Figure 4 - available via license: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
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A) Proportion of tracked lineages over time. The proportions were calculated with a deconvolution model based on the signature mutation frequencies. "WT" denotes a set of reference mutations derived from the deconvolution matrix. Sample results were pooled from 4 different wastewater treatment plants using weighted mean with read number as weights. In case of undistinguishable lineages the proportion derived for the group was distributed equally for the affected lineages. Only samples passing the sample quality scoring (>=90% mutation coverage) were considered. B) Comparison of deconvolution results (dark color) with lineage frequency analysis data from the Robert Koch-Institute (RKI) (light color). Deconvolution results were pooled by weeks using weighted mean using sample read numbers as weights. Only samples passing the sample quality scoring (>=90% mutation coverage) were used.
Source publication
The use of RNA sequencing from wastewater samples is proven to be a valuable way for estimating infection dynamics and circulating lineages of SARS-CoV-2. This approach has the advantage of being independent from patient population testing and symptomatic disease courses. However, it is equally important to develop easily accessible and scalable to...
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Background
The accurate identification of SARS-CoV-2 (SC2) variants and estimation of their abundance in mixed population samples ( e.g. , air or wastewater) is imperative for successful surveillance of community level trends. Assessing the performance of SC2 variant composition estimators (VCEs) should improve our confidence in public health decis...
Citations
... Another approach utilizes diverse deconvolution steps for the assignment of SARS-CoV-2 lineage based on the frequencies of identified mutations in a dataset. These are typically implemented using custom-designed scripts and pipelines [20,21,42,43,62]. Alternatively, consensus sequences can be prepared (for example, in medaka [63]. ...
The SARS-CoV-2 outbreak has already affected more than 555 million people, and 6.3 million people have died. Due to its high infectivity, it is crucial to track SARS-CoV-2 outbreaks early to prevent the spread of infection. Wastewater monitoring appears to be a powerful and effective tool for managing epidemiological situations. Due to emerging mutations of SARS-CoV-2, there is a need to monitor mutations in order to control the pandemic. Since the sequencing of randomly chosen individuals is time-consuming and expensive, sequencing of wastewater plays an important role in revealing the dynamics of infection in a population. The sampling method used is a crucial factor and significantly impacts the results. Wastewater can be collected as a grab sample or as a 24 h composite sample. Another essential factor is the sample volume, as is the method of transport used. This review discusses different pretreatment procedures and RNA extraction, which may be performed using various methods, such as column-based extraction, TRIzol, or magnetic extraction. Each of the methods has its advantages and disadvantages, which are described accordingly. RT-qPCR is a procedure that confirms the presence of SARS-CoV-2 genes before sequencing. This review provides an overview of currently used methods for preparing wastewater samples, from sampling to sequencing.
... Replicate and spike-in experiments (Methods) indicate that the relative prevalence of genomic variants can be quantified from the NGS data, although precision is limited at low prevalence. Replication 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Amplicon 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29) December 2020), consistent with the popularity of the ski resort with British tourists as a holiday destination (Fig. 2). Unlike for Alpha, we found almost no evidence for the distinctive signature mutations of Beta or Gamma (Supplementary Information), which is consistent with the observation that neither of the two variants was able to establish itself in the Swiss population. ...
... At later timepoints, we also observe evidence based on the other three Alpha-specific amplicons both in Lausanne and Zurich. In the ski resort, evidence for the presence of Alpha has already been strong based on the analysis of individual mutations over the entire period of observation (20)(21)(22)(23)(24)(25)(26)(27)(28)(29), and the amplicon-based analysis further supports this observation. For Beta and Gamma, the evidence based on co-occurrence is similarly weak as for the analysis of individual mutations (Supplementary Information). ...
The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK.
Zusammenfassung
Indikatoren sollen der Steuerung von (sozialen) Prozessen dienen. Sie beschreiben jedoch die Realität in der Regel nur deskriptiv und unvermeidlich mit mehr oder weniger großen und systematischen Messfehlern behaftet. Insofern ist es im Allgemeinen alles andere als einfach mit Hilfe von Indikatoren zu steuern; insbesondere dann, wenn für Problembereiche (fehlerbehaftete) Zielwerte vorgegeben werden, für deren Erreichung die Indikatoren keine kausalen Pfade aufzeigen. Am Beispiel der Corona-Pandemie werden statistisch-methodische Probleme von (sozialen) Indikatoren aufgezeigt und auf ein zentrales Defizit der vorhandenen bzw. rasch entwickelten Pandemie-Indikatoren hingewiesen: fehlende Indikatoren für Instrumente der Risikokommunikation. Als eine positive Nebenwirkung der Pandemie wird die verbesserte „statistical literacy“ hervorgehoben und diskutiert.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease 2019 (COVID-19) pandemic has highlighted an important role for efficient surveillance of microbial pathogens. High-throughput sequencing technologies provide valuable surveillance tools, offering opportunities to conduct high-resolution monitoring from diverse sample types, including from environmental sources. However, given their large size and potential to contain mixtures of lineages within samples, such genomic data sets can present challenges for analyzing the data and communicating results with diverse stakeholders. Here, we report MixviR, an R package for exploring, analyzing, and visualizing genomic data from potentially mixed samples of a target microbial group. MixviR characterizes variation at both the nucleotide and amino acid levels and offers the RShiny interactive dashboard for exploring data. We demonstrate MixviR’s utility with validation studies using mixtures of known lineages from both SARS-CoV-2 and Mycobacterium tuberculosis and with a case study analyzing lineages of SARS-CoV-2 in wastewater samples over time at a sampling location in Ohio, USA.
IMPORTANCE High-throughput sequencing technologies hold great potential for contributing to genomic-based surveillance of microbial diversity from environmental samples. However, the size of the data sets, along with the potential for environmental samples to contain multiple evolutionary lineages of interest, present challenges around analyzing and effectively communicating inferences from these data sets. The software described here provides a novel and valuable tool for exploring such data. Though originally designed and used for monitoring SARS-CoV-2 lineages in wastewater, it can also be applied to analyses of genomic diversity in other microbial groups.
