Metagenomic sequencing of municipal wastewater provides a near-complete SARS-CoV-2 genome sequence identified as the B.1.1.7 variant of concern from a Canadian municipality concurrent with an outbreak
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Abstract
Laboratory-based wastewater surveillance for SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, can be conducted using RT-qPCR-based screening of municipal wastewater samples. Although it provides rapid viral detection and can inform SARS-CoV-2 abundance in wastewater, this approach lacks the resolution required for viral genotyping and does not support tracking of viral genome evolution. The recent emergence of several variants of concern, a result of mutations across the genome including the accrual of important mutations within the viral spike glycoprotein, has highlighted the need for a method capable of detecting the cohort of mutations associated with these and newly emerging genotypes. Here we provide an innovative methodology for the recovery of a near-complete SARS-CoV-2 sequence from a wastewater sample collected from across Canadian municipalities including one that experienced a significant outbreak attributable to the SARS-CoV-2 B.1.1.7 variant of concern. Our results demonstrate that a combined interrogation of genome consensus-level sequences and alternative alleles enables the identification of a SARS-CoV-2 variant of concern and the detection of a new allele within a viral accessory gene that may be representative of a recently evolved B.1.1.7 sublineage.
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SciScore for 10.1101/2021.03.11.21253409: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization To reduce the computational resources required for phylogenetic analysis, the Canadian sequences were subsampled by randomly selecting two sequences per Pangolin lineage from each Canadian province. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources The sequencing run was executed using MinKNOW v 3.4.1 Flongle flow cell (ONT; FLO-FLG001) on a Mk1b MinION sequencer with default settings, except live basecalling was disabled, and the .fast5 files were written to disk after every 1000 reads sequenced. MinKNOWsuggested: NoneSummary quality … SciScore for 10.1101/2021.03.11.21253409: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization To reduce the computational resources required for phylogenetic analysis, the Canadian sequences were subsampled by randomly selecting two sequences per Pangolin lineage from each Canadian province. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources The sequencing run was executed using MinKNOW v 3.4.1 Flongle flow cell (ONT; FLO-FLG001) on a Mk1b MinION sequencer with default settings, except live basecalling was disabled, and the .fast5 files were written to disk after every 1000 reads sequenced. MinKNOWsuggested: NoneSummary quality statistics were calculated using the basecalled .fastq file output by Guppy using NanoPlot [35] with default settings. NanoPlotsuggested: NoneThe subsampled Canadian (n=536) and B.1.1.7 (n=49) sequences, wastewater consensus sequence and SARS-CoV-2 Wuhan-Hu-1 (MN908947.3) reference sequence were re-aligned using MAFFT v7.475 with the -add command using default settings [36], [37]. MAFFTsuggested: (MAFFT, RRID:SCR_011811)bam files outputted by the Virontus pipeline using SAMtools v 1.7 [40] and iVar v 1.3 [41] as described in their documentation files, except the minimum quality score used to determine whether or not to count a base (-q) was set to 10, the minimum frequency threshold (-t) was set to 0.01, and the minimum read depth to call variants (-m) was set to 10. SAMtoolssuggested: (SAMTOOLS, RRID:SCR_002105)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.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.
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