Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila.

Regulatory decisions in Drosophila require Polycomb group (PcG) proteins to maintain the silent state and Trithorax group (TrxG) proteins to oppose silencing. Since PcG and TrxG are ubiquitous and lack apparent sequence specificity, a long-standing model is that targeting occurs via protein interactions; for instance, between repressors and PcG proteins. Instead, we found that Pc-repressive complex 1 (PRC1) purifies with coactivators Fs(1)h [female sterile (1) homeotic] and Enok/Br140 during embryogenesis. Fs(1)h is a TrxG member and the ortholog of BRD4, a bromodomain protein that binds to acetylated histones and is a key transcriptional coactivator in mammals. Enok and Br140, another bromodomain protein, are orthologous to subunits of a mammalian MOZ/MORF acetyltransferase complex. Here we confirm PRC1-Br140 and PRC1-Fs(1)h interactions and identify their genomic binding sites. PRC1-Br140 bind developmental genes in fly embryos, with analogous co-occupancy of PRC1 and a Br140 ortholog, BRD1, at bivalent loci in human embryonic stem (ES) cells. We propose that identification of PRC1-Br140 "bivalent complexes" in fly embryos supports and extends the bivalency model posited in mammalian cells, in which the coexistence of H3K4me3 and H3K27me3 at developmental promoters represents a poised transcriptional state. We further speculate that local competition between acetylation and deacetylation may play a critical role in the resolution of bivalent protein complexes during development.

Molluscan Genomes Reveal Extensive Differences in Photopigment Evolution Across the Phylum.

In animals, opsins and cryptochromes are major protein families that transduce light signals when bound to light-absorbing chromophores. Opsins are involved in various light-dependent processes, like vision, and have been co-opted for light-independent sensory modalities. Cryptochromes are important photoreceptors in animals, generally regulating circadian rhythm, they belong to a larger protein family with photolyases, which repair UV-induced DNA damage. Mollusks are great animals to explore questions about light sensing as eyes have evolved multiple times across, and within, taxonomic classes. We used molluscan genome assemblies from 80 species to predict protein sequences and examine gene family evolution using phylogenetic approaches. We found extensive opsin family expansion and contraction, particularly in bivalve xenopsins and gastropod Go-opsins, while other opsins, like retinochrome, rarely duplicate. Bivalve and gastropod lineages exhibit fluctuations in opsin repertoire, with cephalopods having the fewest number of opsins and loss of at least 2 major opsin types. Interestingly, opsin expansions are not limited to eyed species, and the highest opsin content was seen in eyeless bivalves. The dynamic nature of opsin evolution is quite contrary to the general lack of diversification in mollusk cryptochromes, though some taxa, including cephalopods and terrestrial gastropods, have reduced repertoires of both protein families. We also found complete loss of opsins and cryptochromes in multiple, but not all, deep-sea species. These results help set the stage for connecting genomic changes, including opsin family expansion and contraction, with differences in environmental, and biological features across Mollusca.

Single-molecule Sequencing of an Animal Mitochondrial Genome Reveals Chloroplast-like Architecture and Repeat-mediated Recombination.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of the mitochondrial genome architecture and recombination. We used PacBio sequencing to reassemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus. These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by an assembly of short reads and likely corresponding to a large noncoding region commonly present in the mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy (LSC) region and a small single-copy (SSC) region separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa-SSC-IRb structure revealed the occurrence of a "flip-flop" recombination. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus, as well as extensive intermolecular recombination between single-nucleotide polymorphisms in the LSC region. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of inverted repeats in cytoplasmic genomes and their role in mitochondrial genome evolution.

Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila.

