Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published September 1, 2022 | public
Journal Article

Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission

Karthikeyan, Smruthi ORCID icon
Levy, Joshua I. ORCID icon
De Hoff, Peter
Humphrey, Greg
Birmingham, Amanda
Jepsen, Kristen
Farmer, Sawyer
Tubb, Helena M.
Valles, Tommy
Tribelhorn, Caitlin E.
Tsai, Rebecca
Aigner, Stefan
Sathe, Shashank
Moshiri, Niema
Henson, Benjamin
Mark, Adam M.
Hakim, Abbas
Baer, Nathan A.
Barber, Tom
Belda-Ferre, Pedro ORCID icon
Chacón, Marisol
Cheung, Willi
Cresini, Evelyn S.
Eisner, Emily R.
Lastrella, Alma L.
Lawrence, Elijah S.
Marotz, Clarisse A.
Ngo, Toan T.
Ostrander, Tyler
Plascencia, Ashley
Salido, Rodolfo A.
Seaver, Phoebe
Smoot, Elizabeth W.
McDonald, Daniel ORCID icon
Neuhard, Robert M.
Scioscia, Angela L.
Satterlund, Alysson M.
Simmons, Elizabeth H. ORCID icon
Abelman, Dismas B.
Brenner, David
Bruner, Judith C.
Buckley, Anne
Ellison, Michael
Gattas, Jeffrey
Gonias, Steven L.
Hale, Matt
Hawkins, Faith
Ikeda, Lydia
Jhaveri, Hemlata
Johnson, Ted
Kellen, Vince
Kremer, Brendan
Matthews, Gary
McLawhon, Ronald W.
Ouillet, Pierre
Park, Daniel
Pradenas, Allorah
Reed, Sharon
Riggs, Lindsay
Sanders, Alison
Sollenberger, Bradley
Song, Angela
White, Benjamin
Winbush, Terri
Aceves, Christine M.
Anderson, Catelyn
Gangavarapu, Karthik
Hufbauer, Emory
Kurzban, Ezra
Lee, Justin ORCID icon
Matteson, Nathaniel L.
Parker, Edyth
Perkins, Sarah A. ORCID icon
Ramesh, Karthik S.
Robles-Sikisaka, Refugio
Schwab, Madison A. ORCID icon
Spencer, Emily
Wohl, Shirlee
Nicholson, Laura ORCID icon
McHardy, Ian H. ORCID icon
Dimmock, David P. ORCID icon
Hobbs, Charlotte A.
Bakhtar, Omid
Harding, Aaron
Mendoza, Art
Bolze, Alexandre ORCID icon
Becker, David
Cirulli, Elizabeth T.
Isaksson, Magnus ORCID icon
Schiabor Barrett, Kelly M. ORCID icon
Washington, Nicole L. ORCID icon
Malone, John D.
Murphy Schafer, Ashleigh ORCID icon
Gurfield, Nikos
Stous, Sarah
Fielding-Miller, Rebecca ORCID icon
Garfein, Richard S.
Gaines, Tommi
Anderson, Cheryl
Martin, Natasha K.
Schooley, Robert ORCID icon
Austin, Brett
MacCannell, Duncan R.
Kingsmore, Stephen F.
Lee, William
Shah, Seema
McDonald, Eric
Yu, Alexander T.
Zeller, Mark
Fisch, Kathleen M.
Longhurst, Christopher ORCID icon
Maysent, Patty
Pride, David
Khosla, Pradeep K.
Laurent, Louise C.
Yeo, Gene W. ORCID icon
Andersen, Kristian G. ORCID icon
Knight, Rob ORCID icon

Abstract

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1–3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Additional Information

