Detecting SARS‑Cov‑2 Lineages And Mutational Load In Municipal Wastewater And A Use‑Case In The Metropolitan Area Of Thessaloniki, Greece

Detecting SARS‑Cov‑2 Lineages And Mutational Load In Municipal Wastewater And A Use‑Case In The Metropolitan Area Of Thessaloniki, Greece

Author(s): Nikolas Pechlivanis, Maria Tsagiopoulou, Maria Christina Maniou, Anastasis Togkousidis, Stamatia Laidou, Elisavet Vlachonikola, Evangelia Mouchtaropoulou, Taxiarchis Chassalevris, Serafeim Chaintoutis, Chrysostomos Dovas, Maria Petala, Margaritis Kostoglou, Thodoris Karapantsios, Aspasia Orfanou, Styliani Christina Fragkouli, Sofoklis Keisaris, Anastasia Chatzidimitriou, Agis Papadopoulos, Nikolaos Papaioannou, Anagnostis Argiriou, Fotis Psomopoulos

Affiliation(s): Centre of Research and Technology Hellas, Greece

Twitter: @npechl

Nearly two years after the first report of SARS-CoV-2 in Wuhan, China, the virus has caused an unprecedented global crisis. The COVID-19 pandemic has affected almost half a billion people, making necessary the emergence of new approaches able to monitor its spread. Most laboratories rely on PCR-based methods for epidemiological investigation and early mutation detection of the virus. Yet, these methods are not easily scalable, especially in large urban areas, or in cases where new mutations arise. Recently, the detection of SARS-CoV-2 RNA in wastewater turned into a useful tool to study the prevalence of the virus in the community. To this end, a novel methodology, called lineagespot, has been developed for the monitoring of mutations and the detection of SARS-CoV-2 lineages in wastewater samples using next-generation sequencing (NGS). The tool accepts as input a VCF file, which contains all nucleotide (and the corresponding amino acid) mutations identified in a sample, along with a file containing all lineage-assignment mutations. After analyzing all inputs, a tab-delimited file (TSV file) is produced containing the identified mutations that are related to SARS-CoV-2 lineages. In addition, it computes the average allele frequencies to quantify lineage abundance metrics. lineagespot is dependent on the source used for retrieving lineage definitions; currently, the package supports two potential sources, i.e. the lineage-characteristic mutation profiles pre-calculated by and the lineage-characteristic mutation profiles derived from the trained Pangolin models. So far, the tool has been evaluated through application to NGS data produced to cover a six-month period for the municipality of Thessaloniki, Greece. Τhe results revealed the presence of SARS-CoV-2 variants in wastewater data, and have been recently published at scientific reports ( It is worth noting that lineagespot was able to record the evolution and rapid domination of the Alpha variant (B.1.1.7) in the community and identified a strong correlation between the mutations evident through our approach and the mutations observed in patients from the same area and time periods. lineagespot has been developed as an open-source tool, implemented in R, and is freely available through GitHub and the Bioconductor repository.

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