The Polycomb group (PcG) proteins are key regulators of development in Drosophila and are strongly implicated in human health and disease. How PcG complexes form repressive chromatin domains remains unclear. Using cross-linked affinity purifications of BioTAP-Polycomb (Pc) or BioTAP-Enhancer of zeste [E(z)], we captured all PcG-repressive complex 1 (PRC1) or PRC2 core components and Sex comb on midleg (Scm) as the only protein strongly enriched with both complexes. Although previously not linked to PRC2, we confirmed direct binding of Scm and PRC2 using recombinant protein expression and colocalization of Scm with PRC1, PRC2, and H3K27me3 in embryos and cultured cells using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing). Furthermore, we found that RNAi knockdown of Scm and overexpression of the dominant-negative Scm-SAM (sterile α motif) domain both affected the binding pattern of E(z) on polytene chromosomes. Aberrant localization of the Scm-SAM domain in long contiguous regions on polytene chromosomes revealed its independent ability to spread on chromatin, consistent with its previously described ability to oligomerize in vitro. Pull-downs of BioTAP-Scm captured PRC1 and PRC2 and additional repressive complexes, including PhoRC, LINT, and CtBP. We propose that Scm is a key mediator connecting PRC1, PRC2, and transcriptional silencing. Combined with previous structural and genetic analyses, our results strongly suggest that Scm coordinates PcG complexes and polymerizes to produce broad domains of PcG silencing.

Asexuality Associated with Marked Genomic Expansion of Tandemly Repeated rRNA and Histone Genes.

How does asexual reproduction influence genome evolution? Although is it clear that genomic structural variation is common and important in natural populations, we know very little about how one of the most fundamental of eukaryotic traits-mode of genomic inheritance-influences genome structure. We address this question with the New Zealand freshwater snail Potamopyrgus antipodarum, which features multiple separately derived obligately asexual lineages that coexist and compete with otherwise similar sexual lineages. We used whole-genome sequencing reads from a diverse set of sexual and asexual individuals to analyze genomic abundance of a critically important gene family, rDNA (the genes encoding rRNAs), that is notable for dynamic and variable copy number. Our genomic survey of rDNA in P. antipodarum revealed two striking results. First, the core histone and 5S rRNA genes occur between tandem copies of the 18S-5.8S-28S gene cluster, a unique architecture for these crucial gene families. Second, asexual P. antipodarum harbor dramatically more rDNA-histone copies than sexuals, which we validated through molecular and cytogenetic analysis. The repeated expansion of this genomic region in asexual P. antipodarum lineages following distinct transitions to asexuality represents a dramatic genome structural change associated with asexual reproduction-with potential functional consequences related to the loss of sexual reproduction.

Additive and epistatic effects influence spectral tuning in molluscan retinochrome opsin.

The relationship between genotype and phenotype is non-trivial because of the often complex molecular pathways that make it difficult to unambiguously relate phenotypes to specific genotypes. Photopigments, comprising an opsin apoprotein bound to a light-absorbing chromophore, present an opportunity to directly relate the amino acid sequence to an absorbance peak phenotype (λmax). We examined this relationship by conducting a series of site-directed mutagenesis experiments of retinochrome, a non-visual opsin, from two closely related species: the common bay scallop, Argopecten irradians, and the king scallop, Pecten maximus. Using protein folding models, we identified three amino acid sites of likely functional importance and expressed mutated retinochrome proteins in vitro. Our results show that the mutation of amino acids lining the opsin binding pocket is responsible for fine spectral tuning, or small changes in the λmax of these light-sensitive proteins. Mutations resulted in a blue or red shift as predicted, but with dissimilar magnitudes. Shifts ranged from a 16 nm blue shift to a 12 nm red shift from the wild-type λmax. These mutations do not show an additive effect, but rather suggest the presence of epistatic interactions. This work highlights the importance of binding pocket shape in the evolution of spectral tuning and builds on our ability to relate genotypic changes to phenotypes in an emerging model for opsin functional analysis.

The Epithelial adhesin 1 tandem repeat region mediates protein display through multiple mechanisms.

