Flushing out SARS-CoV-2 variants from wastewater samples using NGS

The secrets lie in the sequence: Why NGS is making a splash in wastewater-based SARS-CoV-2 epidemiology

As the COVID-19 pandemic continues to evolve unpredictably with the rise of vaccine escape mutants and breakthrough infections, the need to stay abreast of potential new variants has never been more critical. The emergence of these “Variants of Concern” (VOC) and “Variants of Interest” (VOI)* has triggered a need for large-scale monitoring of populations to detect new variants and determine the prevalence of existing variants. Wastewater surveillance for SARS-CoV-2 is gaining momentum as it enables large populations to be monitored at different levels of scale in a passive and non-invasive way. It also helps determine the abundance of the virus in wastewater samples over time and the relative amounts of mutations associated with VOC.

NGS gives an edge when it comes to wastewater-based SARS-CoV-2 epidemiology as it allows simultaneous analysis of many samples. 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 (1). Whole genome sequencing, in particular, provides unprecedented insights into the viral genome – and helps pinpoint new mutations as they arise.

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 (2). We chatted with Davida in mid-2021 to discuss her experiences with wastewater surveillance and find out how she and her colleagues navigated the challenges of SARS-CoV-2 NGS from wastewater samples.

QIAGEN: What are the key challenges when analyzing wastewater samples for SARS-CoV-2?

Dr. Davida Smyth (DS): The challenge with wastewater is that each sample is very different. And because the samples are different, there're all sorts of inhibitors in the mix. You’re getting a mixture of genetic material. You're getting viruses and bacteria and genomic signatures from the hosts that have also contributed to the water. I'm working in New York, where we have high caseloads. But across the US, everybody's trying the same thing, and everybody's having different levels of success. Not everybody's wastewater is the same. People are having very different experiences with it. The major issue for us is that we don't have an unlimited supply of RNA from wastewater samples. Each RNA sample that we get is different, and we have only a small volume of it. So, normally when you're doing optimization, you can redo the same experiment, altering the parameters of the experiments. But with these samples, we're repeating the same experiment, but every time the RNA sample is going to be slightly different, so that's the problem. I don't have 200 microliters of RNA that I can split into aliquots.

At the time when we were starting this work, we had limited resources. For example, our lab didn't have funding to do this work, so we were working with a very small amount of money and using whatever resources we had in the lab. The plasticware shortage was a key issue for us.

QIAGEN: Talk us through the different solutions you tried and the results you achieved.

DS: With some of the solutions we tried, we got very limited results. We initially tried two ARTIC-based protocols. One of the challenges of ARTIC-based protocols is that they are time-consuming, with a multitude of steps necessary for library prep. The protocol was just so hard. With the ARTIC-based QIAseq SARS-CoV-2 Primer Panel Kit, the protocol was very labor intensive. It was such a long prep to do – it took me nearly three days of intense work, and I was only doing a small number of preps. But it took me almost three days solely doing that. And I burned through so many filter tips. Using so many filter tips is a problem because they're expensive, but the main problem was that we didn’t have them. There just weren’t enough filter tips, so we were scrambling to get filter tips during the pandemic. QIAGEN then sent me the QIAseq DIRECT SARS-CoV-2 Kit to beta test which dramatically cut down the workflow to just 4–5 hours from extracted RNA to sequencer-ready libraries.

QIAGEN: What sort of coverage did you get with ARTIC-based methods?

DS: We didn't get sufficient coverage [using the ARTIC-based QIAseq SARS-CoV-2 Primer Panel^†] of the entire genome, even though we got good depth. It was spotty across samples. And that's the problem. If you are doing wastewater epidemiology for surveillance, you need to be able to compare across samples. So, in the interim, we had developed a targeted sequencing approach. We made our own primer for the spike protein gene, and we had started doing targeted sequencing. The benefit of a targeted sequencing approach was that we got uniform coverage, decent depth and we could compare across samples.

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.

With targeted sequencing, you just see the spike protein gene. With whole genome sequencing, you see everything. So I hadn't given up on whole genome sequencing, and I was hopeful that I could get something to work.

QIAGEN: Which sequencer are you using?

DS: I'm using an Illumina iSeq instrument. The iSeq 100 is a bench-top sequencer and while it's got much less capacity than a MiSeq, it's fast, cheap and affordable.

QIAGEN: Tell us a bit about your experience with whole genome sequencing using the QIAseq DIRECT SARS-CoV-2 Kit.

DS: QIAGEN gave us the QIAseq DIRECT SARS-CoV-2 Kit to beta test. We worked with the QIAGEN team to optimize the workflow, and it's been a lovely experience. Because we're under so much pressure from everybody, it was great to have QIAGEN’s expertise and resources at our beck and call.

QIAGEN never made me feel like I was a burden – even if I messed up. They sent me replacements at one point when I had accidentally used the wrong volume. Together with the QIAGEN team, we’ve optimized our workflow for our samples over time. Pretty much each week, I would meet with them, and I would inform them about my progress. And then, we went through a series of optimization steps for me to try different QIAGEN sample prep kits. We tried preps with different RNA kits to see if we could improve the yield [of sequencing-ready viral genomes] that way. Then we tried all the usual tips and tricks from the field. I would meet with Jennifer from the QIAGEN North America Genomics field application specialist team. She would suggest various optimization steps I could implement to improve the results I was getting. Together, we optimized the whole genome sequencing workflow.

QIAGEN: What are your latest results?

DS: I’m pleased to report that the sets of data that I just ran were beautiful! We incorporated QIAGEN’s RNA inhibitor removal technology. I processed the RNA using QIAGEN’s spin column first, and I got much better results than I've ever gotten before!

Based on the various whole genome sequencing solutions we tried, with the QIAseq DIRECT SARS-CoV-2 Kit, the coverage was far better. And it was in multiple samples, which gives me a lot of hope that I'll be able to get it to work more reproducibly.

I still see that we will probably have to continue to do targeted sequencing because it gives me more reliable data. But I'm hopeful that we'll be able to get whole genome sequencing to work so that I can do that in addition to targeted sequencing.

The whole genome sequencing approach will tell me which areas of the genome are of interest, and then I will then use targeted sequencing to focus on those bits of the genome. That's more efficient and more cost-effective for us.

I'm very excited and happy that I saw these results. I think it's going to be useful, clinically, and otherwise, especially in cities (3).

Why wastewater testing is making waves worldwide

There's growing evidence that wastewater-based SARS-CoV-2 epidemiology is a powerful surveillance tool to prevent a surge in COVID-19 infections, specifically of new variants that are gaining ground in specific communities. Applying wastewater-based epidemiology could reduce pressure on the public health system to manageable levels. It would also empower public health authorities to make informed decisions for better and timely treatments or vaccines. While technologies such as PCR are efficient at detecting existing variants, NGS provides unparalleled resolution into the secrets of viral mutation, evolution and new variant development.

References

1. Agrawal et al. (2021) A pan-European study of SARS-CoV-2 variants in wastewater under the EU Sewage Sentinel System. medRxiv 2021.06.11.21258756.
2. Trujillo et al. (2021) Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater. PLoS ONE 16(9): e0257454.
3. (3) Smyth et al. (2022) Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater. Nature communications (635).
   
   * 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.
   ^† The QIAseq SARS-CoV-2 Primer Panel is now available with a Booster Kit (available upon request) to support increased coverage of emerging variants.