CASPER
A collaboration piloting wastewater sequencing for pathogen surveillance
Scaling a robust approach to pathogen early warning
Reliable monitoring of emerging pathogens is central to biosurveillance, but we currently lack systems that provide sensitive real-time monitoring of many known and unknown pathogens. Multi-city wastewater sequencing could provide the foundation for robust biosurveillance.
Based on extensive research by the NAO and others, untargeted metagenomic sequencing (MGS) of wastewater can identify many human pathogens, including respiratory pathogens. Building on our findings, we’ve partnered with groups across the United States to form the Coalition for Agnostic Sequencing of Pathogens from Environmental Reservoirs (CASPER). In addition to the NAO, CASPER consists of:
- Marc Johnson's lab at the University of Missouri
- Dave O'Connor's lab, at the University of Wisconsin-Madison
- Jason Rothman's lab, at the University of California: Riverside
- Rachel Poretsky's lab, at the University of Illinois Chicago
- John Dennehy's lab, at Queens College, City University of New York
- Helena Solo-Gabriele's lab, at the University of Miami
- Oklahoma Water Survey at the University of Oklahoma
- Center for Predictive Bioresilience at Lawrence Livermore National Laboratory
- Michael Tisza's lab, at Baylor College of Medicine
CASPER provides an initial layer of detection for new biological threats that we and our partners are working to strengthen and extend over time.
Sample Collection
Through the CASPER system, partners collect wastewater from across the US. Some samples are sequenced at Marc Johnson's lab at the University of Missouri, others at the Nucleic Acid Observatory. As of October 30th, 2025, CASPER processes samples from twenty cities, spanning both coasts of the United States and covering several million-person catchments including:
- Boise, ID
- Greater Boston, MA (two sites)
- Chicago, IL (five sites)
- Columbia, MO
- Sacramento, CA
- St Louis, MO
- Monett, MO
- Milan, MO
- New York, NY (several hospitals)
- Ottumwa, IA
- Riverside, CA
We are continuing to onboard sites: if you would be interested in sharing samples please reach out to info@naobservatory.org.
Through regular weekly sample collection and sequencing, we and our partners routinely generate billions of sequencing reads each week, creating the largest collection of untargeted wastewater MGS data ever assembled. A large fraction of this data is publicly available and we will continue to release additional data over time.
Sample Processing and Sequencing
The majority of our samples are processed through two methods, with protocols developed by:
Marc Johnson's lab, providing RNA and DNA sequencing.
The NAO, providing RNA sequencing (protocol).
Both protocols are focused on viruses, but are otherwise untargeted.
We sequence on a NovaSeq X+, to a typical depth of ~1B read pairs per sample.
Data Analysis
Using CASPER for pathogen detection
CASPER already provides data that supports ongoing pathogen-agnostic early detection. At the Nucleic Acid Observatory, we use CASPER data both for identifying known pathogens and for detecting genetically engineered constructs.
For known pathogens, we raise automated alerts if incoming MGS data contains pathogens of concern. For genetically engineered threats, we use chimera detection to flag reads with signs of engineering. Once flagged, we investigate the context of the read through our Outward Assembly algorithm. Using chimera detection, we previously detected a benign genetically engineered virus sequence (cytomegalovirus), which originated from vaccine development work at one of our covered sites.
Using CASPER for public health
The wastewater MGS data generated by CASPER also unlocks many valuable public health applications. The Johnson and O'Connor labs maintain dashboards that show the spread of many different organisms and pathogens relevant for human health. We and our partners are further collaborating with public health departments to integrate CASPER data into their work. Many additional use cases are likely to be discovered as more researchers analyze the data, such as tracking antibiotic resistance and seasonal disease spread.
We are always looking for academics or local public health groups who want to partner on CASPER. If you are interested in learning more, please reach out to info@naobservatory.org!
Funding
CASPER is supported by a range of funders committed to advancing pathogen-agnostic biosurveillance. Among these:
Work at the Johnson and O’Connor labs is supported by Inkfish.
Sequencing efforts at the University of Missouri core facility are supported by SecureBio, through a grant from Open Philanthropy.
Work at the Nucleic Acid Observatory is primarily funded by Open Philanthropy.
Work at the Tisza lab is supported by Baylor College of Medicine and Alkek Foundation Seed Funds
Work at the Rothman lab is supported by The National Science Foundation Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program.
We gratefully acknowledge these organizations for their continued support in expanding pathogen detection and wastewater-based surveillance.