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Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines.
Viruses. 2021 09 18; 13(9)V

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.

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Authors+Show Affiliations

Brandt D
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
Simunovic M
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
Busche T
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
Haak M
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
Belmann P
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany. Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany.
Jünemann S
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany. Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany.
Schulz T
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany. Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany.
Klages LJ
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
Vinke S
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.
Beckstette M
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany. Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany.
Pohl E
Department of Biosciences, Durham University, Durham DH1 3LE, UK.
Scherer C
Evangelisches Klinikum Bethel, Institut für Laboratoriumsmedizin, Mikrobiologie und Hygiene, 33617 Bielefeld, Germany. Universitätsklinikum OWL der Universität Bielefeld, 33615 Bielefeld, Germany.
Sczyrba A
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany. Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany.
Kalinowski J
Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.

MeSH

COVID-19Genes, ViralGenetic VariationHumansNanopore SequencingOpen Reading FramesSARS-CoV-2Sequence Analysis, RNASequence DeletionWhole Genome Sequencing

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

34578452

Citation

Brandt, David, et al. "Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed By Standard Amplicon Sequencing and Variant Calling Pipelines." Viruses, vol. 13, no. 9, 2021.
Brandt D, Simunovic M, Busche T, 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).
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). https://doi.org/10.3390/v13091870
Brandt D, 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 09 18;13(9) PubMed PMID: 34578452.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines. AU - Brandt,David, AU - Simunovic,Marina, AU - Busche,Tobias, AU - Haak,Markus, AU - Belmann,Peter, AU - Jünemann,Sebastian, AU - Schulz,Tizian, AU - Klages,Levin Joe, AU - Vinke,Svenja, AU - Beckstette,Michael, AU - Pohl,Ehmke, AU - Scherer,Christiane, AU - Sczyrba,Alexander, AU - Kalinowski,Jörn, Y1 - 2021/09/18/ PY - 2021/07/13/received PY - 2021/09/14/revised PY - 2021/09/15/accepted PY - 2021/9/28/entrez PY - 2021/9/29/pubmed PY - 2021/10/14/medline KW - ORF8 deletion KW - SARS-CoV-2 KW - genomic surveillance KW - nanopore sequencing KW - viral genomics JF - Viruses JO - Viruses VL - 13 IS - 9 N2 - 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. SN - 1999-4915 UR - https://www.unboundmedicine.com/medline/citation/34578452/Multiple_Occurrences_of_a_168_Nucleotide_Deletion_in_SARS_CoV_2_ORF8_Unnoticed_by_Standard_Amplicon_Sequencing_and_Variant_Calling_Pipelines_ DB - PRIME DP - Unbound Medicine ER -