Detection of Mutations Associated with Variants of Concern Via High Throughput Sequencing of SARS-CoV-2 Isolated from NYC Wastewater

  1. Davida S. Smyth
  2. Monica Trujillo
  3. Kristen Cheung
  4. Anna Gao
  5. Irene Hoxie
  6. Sherin Kannoly
  7. Nanami Kubota
  8. Michelle Markman
  9. Kaung Myat San
  10. Geena Sompanya
  11. John J. Dennehy

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Abstract

Monitoring SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To date, most data on SARS-CoV-2 genetic diversity has come from the sequencing of clinical samples, but such studies may suffer limitations due to costs and throughput. Wastewater-based epidemiology may provide an alternative and complementary approach for monitoring communities for novel variants. Given that SARS-CoV-2 can infect the cells of the human gut and is found in high concentrations in feces, wastewater may be a valuable source of SARS-CoV-2 RNA, which can be deep sequenced to provide information on the circulating variants in a community. Here we describe a safe, affordable protocol for the sequencing of SARS-CoV-2 RNA using high-throughput Illumina sequencing technology. Our targeted sequencing approach revealed the presence of mutations associated with several Variants of Concern at appreciable frequencies. Our work demonstrates that wastewater-based SARS-CoV-2 sequencing can inform surveillance efforts monitoring the community spread of SARS-CoV-2 Variants of Concern and detect the appearance of novel emerging variants more cheaply, safely, and efficiently than the sequencing of individual clinical samples.

IMPORTANCE

The SARS-CoV-2 pandemic has caused millions of deaths around the world as countries struggle to contain infections. The pandemic will not end until herd immunity is reached, that is, when most of the population has either recovered from SARS-CoV-2 infection or is vaccinated against SARS-CoV-2. However, the emergence of new SARS-CoV-2 variants of concern threatens to erase gains. Emerging new variants may re-infect persons who have recovered from COVID-19 or may evade vaccine-induced immunity. However, scaling up SARS-CoV-2 genetic sequencing to monitor Variants of Concern in communities around the world is challenging. Wastewater-based sequencing of SARS-CoV-2 RNA can be used to monitor the presence of emerging variants in large communities to enact control measures to minimize the spread of these variants. We describe here the identification of alleles associated with several variants of concern in wastewater obtained from NYC watersheds.

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    SciScore for 10.1101/2021.03.21.21253978: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Variants were called using the Annotate and Predict Find Variations/SNPs in Geneious and verified by using the V-PIPE SARS-CoV-2 application (https://cbg-ethz.github.io/V-pipe/sars-cov-2/)(28).
    Geneious
    suggested: (Geneious, RRID:SCR_010519)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A caveat with our approach, however, is that we cannot conclusively identify the presence of a Variant of Concern since our sequencing assay targets only a region of the receptor binding domain, and some significant mutations are outside the sequenced region. Furthermore, additional mutations occurring in the primer binding region may allow some mutations to go undetected because their DNA could not be amplified. We are expanding our targeted sequencing approach to include additional regions of interest to minimize the chance of missing important variants. Additionally, we intend to generate cDNA with random hexamers, and to incorporate a level of degeneracy in the sequencing primers to increase the breadth of our targeted sequencing. Our most recent data from March 14th suggests a slight decrease in the prevalence of the E484K variant, but we cannot draw firm conclusions due to the nature of our sequencing assay, which relies on the collective sequencing of a large pool of individuals. Nevertheless, our frequency data agrees with that recently observed in human clinical samples from NYC (23, 24, 27). We intend to supplement our targeted sequencing approach with whole genome amplicon sequencing in the future. We believe that our approach offers a viable alternative to whole genome sequencing for the detection of known variants and can be rapidly deployed to detect additional emerging variants of concern. Importantly as a cost saving measure, labs can generate the libraries th...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2021.03.21.21253978 on medRxiv