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Volume 13
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10.3390/v13091870
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Article

Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines

by
David Brandt
1,
Marina Simunovic
1,
Tobias Busche
1,
Markus Haak
1,
Peter Belmann
1,2,
Sebastian Jünemann
1,2,
Tizian Schulz
1,2,
Levin Joe Klages
1,
Svenja Vinke
1,
Michael Beckstette
1,2,
Ehmke Pohl
3,
Christiane Scherer
4,5,
Alexander Sczyrba
1,2 and
Jörn Kalinowski
1,*
1
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany
2
Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany
3
Department of Biosciences, Durham University, Durham DH1 3LE, UK
4
Evangelisches Klinikum Bethel, Institut für Laboratoriumsmedizin, Mikrobiologie und Hygiene, 33617 Bielefeld, Germany
5
Universitätsklinikum OWL der Universität Bielefeld, 33615 Bielefeld, Germany
*
Author to whom correspondence should be addressed.
Viruses 2021, 13(9), 1870; https://doi.org/10.3390/v13091870
Submission received: 13 July 2021 / Revised: 14 September 2021 / Accepted: 15 September 2021 / Published: 18 September 2021
(This article belongs to the Collection Coronaviruses)
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Abstract

Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism.
Keywords: genomic surveillance; SARS-CoV-2; nanopore sequencing; ORF8 deletion; viral genomics genomic surveillance; SARS-CoV-2; nanopore sequencing; ORF8 deletion; viral genomics

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MDPI and ACS Style

Brandt, D.; Simunovic, M.; Busche, T.; Haak, M.; Belmann, P.; Jünemann, S.; Schulz, T.; Klages, L.J.; Vinke, S.; Beckstette, M.; et al. Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines. Viruses 2021, 13, 1870. https://doi.org/10.3390/v13091870

AMA Style

Brandt D, Simunovic M, Busche T, Haak M, Belmann P, Jünemann S, Schulz T, Klages LJ, Vinke S, Beckstette M, et al. Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines. Viruses. 2021; 13(9):1870. https://doi.org/10.3390/v13091870

Chicago/Turabian Style

Brandt, David, Marina Simunovic, Tobias Busche, Markus Haak, Peter Belmann, Sebastian Jünemann, Tizian Schulz, Levin Joe Klages, Svenja Vinke, Michael Beckstette, and et al. 2021. "Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines" Viruses 13, no. 9: 1870. https://doi.org/10.3390/v13091870

APA Style

Brandt, D., Simunovic, M., Busche, T., Haak, M., Belmann, P., Jünemann, S., Schulz, T., Klages, L. J., Vinke, S., Beckstette, M., Pohl, E., Scherer, C., Sczyrba, A., & Kalinowski, J. (2021). Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines. Viruses, 13(9), 1870. https://doi.org/10.3390/v13091870

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