The pathogenic yeast Candida glabrata is reliant on a suite of cell surface adhesins that play a variety of roles necessary for transmission, establishment and proliferation during infection. One particular adhesin, Epithelial Adhesin 1 [Epa1p], is responsible for binding to host tissue, a process which is essential for fungal propagation. Epa1p structure consists of three domains: an N-terminal intercellular binding domain responsible for epithelial cell binding, a C-terminal GPI anchor for cell wall linkage and a serine/threonine-rich linker domain connecting these terminal domains. The linker domain contains a 40-amino acid tandem repeat region, which we have found to be variable in repeat copy number between isolates from clinical sources. We hypothesized that natural variation in Epa1p repeat copy may modulate protein function. To test this, we recombinantly expressed Epa1p with various repeat copy numbers in S. cerevisiae to determine how differences in repeat copy number affect Epa1p expression, surface display and binding to human epithelial cells. Our data suggest that repeat copy number variation has pleiotropic effects, influencing gene expression, protein surface display and shedding from the cell surface of the Epa1p adhesin. This study serves to demonstrate repeat copy number variation can modulate protein function through a number of mechanisms in order to contribute to pathogenicity of C. glabrata.

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry.

Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.

Genomic evidence for population-specific responses to co-evolving parasites in a New Zealand freshwater snail.

Reciprocal co-evolving interactions between hosts and parasites are a primary source of strong selection that can promote rapid and often population- or genotype-specific evolutionary change. These host-parasite interactions are also a major source of disease. Despite their importance, very little is known about the genomic basis of co-evolving host-parasite interactions in natural populations, especially in animals. Here, we use gene expression and sequence evolution approaches to take critical steps towards characterizing the genomic basis of interactions between the freshwater snail Potamopyrgus antipodarum and its co-evolving sterilizing trematode parasite, Microphallus sp., a textbook example of natural coevolution. We found that Microphallus-infected P. antipodarum exhibit systematic downregulation of genes relative to uninfected P. antipodarum. The specific genes involved in parasite response differ markedly across lakes, consistent with a scenario where population-level co-evolution is leading to population-specific host-parasite interactions and evolutionary trajectories. We also used an FST -based approach to identify a set of loci that represent promising candidates for targets of parasite-mediated selection across lakes as well as within each lake population. These results constitute the first genomic evidence for population-specific responses to co-evolving infection in the P. antipodarum-Microphallus interaction and provide new insights into the genomic basis of co-evolutionary interactions in nature.

Opsin expression varies across larval development and taxa in pteriomorphian bivalves.

Introduction: Many marine organisms have a biphasic life cycle that transitions between a swimming larva with a more sedentary adult form. At the end of the first phase, larvae must identify suitable sites to settle and undergo a dramatic morphological change. Environmental factors, including photic and chemical cues, appear to influence settlement, but the sensory receptors involved are largely unknown. We targeted the protein receptor, opsin, which belongs to large superfamily of transmembrane receptors that detects environmental stimuli, hormones, and neurotransmitters. While opsins are well-known for light-sensing, including vision, a growing number of studies have demonstrated light-independent functions. We therefore examined opsin expression in the Pteriomorphia, a large, diverse clade of marine bivalves, that includes commercially important species, such as oysters, mussels, and scallops. Methods: Genomic annotations combined with phylogenetic analysis show great variation of opsin abundance among pteriomorphian bivalves, including surprisingly high genomic abundance in many species that are eyeless as adults, such as mussels. Therefore, we investigated the diversity of opsin expression from the perspective of larval development. We collected opsin gene expression in four families of Pteriomorphia, across three distinct larval stages, i.e., trochophore, veliger, and pediveliger, and compared those to adult tissues. Results: We found larvae express all opsin types in these bivalves, but opsin expression patterns are largely species-specific across development. Few opsins are expressed in the adult mantle, but many are highly expressed in adult eyes. Intriguingly, opsin genes such as retinochrome, xenopsins, and Go-opsins have higher levels of expression in the later larval stages when substrates for settlement are being tested, such as the pediveliger. Conclusion: Investigating opsin gene expression during larval development provides crucial insights into their intricate interactions with the surroundings, which may shed light on how opsin receptors of these organisms respond to various environmental cues that play a pivotal role in their settlement process.

