Sightline is Verily's public health business unit. Sightline's wastewater program provides the end-to-end laboratory, logistics, and data infrastructure for wastewater monitoring – a critical tool for providing an unbiased, comprehensive, and cost-effective early warning signal for public health.

Our infrastructure provides the laboratory and logistics services for two of the largest and most prominent wastewater monitoring programs in the United States:

1. The Centers for Disease Control and Prevention's (CDC) National Wastewater Surveillance System (NWSS), for which we test samples from up to 400 facilities nationwide.
2. WastewaterSCAN, a national research project led by Stanford University, which includes approximately 150 participating sites.

Our program began in 2020 in response to the COVID-19 pandemic, and has grown substantially in scale and scope since. During that time, we also worked as a direct, end-to-end partner for state and local health departments, including the California Department of Health (CDPH) and Alameda County.

We support several hundred wastewater treatment facilities across the United States and its territories. Our state-of-the-art lab in South San Francisco, California, has processed to date more than 100,000 samples, delivering more than 1,000,000 results.

We can test for a wide variety of pathogens, and our scientific team continually develops and validates new assays. We use highly multiplexed digital PCR for efficiency.

Pathogens we currently test for include, but are not limited to:

• Respiratory viruses: SARS-CoV-2; influenza A (IAV); influenza B (IBV); IAV subtypes (H1, H3, and H5); RSV A & B; human metapneumovirus; human parvovirus B19; parainfluenza 1, 2, 3, 4A, 4B; enterovirus D68; measles
• Gastroenteric viruses: Norovirus GII; adenovirus 40/41; rotavirus
• Other viruses: Mpox (clade I & II), non-variola orthopoxvirus; Oropouche; hepatitis A; dengue (1, 2, 3, & 4); West Nile virus
• Fungi: Candida auris

Our service is highly customizable. Each client selects which pathogens we test their samples for. Customers can change their panels over time, as public health priorities change, or as new, validated assays become available.

We are fully equipped to add new targets. Our team has extensive experience in rapidly developing and validating assays to address emerging public health threats.

• For existing, validated assays: If we have already validated an assay, we can typically add it to a given testing program in about one week.
• For new assays: Development, procurement, and validation of a brand-new assay for use in multiplexed testing is typically done in 6-8 weeks.

Our dedicated wastewater lab in South San Francisco is fully equipped with state-of-the-art automation and detection equipment, including:

• Automation: Liquid handlers and automated extraction systems to improve quality and reduce variability.
• Quantification: digital PCR systems that allow for multiplexing, enabling us to test for multiple targets simultaneously.
• Sequencing: A suite of sequencers to minimize turnaround time.

We use digital PCR because it offers significant advantages for the complex and challenging wastewater matrix:

1. Absolute Quantification: digital PCR directly counts positive droplets, providing an absolute concentration, without relying on a standard curve. This improves accuracy and reduces variability.
2. High Sensitivity: By partitioning the sample into approximately tens to hundreds of thousands of droplets, digital PCR increases the signal-to-noise ratio, making it highly sensitive for detecting low concentrations of a pathogen.
3. Inhibitor Resistance: The partitioning process also makes digital PCR more resilient to PCR inhibitors that are common in wastewater samples.

These are two quality control targets Verily runs on every sample to ensure data is reliable:

• Pepper mild mottle virus (PMMoV): A plant virus found in human feces. We measure it as a fecal normalization control. This helps us account for variations in the human population size contributing to the sewershed or dilution from stormwater runoff, allowing for more accurate comparisons of pathogen concentration over time.
• Bovine coronavirus (BCoV): A process control. We spike a known quantity of BCoV into every sample at the beginning of the sample testing process. By measuring how much we recover at the end, we can verify that the extraction and testing process was successful and efficient.

Yes. We can perform amplicon sequencing for SARS-CoV-2 and MPXV to identify and track variants. Our sequencing protocol is optimized for the unique challenges of wastewater – fragmented genomes and high diversity.

We have rigorous, multi-step quality control procedures for every sample. Every result is reviewed by our scientific team before it is released. Our key metrics involve:

• Extraction controls
• PCR controls
• Process controls
• Inhibition

If a sample fails a quality control metric, it is immediately flagged for re-preparation and re-testing to ensure only valid data is reported.

Each client is assigned a dedicated Program Manager who serves as their point of contact. They will facilitate regular meetings and are supported by our scientific, logistics, and other teams. We offer flexible data reporting options, including:

• Direct API Integration
• A website configured for your program, which can visualize data trends and supports CSV downloads
• Custom reports

We have a demonstrated track record of rapid turnaround times, which are critical for public health action.

• Quantification Data: We typically return concentration results within 48 hours of sample receipt. Many results are returned sooner.
• Sequence Data: We typically return sequence data within 10 calendar days of sample receipt.

Data ownership and data sharing rights are defined in the legal agreements established at the start of the program. These terms are typically defined in the primary contract with the program sponsor (such as a public health department), and, where applicable, further outlined in material transfer agreements with the sampling facility (e.g., the wastewater facility). We work closely with all partners to ensure these agreements reflect the program's data needs and privacy standards.

Verily turns raw concentration data, like copies of a pathogen nucleic acid per gram of waste, into simple, actionable abundance categories. We provide two key pieces of information:

1. Abundance: How much of the pathogen nucleic acid is currently in the wastewater compared to historical levels?
2. Trend: Is the abundance of the pathogen currently going up, going down, or staying the same?

We examine all wastewater sample concentration results from a specific site over the last two years, and line them up from the lowest concentration to the highest. We then divide that list into five equal groups, or quintiles. We then see where the current pathogen abundance falls along that list.

• 1st quintile, or bottom 20%: The virus levels are among the lowest historically seen at this site.
• 2nd quintile Levels are below the median for this site.
• 3rd quintile Levels are average/median for this site.
• 4th quintile Levels are above the median for this site.
• 5th quintile, or top 20%: The pathogen levels are among the highest historically seen at this site.

This method provides context for the current data. For example, a result in the 5th quintile means current abundance is higher than that in 80% of the samples collected at this location over the past two years. If a site is new and lacks sufficient history, we temporarily use a national benchmark to make this comparison.

Abundance helps us categorize pathogen nucleic acid concentration levels. We then analyze data from the last 21 days to determine the direction, or trend, of pathogen levels.

• Upward: There is a statistically significant increase in pathogen levels over the last three weeks.
• Downward: There is a statistically significant decrease in pathogen levels over the last three weeks.
• No Trend: The levels are stable, or the data is not clear enough to point a direction.

Different pathogens behave differently. We categorize them into three groups to ensure our analysis is statistically accurate for each type:

• Common pathogens (e.g., SARS-CoV-2): These circulate year-round or very frequently. We constantly track their abundance and trends.
• Seasonal pathogens (e.g., influenza): These viruses have distinct seasons. During periods of absence, standard trend calculations can be misleading, as even minor fluctuations in abundance can look like large statistical jumps. To prevent false alarms, we flag these spikes only after detecting a sustained, significant increase in the pathogen's abundance.
• Rare pathogens (e.g., mpox): These are rarely detected. We use a simpler present/absent approach – if we detect it, we flag it immediately.