Metatranscriptomic Analysis Reveals SARS-Cov-2 Mutations in Wastewater of the Frankfurt Metropolitan Area in Southern Germany
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OMICS DATA SETS Metatranscriptomic Analysis Reveals SARS-CoV-2 Mutations in Wastewater of the Frankfurt Metropolitan Area in Southern Germany Shelesh Agrawal,a Laura Orschler,a Susanne Lacknera aTechnical University of Darmstadt, Institute IWAR, Chair of Wastewater Engineering, Darmstadt, Germany ABSTRACT We report a sequencing analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples collected in the Frankfurt metropolitan area of Germany. The majority of the detected mutations have been identified only in clinical genomes outside Frankfurt, indicating that the sequenc- ing of SARS-CoV-2 RNA in wastewater can provide insights into emerging variants in a city. astewater-based epidemiology (WBE) can provide complementary information Wabout the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) infections at the community level because SARS-CoV-2 (genus Betacoronavirus, family Coronaviridae) RNA has been detected in human urine and feces (1, 2). However, most of the WBE studies have focused primarily on reverse transcription-quantitative PCR analysis (3–5). Since the onset of the coronavirus disease 2019 (COVID-19) pan- demic, several clinically relevant mutations have been reported, resulting in various SARS-CoV-2 variants (6–9). A few studies showed that the sequencing of SARS-CoV-2 RNA in wastewater could help to determine which SARS-CoV-2 variants are circulating in the studied areas (10, 11). We collected 24-h composite samples on 4 December 2020 at three sampling points in the Frankfurt metropolitan area of Germany. The three sampling points were (i) influent of the wastewater treatment plant (WWTP) Niederrad (NR) (50.08N, 8.63E), (ii) influent of the WWTP Sindlingen (SL) (50.09N, 8.51E), and (iii) a sewage sample from Griesheim (GR) (50.09N, 8.60E). One liter of the untreated wastewater was filtered through a 0.45-mm electronegative membrane filter to concentrate the virus-like particles, followed by extraction using the FastRNA Pro Blue kit (MP Biomedicals) according to the manufacturer’s protocol. cDNA synthesis was per- formed using SuperScript VILO Master Mix (Thermo Fisher Scientific), followed by Citation Agrawal S, Orschler L, Lackner S. 2021. library preparation using the Ion AmpliSeq SARS-CoV-2 research panel (Thermo Metatranscriptomic analysis reveals SARS-CoV-2 Fisher Scientific) according to the manufacturer’s instructions. This panel consists of mutations in wastewater of the Frankfurt 237 primer pairs, resulting in an amplicon length range of 125 to 275 bp and covering metropolitan area in southern Germany. Microbiol Resour Announc 10:e00280-21. https:// the nearly full genome of SARS-CoV-2. Libraries were multiplexed and sequenced doi.org/10.1128/MRA.00280-21. using an Ion Torrent 530 chip on an Ion S5 sequencer (Thermo Fisher Scientific) Editor Simon Roux, DOE Joint Genome according to the manufacturer’s instructions. Institute Ion Torrent Suite software (v 5.12.2) of the Ion S5 sequencer was used to map the Copyright © 2021 Agrawal et al. This is an open-access article distributed under the terms generated reads to a SARS-CoV-2 reference genome (Wuhan-Hu-1 [GenBank accession of the Creative Commons Attribution 4.0 numbers NC_045512 and MN908947.3]), using TMAP software included in the Torrent International license. Suite. The sequencing data for the samples are summarized in Table 1. For mutation Address correspondence to Shelesh Agrawal, [email protected]. calls, additional Ion Torrent plugins were used. First, all single-nucleotide variants (SNVs) “ Received 16 March 2021 were called using Variant Caller (v5.12.0.4) with Generic - S5/S5XL (510/520/530) - Somatic - Accepted 22 March 2021 Low Stringency” default parameters. Then, for annotation and determination of the base Published 15 April 2021 substitution effect, COVID19AnnotateSnpEff (v1.0.0.1), a plugin developed explicitly for Volume 10 Issue 15 e00280-21 mra.asm.org 1 Agrawal et al. FIG 1 Locations of mutations detected in samples (NR, Niederrad; SL, Sindlingen; GR, Griesheim), corresponding to the locations reporting the mutations in clinical samples. The detected mutations are organized according to the corresponding gene (M, N, open reading frame 1ab [Orf1ab], Orf3a, and S) of the SARS-CoV-2 genome. Points indicate the locations reporting the detected mutations. Volume 10 Issue 15 e00280-21 mra.asm.org 2 Microbiology Resource Announcement TABLE 1 Summary of sequencing data for the samples No. of mapped Avg target base Avg read identity Sample Sample location Total no. of reads reads coverage depth (×) to target (%)a GC content (%) Accession no. NR Niederrad 2,781,914 854,804 817.9 95.68 48.3 SAMN18310569 SL Sindlingen 2,143,766 675,006 680.8 94.01 46.3 SAMN18310571 GR Griesheim 2,349,476 640,702 215.1 94.84 47.4 SAMN18310570 a The target sequence was the SARS-CoV-2 reference genome (Wuhan-Hu-1 [GenBank accession numbers NC_045512 and MN908947.3]). SARS-CoV-2, was used. To determine whether the mutations detected in these samples had already been reported in clinical samples at different locations, we downloaded the clinically reported mutations and their corresponding locations from COVID CG (https://covidcg.org) (12) on 7 March 2021. We detected 75 unique mutations across all of the samples, including 5 muta- tions reported outside Europe, 35 reported in Europe, 13 reported in Germany, 18 reported in the Frankfurt region, and 4 not reported (Fig. 1). In the SL sample, we also observed D614G, a predominant mutation reported around the globe (13) and a key mutation associated with the B.1.1.7, B.1.351, and P.1 variants (14). The 69/70 deletion mutation, a key mutation associated with B.1.1.7 (14), was also found in the SL sample. Due to the plausible presence of SARS-CoV-2 RNA of multiple var- iants in wastewater, WBE analysis can provide only indirect information about the variants. However, observations from this study suggest that an integration of genomic epidemiology into WBE may support identification of variants that have already been detected in a city, as well as those that have not yet been detected in clinical samples. Data availability. The raw metagenomic sequence data are available in the NCBI Sequence Read Archive (SRA) under BioProject number PRJNA714504 and SRA acces- sion numbers SRX10340342 (NR), SRX10340344 (SL), and SRX10340343 (GR). ACKNOWLEDGMENTS This work was funded by the Hessian Ministry of Economics, Energy, Transport, and Housing within the framework of the IWB-EFRE program for knowledge and technology transfer under grant 20007030. We thank the employees of the Stadtentwässerung Frankfurt (SEF) and members of our research group for their support with sample collection. REFERENCES 1. Chen Y, Chen L, Deng Q, Zhang G, Wu K, Ni L, Yang Y, Liu B, Wang W, Wei 7. Pfefferle S, Günther T, Kobbe R, Czech-Sioli M, Nörz D, Santer R, Oh J, C, Yang J, Ye G, Cheng Z. 2020. The presence of SARS-CoV-2 RNA in the Kluge S, Oestereich L, Peldschus K, Indenbirken D, Huang J, Grundhoff A, feces of COVID-19 patients. J Med Virol 92:833–840. https://doi.org/10 Aepfelbacher M, Knobloch JK, Lütgehetmann M, Fischer N. 2021. 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