Ocean state rising: Storm simulation and vulnerability mapping to predict hurricane impacts for Rhode Island's critical infrastructure.

Predicting the consequences of a major coastal storm is increasingly difficult as the result of global climate change and growing societal dependence on critical infrastructure (CI). Past storms are no longer a reliable predictor of future weather events, and the traditional approach to vulnerability assessment presents accumulated loss in largely quantitative terms that lack the specificity local emergency managers need to develop effective plans and mitigation strategies. The Rhode Island Coastal Hazards Modeling and Prediction (RI-CHAMP) system is a geographic information system (GIS)-based modeling tool that combines high-resolution storm simulations with geolocated vulnerability data to predict specific consequences based on local concerns about impacts to CI. This case study discusses implementing RI-CHAMP for the State of Rhode Island to predict impacts of wind and inundation on its CI during a hurricane, tropical storm, or nor'easter. This paper addresses the collection and field verification of vulnerability data, along with RI-CHAMP's process for integrating those data with storm models. The project deeply engaged end-users (emergency managers, facility managers, and other stakeholders) in developing RI-CHAMP's ArcGIS Online dashboard to ensure it provides specific, actionable data. The results of real and synthetic storm models are presented along with discussion of how the data in these simulations are being used by state and local emergency managers, facility owners, and others.

A chromosome-level genome assembly of the disco clam, Ctenoides ales.

The bivalve subclass Pteriomorphia, which includes the economically important scallops, oysters, mussels, and ark clams, exhibits extreme ecological, morphological, and behavioral diversity. Among this diversity are five morphologically distinct eye types, making Pteriomorphia an excellent setting to explore the molecular basis for the evolution of novel traits. Of pteriomorphian bivalves, Limida is the only order lacking genomic resources, greatly limiting the potential phylogenomic analyses related to eyes and phototransduction. Here, we present a limid genome assembly, the disco clam, Ctenoides ales, which is characterized by invaginated eyes, exceptionally long tentacles, and a flashing light display. This genome assembly was constructed with PacBio long reads and Dovetail Omni-CTM proximity-ligation sequencing. The final assembly is ∼2.3Gb and over 99% of the total length is contained in 18 pseudomolecule scaffolds. We annotated 41,064 protein coding genes and report a BUSCO completeness of 91.9% for metazoa_obd10. Additionally, we report a complete and annotated mitochondrial genome, which also had been lacking from Limida. The ∼20Kb mitogenome has 12 protein coding genes, 22 tRNAs, 2 rRNA genes, and a 1,589 bp duplicated sequence containing the origin of replication. The C. ales nuclear genome size is substantially larger than other pteriomorphian genomes, mainly accounted for by transposable element sequences. We inventoried the genome for opsins, the signaling proteins that initiate phototransduction, and found that, unlike its closest eyed-relatives, the scallops, C. ales lacks duplication of the rhabdomeric Gq-protein coupled opsin that is typically used for invertebrate vision. In fact, C. ales has uncharacteristically few opsins relative to the other pteriomorphian families, all of which have unique expansions of xenopsins, a recently discovered opsin subfamily. This chromosome-level assembly, along with the mitogenome, will be valuable resources for comparative genomics and phylogenetics in bivalves and particularly for the understudied but charismatic limids.

Genome Evolution and Introgression in the New Zealand mud Snails Potamopyrgus estuarinus and Potamopyrgus kaitunuparaoa.

