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

Karthikeyan, Smruthi and Levy, Joshua I. and De Hoff, Peter and Humphrey, Greg and Birmingham, Amanda and Jepsen, Kristen and Farmer, Sawyer and Tubb, Helena M. and Valles, Tommy and Tribelhorn, Caitlin E. and Tsai, Rebecca and Aigner, Stefan and Sathe, Shashank and Moshiri, Niema and Henson, Benjamin and Mark, Adam M. and Hakim, Abbas and Baer, Nathan A. and Barber, Tom and Belda-Ferre, Pedro and Chacón, Marisol and Cheung, Willi and Cresini, Evelyn S. and Eisner, Emily R. and Lastrella, Alma L. and Lawrence, Elijah S. and Marotz, Clarisse A. and Ngo, Toan T. and Ostrander, Tyler and Plascencia, Ashley and Salido, Rodolfo A. and Seaver, Phoebe and Smoot, Elizabeth W. and McDonald, Daniel and Neuhard, Robert M. and Scioscia, Angela L. and Satterlund, Alysson M. and Simmons, Elizabeth H. and Abelman, Dismas B. and Brenner, David and Bruner, Judith C. and Buckley, Anne and Ellison, Michael and Gattas, Jeffrey and Gonias, Steven L. and Hale, Matt and Hawkins, Faith and Ikeda, Lydia and Jhaveri, Hemlata and Johnson, Ted and Kellen, Vince and Kremer, Brendan and Matthews, Gary and McLawhon, Ronald W. and Ouillet, Pierre and Park, Daniel and Pradenas, Allorah and Reed, Sharon and Riggs, Lindsay and Sanders, Alison and Sollenberger, Bradley and Song, Angela and White, Benjamin and Winbush, Terri and Aceves, Christine M. and Anderson, Catelyn and Gangavarapu, Karthik and Hufbauer, Emory and Kurzban, Ezra and Lee, Justin and Matteson, Nathaniel L. and Parker, Edyth and Perkins, Sarah A. and Ramesh, Karthik S. and Robles-Sikisaka, Refugio and Schwab, Madison A. and Spencer, Emily and Wohl, Shirlee and Nicholson, Laura and McHardy, Ian H. and Dimmock, David P. and Hobbs, Charlotte A. and Bakhtar, Omid and Harding, Aaron and Mendoza, Art and Bolze, Alexandre and Becker, David and Cirulli, Elizabeth T. and Isaksson, Magnus and Schiabor Barrett, Kelly M. and Washington, Nicole L. and Malone, John D. and Murphy Schafer, Ashleigh and Gurfield, Nikos and Stous, Sarah and Fielding-Miller, Rebecca and Garfein, Richard S. and Gaines, Tommi and Anderson, Cheryl and Martin, Natasha K. and Schooley, Robert and Austin, Brett and MacCannell, Duncan R. and Kingsmore, Stephen F. and Lee, William and Shah, Seema and McDonald, Eric and Yu, Alexander T. and Zeller, Mark and Fisch, Kathleen M. and Longhurst, Christopher and Maysent, Patty and Pride, David and Khosla, Pradeep K. and Laurent, Louise C. and Yeo, Gene W. and Andersen, Kristian G. and Knight, Rob (2022) Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission. Nature, 609 (7925). pp. 101-108. ISSN 0028-0836. PMCID PMC9433318. doi:10.1038/s41586-022-05049-6.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle ItemDiscussion Paper
Karthikeyan, Smruthi0000-0001-6226-4536
Levy, Joshua I.0000-0001-7573-7793
Belda-Ferre, Pedro0000-0001-6532-1161
McDonald, Daniel0000-0003-0876-9060
Simmons, Elizabeth H.0000-0002-0646-3458
Lee, Justin0000-0002-3657-4386
Perkins, Sarah A.0000-0002-0557-3102
Schwab, Madison A.0000-0003-0748-1858
Nicholson, Laura0000-0002-1761-0951
McHardy, Ian H.0000-0002-2121-5460
Dimmock, David P.0000-0001-6690-2523
Bolze, Alexandre0000-0001-7399-2766
Isaksson, Magnus0000-0001-6278-0522
Schiabor Barrett, Kelly M.0000-0001-6194-787X
Washington, Nicole L.0000-0001-8936-9143
Murphy Schafer, Ashleigh0000-0002-5131-8399
Fielding-Miller, Rebecca0000-0002-5099-0589
Schooley, Robert0000-0002-2498-8426
Longhurst, Christopher0000-0003-4908-6856
Yeo, Gene W.0000-0002-0799-6037
Andersen, Kristian G.0000-0001-6431-5982
Knight, Rob0000-0002-0975-9019
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 ( The UCSD campus dashboard can be accessed at The county wastewater data from Point Loma are available through the public dashboard at The SEARCH genomic surveillance dashboard is available at Code availability. Freyja is hosted publicly on GitHub ( and is available under a BSD-2-Clause license (, all versions). Freyja is accessible as a package via bioconda ( in container form via dockerhub ( COVID-19 Viral Epidemiology Workflow (C-VIEW) is available at 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.
Funding AgencyGrant Number
University of California, San DiegoUNSPECIFIED
Centers for Disease Control and Prevention75D30121P10258
Centers for Disease Control and Prevention75D30120C09795
Conrad Prebys FoundationUNSPECIFIED
NIH Predoctoral Fellowship5T32AI007244-38
Issue or Number:7925
PubMed Central ID:PMC9433318
Record Number:CaltechAUTHORS:20221215-539953000.2
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:118343
Deposited By: George Porter
Deposited On:17 Dec 2022 00:48
Last Modified:17 Dec 2022 00:48

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