key: cord-0972747-goer8eqg
authors: Vo, Van; Tillett, Richard L.; Chang, Ching-Lan; Gerrity, Daniel; Betancourt, Walter Q.; Oh, Edwin C.
title: SARS-CoV-2 variant detection at a university dormitory using wastewater genomic tools
date: 2021-09-07
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2021.149930
sha: a019e27555d5965c13c10273c0d0873de410cecd
doc_id: 972747
cord_uid: goer8eqg

In the Fall of 2020, university campuses in the United States resumed on-campus instruction and implemented wastewater monitoring for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While quantitative polymerase chain reaction (qPCR) tests were deployed successfully to detect viral RNA in wastewater across campuses, the feasibility of detecting viral variants from a residential building like a dormitory was unclear. Here, we demonstrate that wastewater surveillance from a dormitory with at least three infected students could lead to the identification of viral genomes with more than 95% coverage. Our results indicate that viral variant detection from wastewater is achievable at a dormitory and that coronavirus disease 2019 (COVID-19) wastewater surveillance programs will benefit from the implementation of viral whole genome sequencing at universities.

Whole genome sequencing (WGS) of SARS-CoV-2 from large wastewater treatment plants has proven to be an emerging technology for tracing viral evolution [1] [2] [3] ; however, the utility of such tools at the level of a single building is only starting to emerge 4 .

Several studies have suggested that successful genome sequencing requires at least 2.8x10 5 viral copies per liter of wastewater [1] [2] [3] , yet this calculation can be variable and dependent on sample preparation and concentration procedures.

To test whether WGS can be used to identify SARS-CoV-2 genomes and emerging variants from a dormitory, we sequenced and analyzed wastewater samples 5 during the reopening of the University of Arizona in the Fall semester of 2020. Using RNA samples that contained on average 1.1 × 10 6 SARS-CoV-2 copies/L, we identified viral signatures in the dormitory wastewater sample that included clade 20C and a 20A+20268G subclade. Our findings intimate that transmission of at least two cluster strains in college students was occurring from the onset of the potential outbreak.

Obtaining wastewater viral genomes from a dormitory with at least three infected individuals out of 311 residents supports the hypothesis that wastewater WGS has the potential to complement other public health resources to monitor patterns of viral transmission at the level of a single building. In addition, the utility of wastewater WGS for variant surveillance may serve an even more important role in 2021/2022 due to 1) the decline of clinical testing, and 2) testing and vaccination hesitancy in communities. 

RNAse free dH2O.

Whole genome sequencing libraries were constructed using the CleanPlex SARS-CoV-2 FLEX Panel from Paragon Genomics by following the manufacturer's instructions.

Total RNA (10ng) was processed for first-strand cDNA synthesis. Libraries were sequenced using an Illumina NextSeq 500 platform and a mid-output v2.5 (300 cycles) flow cell. Illumina adapter sequences were trimmed from reads using cutadapt v3.2 6 . All sequencing reads were mapped to SARS-CoV-2 genome (NC_045512.2) using bwa mem v0.7.17-r1188 7 . Amplicon primers were trimmed from aligned reads using fgbio TrimPrimers v1.3.0 and variants were called with iVar variants v1.3 8 . Genome coverages were calculated by samtools coverage v1.10 9 . Coverage of the SARS-CoV-2 genomes was above 95% with a median depth above 50X. Phylogenetic analysis was conducted on March 1, 2021, by using Nextstrain and open-source tools for visualizing viral genomes 10 . Genome sequences of 3988 isolates were obtained from GISAID and aligned to produce a phylogenetic tree using the NGphylogeny.fr FastME workflow 11 (Supplementary Table 1 ). Nextstrain clade memberships of genomes were inferred using nextstrain-cli v1.16.5 and applied to the FastME tree.

As part of a larger public health surveillance effort, morning wastewater samples were WGS of SARS-CoV-2 genomes from municipal sewage treatment plants servicing large residential and commercial area has been achieved previously [1] [2] [3] . In these studies, hybrid capture (Illumina respiratory virus panel) 2 and amplicon-based (Oxford Nanopore) 1,3 library preparation kits were used to amplify the target genomes before sequencing on an Illumina instrument. Here, we demonstrate that our processing 5,13 and genomic tools, using another amplicon-based approach (Paragon Genomics), have the specificity and sensitivity to sequence single viral genomes from an individual housing unit like a university dormitory. Interestingly, the observation of two viral signatures in the wastewater sample (Figure 1 ) suggests that transmission of at least two cluster strains in college students was occurring from the onset of the potential outbreak 5 . However, our study did not compare clinical genomes from infected students and our conclusions about potential transmission remain limited.

The findings in this study demonstrate that WGS of wastewater samples can be performed in a laboratory setting and does not require extensive resources that are typically found in large universities or biotechnology companies with comprehensive core services. In addition, due to the relatively small size of the SARS-CoV-2 genome, smaller desktop Illumina instruments can be utilized for sequencing hundreds of viral genomes using a single flow cell. Given the availability of such resources, we propose the inclusion of whole genome sequencing in wastewater surveillance programs with J o u r n a l P r e -p r o o f other public health measures to identify viral variants that may be present in a university dormitory. Currently, both qPCR and digital PCR (dPCR) strategies are utilized to determine whether viral load can be detected and whether potential variants of concern may be circulating in a community; while costs can be somewhat limited by using PCR techniques to identify signature mutations in viral strains, the implementation of WGS enables public health authorities to definitively identify viral strains and manage local outbreaks. Such sequencing techniques are especially important given the emergence and evolution of new lineages and sub-lineages of variants over the last 12 months 14, 15 .

Moving forward, we can envision a situation when qPCR/dPCR strategies will be used to triage samples for more detailed analyses like WGS. In addition, the combination of such genomic resources, especially when diagnostic testing may be limited, will have the potential to link the earliest encounter of a viral variant to the index case(s), thereby limiting the spread of an infectious disease. 

Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater

Genome Sequencing of Sewage Detects Regionally Prevalent SARS-CoV-2 Variants. mBio

Monitoring SARS-CoV-2 Circulation and Diversity through Community Wastewater Sequencing, the Netherlands and Belgium. Emerg Infect Dis

COVID-19 containment on a college campus via wastewater-based epidemiology, targeted clinical testing and an intervention

Cutadapt removes adapter sequences from high-throughput sequencing reads

Aligning sequence reads, clone sequences and assembly contigs with

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

Genome biology

The sequence alignment/map format and SAMtools

Nextstrain: real-time tracking of pathogen evolution

fr: new generation phylogenetic services for non-specialists

Introduction and Characteristics of SARS-CoV-2 in North-East of Romania During the First

Early-pandemic wastewater surveillance of SARS-CoV-2 in Southern Nevada: Methodology, occurrence, and incidence/prevalence considerations

COVID-19 CG enables SARS-CoV-2 mutation and lineage tracking by locations and dates of interest

Credit authorship contribution statement Van Vo: Methodology, Supervision, Formal analysis, Writing -original draft. Richard L. Tillett: Methodology, Supervision, Formal analysis, Writing -original draft. Ching-Lan Chang: Resource, Writing -reviewing & editing. Daniel Gerrity: Resource, Writingreviewing & editing. Walter W. Betancourt: Formal analysis, Visualization, Writing -original draft

Tillett are co-first authors and contributed equally

Betancourt and Edwin Oh are co-corresponding authors