How does sequencing SARS-CoV-2 variants from wastewater work?
Similar to our qPCR methods, we start by capturing the SARS-CoV-2 virus, extracting viral RNA, and converting the RNA into DNA. Next, we use PCR to amplify the SARS-CoV-2 genome. Amplification means that multiple copies of the genome are generated from the original template genome. This is necessary to meet sequencer input requirements and for reliable detection of SARS-CoV-2. Then the amplified sample is sequenced. Since there are multiple SARS-CoV-2 viruses in our samples we end up with multiple SARS-CoV-2 genome segments with different sets of mutations. We look for particular combinations of mutations that are specific to a given SARS-CoV-2 variant to identify which variants are present in a sample. Then, based on the frequency of these patterns of mutations, we can estimate relative percentages of the various variants in a sample.
What is the difference between qPCR and whole genome sequencing?
qPCR only targets a specific region of the SARS-CoV-2 genome and measures its abundance. Whole genome sequencing looks at the entire genome of the SARS-CoV-2 virus and lets us identify different variants of SARS-CoV-2 in a sample and their relative frequencies.
There are other methods (e.g., variant-specific qPCR) that target specific variants, but these methods must be updated whenever there’s a new variant. Our method is much more flexible and allows us to detect new variants as soon as they begin circulating.
What is the expected turnaround time for sequencing samples?
We process sequencing samples in weekly batches, so turnaround time for an individual sample may be 11 to 15 days from sample receipt in our lab to results.
Why is the expected turnaround time for sequencing slower than for qPCR?
Compared to qPCR, sequencing requires additional laboratory procedures and more complex data analysis. This is true of both wastewater sequencing and clinical specimen sequencing.
How can I use my sequencing data to inform public health related decisions?
Each variant has an associated set of characteristics related to transmissibility, virulence, treatment efficacy, etc. tied to their specific mutations. Identifying these variants and their relative percentages can influence how a public health department or an employer responds.
For example, Omicron’s increased transmissibility led to dramatic increases in case counts in winter 2021-2022. Even though at an individual level Omicron infections were less likely to result in hospitalization than infections from Delta, hospitals had to prepare for an influx of cases because there were so many cases overall that the absolute number of severe cases reached new heights. Employers, including hospitals and healthcare facilities, had to contend with high rates of absenteeism due to illness. Officials had to make hard choices about mask wearing in indoor settings and quarantining because case counts were so high. In addition, tests were not readily available. During this time, testing the amount of virus circulating in wastewater via qPCR and identifying which variants were circulating via sequencing was an important leading indicator that gave cities and employers a 1-2 week head start to begin planning how they would handle the much more contagious Omicron variant.
For more information on how to use your data to inform decision making see our 5 Uses of Wastewater Data for Covid-19 Responses.
Can your methods tell me if you have recombinants?
Detection of recombinant variants is challenging. Due to the fact that wastewater samples contain viruses from many people, and because we amplify segments of the whole genome, it is difficult to determine if the relevant mutations belong to variant A, variant B, or the recombinant variant C. However, in some cases, such as XBB, the lineages that combined to form the recombinant variant may not be widely circulating but the recombinant is abundant. In such cases, it is much easier to positively identify the recombinant variant.
What if there is a new variant that begins spreading?
As part of our bioinformatics pipeline, we use UShER, an up-to-date database of variants and their specific mutations. This allows us to track variants even before they are listed as variants of concern by CDC or WHO. We regularly review CDC and WHO’s list of variants and update which variants we report on based on what is circulating widely or of concern.