We thank L. Asato and the Microbiology Laboratory at the SD Public Utilities Department for providing us with county wastewater samples; UCSD's Return to Learn (RTL) program for funding the campus-wide wastewater surveillance efforts; J. Kayne, R. Cota, J. Ortiz and the Facilities management team at the UCSD; J. Mayer from the Center for Aerosol Impacts on Chemistry of the Environment (CAICE); L. Arnold of the Campus Research Machine Shop (CRMS) for assistance with the installation and operation of the autosamplers; R. Jacobs, S. Knepple and their team at UCSD Logistics for assisting with our daily sampling efforts; B. Pollak and the UCSD Information Technology Services team for assisting with the daily notifications; the Office of Academic Affairs for contact tracing and targeted campus messaging assistance; J. Severson, P. Hochstein, the UCSD HDH team and the UCSD Environmental Health and Safety personnel; J. Gilbert and the Microbiome Sample Processing Core at UCSD for access to qPCR equipment; the CDC SPHERES consortium, SEARCH (San Diego Epidemiology and Research for COVID Health) Alliance and members of the Andersen laboratory for discussion and help with logistics; and the healthcare workers, frontline workers and patients who made the collection of this SARS-CoV-2 dataset possible and all those who made genomic data available for analysis via GISAID. This work has been funded by CDC BAA contracts 75D30121P10258 (Helix) and 75D30120C09795 (to G.W.Y., R.K., L.C.L. and K.G.A.), NIH NIAID 3U19AI135995-03S2 (to K.G.A.), U19AI135995 (to K.G.A.), U01AI151812 (to K.G.A.), NIH NCATS UL1TR002550 (to K.G.A.), the Conrad Prebys Foundation (to K.G.A.), NIH 5T32AI007244-38 (to J.I.L.), NIH Pioneer grant 1DP1AT010885 (to R.K), NSF RAPID 2029069 (to R.K.), San Diego County Health and Human Services Agency (to R.F.-M.) and NIH S10OD026929 (to K.J.). The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC and California Department of Public Health or the California Health and Human Services Agency. Use of trade names is for identification only and does not imply endorsement by the CDC. These authors contributed equally: Smruthi Karthikeyan, Joshua I. Levy. These authors jointly supervised this work: Kristian G. Andersen, Rob Knight. Data availability. All raw wastewater sequencing data are available via the NCBI Sequence Read Archive under the BioProject ID PRJNA819090. Consensus sequences from clinical and wastewater surveillance are all available on GISAID. Spike-in sequencing data are available via Google cloud (https://console.cloud.google.com/storage/browser/search-reference_data). The UCSD campus dashboard can be accessed at https://returntolearn.ucsd.edu/dashboard/. The county wastewater data from Point Loma are available through the public dashboard at https://searchcovid.info/dashboards/wastewater-surveillance/. The SEARCH genomic surveillance dashboard is available at https://searchcovid.info/dashboards/sequencing-statistics/. Code availability. Freyja is hosted publicly on GitHub (https://github.com/andersen-lab/Freyja) and is available under a BSD-2-Clause license (https://doi.org/10.5281/zenodo.6585067, all versions). Freyja is accessible as a package via bioconda (https://bioconda.github.io/recipes/freyja/README.html) in container form via dockerhub (https://hub.docker.com/r/andersenlabapps/freyja). COVID-19 Viral Epidemiology Workflow (C-VIEW) is available at https://github.com/ucsd-ccbb/C-VIEW as an open-source, end-to-end workflow for viral epidemiology focused on SARS-CoV-2 lineage assignment and phylogenetics. C-VIEW uses minimap2 (v2.17), samtools(v1.11), iVar(v1.3.1) and pangolin (varying versions). Contributions. R.K. and K.G.A. conceptualized the study. S.K., J.I.L., N.K.M., P.D.H., A. Birmingham, S. Sathe, K.M.F., L.C.L., G.W.Y., K.G.A. and R.K. contributed the methodology. J.I.L., D.M., N.M., K.M.F., A. Birmingham, K.J., B.H., S. Sathe, K.G., N.L.M., K.S.R., C.M.A., E.H. and A.M.M. provided software. S.K. and J.I.L. performed formal analysis. S.K., J.I.L., N.M., S.F., H.M.T., T.V., C.E.T., R.T., N.A.B., T.B., M.C., W.C., E.S.C., E.R.E., A. Hakim, G.H., A.L.L., E.S.L., T.T.N., T.O., A. Plascencia, R.A.S., P.S., P.B.-F., E.W.S., S.A., P.D.H., C.A.M., L.C.L., G.W.Y., Cheryl Anderson, E.K., M.A.S., S.A.P., J.L., E.P., M.Z., E.S., R.F.-M., T.G., R.S.G., K.G.A. and R.K. conducted the investigation. Catelyn Anderson, N.K.M., R.M.N., R.S., E.H.S., A.M. Schafer, S.F.K., D.P.D., C.A.H., A.M., S. Strous, B.A., S. Shah, N.G., J.D.M., E.M., I.H.M., A. Harding, O.B., A.M., A. Bolze, K.M.S.B., E.T.C., N.L.W., W.L., M.I., D. Becker, L.N., S.W., M.Z., R.R.-S., R.F.-M., T.G., R.S.G., D.B.A., D. Brenner, J.C.B., A. Buckley, M.E., J.G., S.L.G., M.H., F.H., L.I., H.J., T.J., V.K., B.K., L.R., C.A.H., G.M., P.M., R.F.-M., P.O., D. Park, A. Pradenas, A.M.S., B.S., A. Song, B.W., T.W., S.R., P.K.K., A.T.Y., D.R.M., F.H., G.M., R.W.M. and C.L. provided resources. S.K., J.I.L., P.D.H., G.H., S.F., H.M.T., C.E.T., R.T., T.V., A. Birmingham, N.M., A.M.M. and K.M.F. curated data. S.K., J.I.L., K.G.A. and R.K. wrote the original draft of the manuscript. All authors reviewed and edited the manuscript. S.K. and J.I.L. performed visualization. R.M.N., N.K.M., R.S., A.L.S., E.H.S., A.M. Satterlund, P.D.H., L.C.L., D. Pride, G.W.Y., K.G.A. and R.K. supervised the study. R.M.N., N.K.M., R.S., A.L.S., E.H.S., A.M. Satterlund, P.D.H., L.C.L., G.W.Y., K.G.A. and R.K. undertook project administration. R.K. and K.G.A. acquired funding. Competing interests. A. Bolze, D. Becker, E.T.C., M.I., K.M.S.B., N.L.W. and W.L. are employees of Helix. K.G.A. has received consulting fees for advising on SARS-CoV-2, variants and the COVID-19 pandemic. Nature thanks Timothy Julian and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional details

Created:
August 22, 2023
Modified:
October 24, 2023