-2 began circulating worldwide earlier than originally believed, making control of the virus a constant game of catch-up. Given that respiratory viruses can spread rapidly while also causing overlapping symptoms, some scientists are eager to use unbiased methods to constantly monitor samples from respiratory disease hospitalizations. PCR and antibody-based tests are well-suited to provide yes/no answers for known infectious agents, but sequencing is much better suited to the type of broad surveillance that would be needed to detect the rapid spread of a novel virus or new variants.

I believe that most scientists involved in sequencing viral samples will be very much in favor of building continued surveillance programs. We are starting to see signs of this with the UK and German governments recently announcing long-term global surveillance programs in collaboration with the WHO. Unlike previous surveillance, these have specific goals around sequencing of pathogens. Something I would not have considered some time ago is wastewater testing. I think this could be another avenue that will help identify and track pathogens. Testing wastewater will not be a patient-level measure but might warn us of focal outbreaks before other forms of surveillance might capture an event. And the complexity of these samples requires the use of NGS platforms and reagents now and into the future.

Nick Downey was speaking with Anna MacDonald, Science Writer for Technology Networks.

*RUO - For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.