key: cord-0795952-vtyuc1yt authors: Lin, X.; Glier, M.; Kuchinski, K.; Ross-Van Mierlo, T.; McVea, D.; Tyson, J. R.; Prystajecky, N.; Ziels, R. M. title: Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater date: 2021-05-29 journal: nan DOI: 10.1101/2021.05.26.21257861 sha: d40a12b352ac0b4a5a7b27f70e0be74bbbe8f3a1 doc_id: 795952 cord_uid: vtyuc1yt Wastewater-based genomic surveillance of the SARS-CoV-2 virus shows promise to complement genomic epidemiology efforts. Multiplex tiled PCR is a desirable approach for targeted genome sequencing of SARS-CoV-2 in wastewater due to its low cost and rapid turnaround time. However, it is not clear how different multiplex tiled PCR primer schemes or wastewater sample matrices impact the resulting SARS-CoV-2 genome coverage. The objective of this work was to assess the performance of three different multiplex primer schemes, consisting of 150bp, 400bp, and 1200bp amplicons, as well as two wastewater sample matrices, influent wastewater and primary sludge, for targeted genome sequencing of SARS-CoV-2. Wastewater samples were collected weekly from five municipal wastewater treatment plants (WWTPs) in the Metro Vancouver region of British Columbia, Canada during a period of increased COVID-19 case counts from February to April, 2021. RNA extracted from clarified influent wastewater provided significantly higher genome coverage (breadth and median depth) than primary sludge samples across all primer schemes. Shorter amplicons appeared more resilient to sample RNA degradation, but were hindered by greater primer pool complexity in the 150bp scheme. The identified optimal primer scheme (400bp) and sample matrix (influent) was capable of detecting the emergence of mutations associated with genomic variants of concern, of which the daily wastewater load significantly correlated with clinical case counts. Taken together, these results provide guidance on best practices for implementing wastewater-based genomic surveillance, and demonstrate its ability to inform epidemiology efforts by detecting genomic variants of concern circulating within a geographic region. February to April, 2021. RNA extracted from clarified influent wastewater provided 31 significantly higher genome coverage (breadth and median depth) than primary sludge 32 samples across all primer schemes. Shorter amplicons appeared more resilient to sample 33 RNA degradation, but were hindered by greater primer pool complexity in the 150bp 34 scheme. The identified optimal primer scheme (400bp) and sample matrix (influent) was 35 capable of detecting the emergence of mutations associated with genomic variants of 36 concern, of which the daily wastewater load significantly correlated with clinical case 37 counts. Taken together, these results provide guidance on best practices for 38 implementing wastewater-based genomic surveillance, and demonstrate its ability to 39 inform epidemiology efforts by detecting genomic variants of concern circulating within a 40 geographic region. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Importance 43 Monitoring the genomic characteristics of the SARS-CoV-2 virus circulating in a 44 population can shed important insights into epidemiological aspects of the COVID-19 45 outbreak. Sequencing every clinical patient sample in a highly populous area is a difficult 46 feat, and thus sequencing SARS-CoV-2 RNA in municipal wastewater offers great 47 promise to augment genomic surveillance by characterizing a pooled population sample 48 matrix, particularly during an escalating outbreak. Here, we assess different approaches 49 and sample matrices for rapid targeted genome sequencing of SARS-CoV-2 in municipal 50 wastewater. We demonstrate that the optimal approach is capable of detecting the 51 emergence of SARS-CoV-2 genomic variants of concern, with strong correlations to 52 clinical case data in the province of British Columbia. These results provide guidance on 53 best practices on, as well as further support for, the application of wastewater genomic 54 surveillance as a tool to augment current genomic epidemiology efforts. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Genomic surveillance of the SARS-CoV-2 virus plays a critical role in tracking its evolution 57 during the current global COVID-19 pandemic (1) (2) (3) . Recently, several emerging lineages 58 of SARS-CoV-2, so-called variants of concern (VoCs), have been associated with 59 increased levels of transmission (4), disease severity (5), and/or immune escape (6, 7). These VoCs have originated from various locations globally (4, 8), but are spreading 61 within new geographic regions due to travel-associated and local transmission (9). Providing rapid detection of VoC infections within a population could thus help to inform 63 effective public health outbreak mitigation strategies. As the SARS-CoV-2 virus is shed in feces during infection (10), viral