Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission
Authors: Karthikeyan Smruthi , Levy Joshua I. , 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 , 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 , Neuhard Robert M. , Scioscia Angela L. , Satterlund Alysson M. , Simmons Elizabeth H. , 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 , Matteson Nathaniel L. , Parker Edyth , Perkins Sarah A. , Ramesh Karthik S. , Robles-Sikisaka Refugio , Schwab Madison A. , Spencer Emily , Wohl Shirlee , Nicholson Laura , McHardy Ian H. , Dimmock David P. , Hobbs Charlotte A. , Bakhtar Omid , Harding Aaron , Mendoza Art , Bolze Alexandre , Becker David , Cirulli Elizabeth T. , Isaksson Magnus , Schiabor Barrett Kelly M. , Washington Nicole L. , Malone John D. , Schafer Ashleigh Murphy , Gurfield Nikos , Stous Sarah , Fielding-Miller Rebecca , Garfein Richard S. , Gaines Tommi , Anderson Cheryl , Martin Natasha K. , Schooley Robert , Austin Brett , MacCannell Duncan R. , Kingsmore Stephen F. , Lee William , Shah Seema , McDonald Eric , Yu Alexander T. , Zeller Mark , Fisch Kathleen M. , Longhurst Christopher , Maysent Patty , Pride David , Khosla Pradeep K. , Laurent Louise C. , Yeo Gene W. , Andersen Kristian G. , Knight Rob 
Published: July 2022

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.