We have sequenced, assembled, and analyzed the nuclear and mitochondrial genomes and transcriptomes of Potamopyrgus estuarinus and Potamopyrgus kaitunuparaoa, two prosobranch snail species native to New Zealand that together span the continuum from estuary to freshwater. These two species are the closest known relatives of the freshwater species Potamopyrgus antipodarum-a model for studying the evolution of sex, host-parasite coevolution, and biological invasiveness-and thus provide key evolutionary context for understanding its unusual biology. The P. estuarinus and P. kaitunuparaoa genomes are very similar in size and overall gene content. Comparative analyses of genome content indicate that these two species harbor a near-identical set of genes involved in meiosis and sperm functions, including seven genes with meiosis-specific functions. These results are consistent with obligate sexual reproduction in these two species and provide a framework for future analyses of P. antipodarum-a species comprising both obligately sexual and obligately asexual lineages, each separately derived from a sexual ancestor. Genome-wide multigene phylogenetic analyses indicate that P. kaitunuparaoa is likely the closest relative to P. antipodarum. We nevertheless show that there has been considerable introgression between P. estuarinus and P. kaitunuparaoa. That introgression does not extend to the mitochondrial genome, which appears to serve as a barrier to hybridization between P. estuarinus and P. kaitunuparaoa. Nuclear-encoded genes whose products function in joint mitochondrial-nuclear enzyme complexes exhibit similar patterns of nonintrogression, indicating that incompatibilities between the mitochondrial and the nuclear genome may have prevented more extensive gene flow between these two species.

Metrics to relate COVID-19 wastewater data to clinical testing dynamics.

Wastewater surveillance has emerged as a useful tool in the public health response to the COVID-19 pandemic. While wastewater surveillance has been applied at various scales to monitor population-level COVID-19 dynamics, there is a need for quantitative metrics to interpret wastewater data in the context of public health trends. 24-hour composite wastewater samples were collected from March 2020 through May 2021 from a Massachusetts wastewater treatment plant and SARS-CoV-2 RNA concentrations were measured using RT-qPCR. The relationship between wastewater copy numbers of SARS-CoV-2 gene fragments and COVID-19 clinical cases and deaths varies over time. We demonstrate the utility of three new metrics to monitor changes in COVID-19 epidemiology: (1) the ratio between wastewater copy numbers of SARS-CoV-2 gene fragments and clinical cases (WC ratio), (2) the time lag between wastewater and clinical reporting, and (3) a transfer function between the wastewater and clinical case curves. The WC ratio increases after key events, providing insight into the balance between disease spread and public health response. Time lag and transfer function analysis showed that wastewater data preceded clinically reported cases in the first wave of the pandemic but did not serve as a leading indicator in the second wave, likely due to increased testing capacity, which allows for more timely case detection and reporting. These three metrics could help further integrate wastewater surveillance into the public health response to the COVID-19 pandemic and future pandemics.

Nationwide Trends in COVID-19 Cases and SARS-CoV-2 RNA Wastewater Concentrations in the United States.

Wastewater-based epidemiology has emerged as a promising technology for population-level surveillance of COVID-19. In this study, we present results of a large nationwide SARS-CoV-2 wastewater monitoring system in the United States. We profile 55 locations with at least six months of sampling from April 2020 to May 2021. These locations represent more than 12 million individuals across 19 states. Samples were collected approximately weekly by wastewater treatment utilities as part of a regular wastewater surveillance service and analyzed for SARS-CoV-2 RNA concentrations. SARS-CoV-2 RNA concentrations were normalized to pepper mild mottle virus, an indicator of fecal matter in wastewater. We show that wastewater data reflect temporal and geographic trends in clinical COVID-19 cases and investigate the impact of normalization on correlations with case data within and across locations. We also provide key lessons learned from our broad-scale implementation of wastewater-based epidemiology, which can be used to inform wastewater-based epidemiology approaches for future emerging diseases. This work demonstrates that wastewater surveillance is a feasible approach for nationwide population-level monitoring of COVID-19 disease. With an evolving epidemic and effective vaccines against SARS-CoV-2, wastewater-based epidemiology can serve as a passive surveillance approach for detecting changing dynamics or resurgences of the virus.

SARS-CoV-2 RNA concentrations in wastewater foreshadow dynamics and clinical presentation of new COVID-19 cases.