genome fragments 66 can be detected in municipal wastewater, and have been associated with clinical case 67 numbers within contributing regions (11) (12) (13) (14) . Previous work has demonstrated the 68 potential to sequence SARS-CoV-2 fragments in municipal wastewater and detect single 69 nucleotide variants (SNVs) that correspond to clinical cases in the contributing sewershed 70 (15-17). As SARS-CoV-2 titers in wastewater are relatively low (11, 13), an enrichment 71 step is typically needed prior to sequencing to improve sensitivity (15). The two main 72 approaches for enriching SARS-CoV-2 RNA in wastewater include oligonucleotide based 73 capture (15), and multiplex tiled PCR based targeted amplification (16, 17). The latter 74 approach is promising for wastewater-based viral genomic surveillance due to its lower 75 reagent cost and potential to be deployed rapidly and in remote locations (18). An 76 important consideration for applying multiplex tiled PCR is the average amplicon length, 77 as this can impact assay sensitivity in the case of RNA degradation (19). This could be 78 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2021. ; https://doi.org/10.1101/2021.05.26.21257861 doi: medRxiv preprint particularly important for its application to wastewater based epidemiology, as SARS- CoV-2 particles and free RNA can undergo variable levels of degradation (20, 21), and 80 may vary based on the type of wastewater sample matrix (e.g. influent versus primary 81 sludge) (22). We therefore hypothesized that there may be an optimal tiled PCR amplicon 82 size and wastewater sample matrix type that enables adequate genome coverage of 83 SARS-CoV-2 for the identification of genomic VoCs. CoV-2 genome coverage. 87 We sequenced a total of 96 wastewater samples collected between February 7 th to April this finding could be that the sludge matrix was inhibitory to RT-PCR (11); however, no 98 inhibition of RT-qPCR on sludge RNA extracts was detected using internal controls (Text 99 S1, Table S2 ). Another potential reason for the lower genome coverage in sludge is that 100 SARS-CoV-2 was more nonintact or its RNA more degraded with the direct sludge 101 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2021. ; https://doi.org/10.1101/2021.05.26.21257861 doi: medRxiv preprint 6 extraction compared to ultrafiltration of influent wastewater, as has been previously 102 hypothesized (22). A third potential cause of discrepancies in genome coverage between 103 sludge and influent wastewater samples could be higher off-target amplification in sludge 104 extracts. Correspondingly, the sample type significantly impacted read mapping rates for 105 all schemes after accounting for Ct values (p<0.01, two-way ANCOVA), with mean 106 mapping rates of sludge samples being over 100-times lower than that of influent samples 107 (0.01% vs. 11.3%, respectively; Table S1 ). Therefore, ultrafiltration of influent wastewater 108 provided more suitable RNA extracts for multiplexed tiled PCR of SARS-CoV-2 than did 109 direct extraction from wastewater sludge, likely due to a combination of greater SARS- 144 The sequence data produced via the 400bp primer scheme and influent wastewater 145 samples was used to measure the frequency of VoC-associated SNVs (Table S3) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Table S1 , along with the sample metadata. Laboratories at BCCDC Public Health Laboratory for materials and access to testing 204 equipment, and the BCCDC and BC Regional Health Authorities for publicly sharing data 205 on clinical case counts and variants of concern. This work was funded by the Natural CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic 212 epidemiology. 11 Cryptic transmission of SARS-CoV-2 in Washington state SARS-CoV-2 Titers in Wastewater Are Higher than Expected 289 from Clinically Confirmed Cases Regionally Prevalent SARS-CoV-2 Variants Temporal Detection and Phylogenetic Assessment of 296 SARS-CoV-2 in Municipal Wastewater Monitoring SARS-CoV-2 Circulation and Diversity through Community Wastewater 300 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Real-time, portable 316 genome sequencing for Ebola surveillance Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus 322 genomes directly from clinical samples 16 Persistence of SARS-CoV-2 in Water and Wastewater Several forms of SARS-CoV-2 RNA can be detected in 328 wastewaters: implication for wastewater-based epidemiology and risk assessment Challenges in Measuring the 331 Recovery of SARS-CoV-2 from Wastewater Improvements to the ARTIC multiplex PCR method for SARS-335 CoV-2 genome sequencing using nanopore Rapid and inexpensive whole-genome 338 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 29, 2021. ; https://doi.org/10.1101/2021.05.26.21257861 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted May 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021