Where can I see Biobot's variant sequencing results?
If your organization has chosen to join the Biobot Network, you can see the sequencing results of your location’s samples on Biobot’s public data visualization at biobot.io/data. These results published on our website are aggregated by region and by county and updated weekly. For more information on the sequencing results reported on the data visualization, please visit FAQ - Data Visualization vs. Standard Reports.
Can I receive variant sequencing data specific to my location?
Yes! To see location-specific variant sequencing data, we can deliver our variant sequencing product directly to you. This product is an add-on to our Covid-19 reports.* If you’re interested in receiving variant sequencing reports in addition to your Covid-19 reports, please contact us at firstname.lastname@example.org.
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.
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.
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.
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.
Can your methods tell me if you have recombinants?
Unfortunately not, given wastewater samples contain viruses from many people and we amplify segments of the whole genome, we don’t have a way to determine if the relevant mutations belong to variant A, variant B, or the recombinant variant C.
Fortunately, recombinants of SAR-CoV-2 currently appear to be exceedingly rare.
FAQ - Variant Sequencing Reports*
What do the percentages of variants mean in my sequencing results?
There are always many different SARS-CoV-2 variants and sub-variants circulating at any one time but only a fraction of variants are considered clinically relevant and impact how public health officials respond. We track a subset of variants of concern designated by the CDC and WHO circulating in the US. See our release notes for the list of variants we are tracking.
We also report % ‘other’, which encapsulates a combination of sequences from variants that are not reported in the results and those sequences that we did not have the resolution to assign to a variant.
What does “Non-detect” mean in my sequencing results?
If there was no SARS-CoV-2 identified by qPCR, we do not send the sample for sequencing.
What does “Concentration too low” mean in my sequencing results?
If there is SARS-CoV-2 in the sample, but the concentration is too low (<30 copies/mL sewage) to attempt sequencing, then your report will show “Concentration too low”.
What does “Sample QC failure” mean in my sequencing results?
Samples were tested for SARS-CoV-2 via qPCR but failed quality control and were not eligible for sequencing. For example, qPCR results appear to be compromised because of the presence of inhibitory compounds within the sample or poor amplification of the molecule we use to gauge human waste abundance.
What does “Pending” mean in my sequencing results?
Results may be pending for a variety of reasons and will be included in a subsequent report. For example, sample results could be delayed if we received the sample later than was expected or if samples required reanalysis. In some rare cases, we reanalyze samples to obtain more certainty regarding the variants present (for instance, if an unusual variant was detected).
What does “Sequencing failed” mean in my sequencing results?
Your report may show “Sequencing failed” which means we were able to detect SARS-CoV-2 but unable to distinguish between and quantify relative frequencies of variants. Occasionally when sequencing any mixed sample, the amplified segments do not contain enough variant-specific mutations to differentiate between variants of an organism. In wastewater this is more of a challenge than other sample types (e.g., nasal swabs) because SARS-CoV-2 occurs in much lower concentrations in wastewater, so there are fewer occurrences of the variant-specific mutations needed to differentiate and quantify the relative percentages of variants.
What can I do to improve sample quality to avoid failing?
We haven’t identified any single step customers can take that will reliably improve sequencing results from wastewater. One inherent challenge with wastewater is the fragmentation of the SARS-CoV-2 genome as well as the presence of chemicals that inhibit the sequencing reaction.
Some sampling sites may have more of these chemicals than others, which could cause consistently poor sequencing results. We are continually improving our laboratory processes in hopes that we may better address inhibition in the future.
My values have changed a lot since my previous report, what does this mean?
Similar to qPCR, if your sampling site represents a smaller population, small changes in which variants are circulating can lead to large week-to-week swings in the data. In addition, new variants can take over quickly when they begin to spread exponentially (e.g., Omicron's explosive spread in winter 2021-2022).
There can also be some variability in lab procedures that can lead to small differences from week-to-week.
How can you be confident in the results?
As part of our bioinformatics pipeline we include numerous quality control checks. If we do not feel that the quality of sequencing data is high enough to support differentiation and relative quantification of variants we will report “sequencing failed”. We use Freyja, a tool developed by Dr. Kristian G. Andersen’s lab at the Scripps Research Institute, to identify and quantify SARS-CoV-2 variants of interest.
*Note: The medium through which you receive your results is dependent on your customer type. Community, Community+, and Building-level customers receive their results as emailed pdf reports. Biobot Network participants instead receive their results through the data visualization on the Biobot website. CDC NWSS Program Participants receive emailed pdf reports as well as data visualized on the CDC COVID Data Tracker. CDC Program Participants can also opt in to the data visualization on the Biobot website. Email email@example.com if you have any questions about your customer type.