Current estimates of COVID-19 prevalence are largely based on symptomatic, clinically diagnosed cases. The existence of a large number of undiagnosed infections hampers population-wide investigation of viral circulation. Here, we quantify the SARS-CoV-2 concentration and track its dynamics in wastewater at a major urban wastewater treatment facility in Massachusetts, between early January and May 2020. SARS-CoV-2 was first detected in wastewater on March 3. SARS-CoV-2 RNA concentrations in wastewater correlated with clinically diagnosed new COVID-19 cases, with the trends appearing 4-10 days earlier in wastewater than in clinical data. We inferred viral shedding dynamics by modeling wastewater viral load as a convolution of back-dated new clinical cases with the average population-level viral shedding function. The inferred viral shedding function showed an early peak, likely before symptom onset and clinical diagnosis, consistent with emerging clinical and experimental evidence. This finding suggests that SARS-CoV-2 concentrations in wastewater may be primarily driven by viral shedding early in infection. This work shows that longitudinal wastewater analysis can be used to identify trends in disease transmission in advance of clinical case reporting, and infer early viral shedding dynamics for newly infected individuals, which are difficult to capture in clinical investigations.

Lineage abundance estimation for SARS-CoV-2 in wastewater using transcriptome quantification techniques.

Effectively monitoring the spread of SARS-CoV-2 mutants is essential to efforts to counter the ongoing pandemic. Predicting lineage abundance from wastewater, however, is technically challenging. We show that by sequencing SARS-CoV-2 RNA in wastewater and applying algorithms initially used for transcriptome quantification, we can estimate lineage abundance in wastewater samples. We find high variability in signal among individual samples, but the overall trends match those observed from sequencing clinical samples. Thus, while clinical sequencing remains a more sensitive technique for population surveillance, wastewater sequencing can be used to monitor trends in mutant prevalence in situations where clinical sequencing is unavailable.

Metrics to relate COVID-19 wastewater data to clinical testing dynamics.

Wastewater surveillance has emerged as a useful tool in the public health response to the COVID-19 pandemic. While wastewater surveillance has been applied at various scales to monitor population-level COVID-19 dynamics, there is a need for quantitative metrics to interpret wastewater data in the context of public health trends. We collected 24-hour composite wastewater samples from March 2020 through May 2021 from a Massachusetts wastewater treatment plant and measured SARS-CoV-2 RNA concentrations using RT-qPCR. We show that the relationship between wastewater viral titers and COVID-19 clinical cases and deaths varies over time. We demonstrate the utility of three new metrics to monitor changes in COVID-19 epidemiology: (1) the ratio between wastewater viral titers and clinical cases (WC ratio), (2) the time lag between wastewater and clinical reporting, and (3) a transfer function between the wastewater and clinical case curves. We find that the WC ratio increases after key events, providing insight into the balance between disease spread and public health response. We also find that wastewater data preceded clinically reported cases in the first wave of the pandemic but did not serve as a leading indicator in the second wave, likely due to increased testing capacity. These three metrics could complement a framework for integrating wastewater surveillance into the public health response to the COVID-19 pandemic and future pandemics.

Wastewater Surveillance of SARS-CoV-2 across 40 U.S. states.

Wastewater-based disease surveillance is a promising approach for monitoring community outbreaks. Here we describe a nationwide campaign to monitor SARS-CoV-2 in the wastewater of 159 counties in 40 U.S. states, covering 13% of the U.S. population from February 18 to June 2, 2020. Out of 1,751 total samples analyzed, 846 samples were positive for SARS-CoV-2 RNA, with overall viral concentrations declining from April to May. Wastewater viral titers were consistent with, and appeared to precede, clinical COVID-19 surveillance indicators, including daily new cases. Wastewater surveillance had a high detection rate (>80%) of SARS-CoV-2 when the daily incidence exceeded 13 per 100,000 people. Detection rates were positively associated with wastewater treatment plant catchment size. To our knowledge, this work represents the largest-scale wastewater-based SARS-CoV-2 monitoring campaign to date, encompassing a wide diversity of wastewater treatment facilities and geographic locations. Our findings demonstrate that a national wastewater-based approach to disease surveillance may be feasible and effective.