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periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data

View ORCID ProfileMatthew D Parker, View ORCID ProfileBenjamin B Lindsey, Shay Leary, Silvana Gaudieri, Abha Chopra, Matthew Wyles, View ORCID ProfileAdrienn Angyal, View ORCID ProfileLuke R Green, Paul Parsons, Rachel M Tucker, Rebecca Brown, Danielle Groves, Katie Johnson, Laura Carrilero, Joe Heffer, David Partridge, Cariad Evans, Mohammad Raza, Alexander J Keeley, Nikki Smith, View ORCID ProfileDennis Wang, View ORCID ProfileSimon Mallal, View ORCID ProfileThushan I de Silva
doi: https://doi.org/10.1101/2020.07.01.181867
Matthew D Parker
1Sheffield Bioinformatics Core, The University of Sheffield, Sheffield, UK
9Neuroscience Institute, The University of Sheffield, Sheffield, UK
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Benjamin B Lindsey
4Sheffield Teaching Hospitals NHS Foundation Trust, Department of Virology/Microbiology, Sheffield, UK
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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Shay Leary
2Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western Australia, Australia
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Silvana Gaudieri
2Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western Australia, Australia
3Division of Infectious Diseases, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA
6School of Human Sciences, University of Western Australia, Crawley, Western Australia, Australia
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Abha Chopra
2Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western Australia, Australia
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Matthew Wyles
9Neuroscience Institute, The University of Sheffield, Sheffield, UK
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Adrienn Angyal
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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Luke R Green
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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Paul Parsons
7Department of Animal and Plant Sciences, Alfred Denny Building, The University of Sheffield, S10 2TN
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Rachel M Tucker
7Department of Animal and Plant Sciences, Alfred Denny Building, The University of Sheffield, S10 2TN
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Rebecca Brown
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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Danielle Groves
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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Katie Johnson
4Sheffield Teaching Hospitals NHS Foundation Trust, Department of Virology/Microbiology, Sheffield, UK
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Laura Carrilero
7Department of Animal and Plant Sciences, Alfred Denny Building, The University of Sheffield, S10 2TN
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Joe Heffer
10IT Services, The University of Sheffield, Sheffield, UK
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David Partridge
4Sheffield Teaching Hospitals NHS Foundation Trust, Department of Virology/Microbiology, Sheffield, UK
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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Cariad Evans
4Sheffield Teaching Hospitals NHS Foundation Trust, Department of Virology/Microbiology, Sheffield, UK
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Mohammad Raza
4Sheffield Teaching Hospitals NHS Foundation Trust, Department of Virology/Microbiology, Sheffield, UK
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Alexander J Keeley
4Sheffield Teaching Hospitals NHS Foundation Trust, Department of Virology/Microbiology, Sheffield, UK
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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Nikki Smith
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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Dennis Wang
1Sheffield Bioinformatics Core, The University of Sheffield, Sheffield, UK
8Department of Computer Science, The University of Sheffield, Sheffield, UK
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Simon Mallal
2Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western Australia, Australia
3Division of Infectious Diseases, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA
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Thushan I de Silva
4Sheffield Teaching Hospitals NHS Foundation Trust, Department of Virology/Microbiology, Sheffield, UK
5The Florey Institute, Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK
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  • For correspondence: t.desilva@sheffield.ac.uk
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Abstract

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA in ARTIC network protocol generated Nanopore SARS-CoV-2 sequence data. We applied periscope to 1155 SARS-CoV-2 sequences from Sheffield, UK. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sub-genomic RNA. We were able to detect all canonical sub-genomic RNAs at expected abundances, with the exception of ORF10, suggesting that this is not a functional ORF. A number of recurrent non-canonical sub-genomic RNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sub-genomic RNA analysis. Finally variants found in genomic RNA are transmitted to sub-genomic RNAs with high fidelity in most cases. This tool can be applied to tens of thousands of sequences worldwide to provide the most comprehensive analysis of SARS-CoV-2 sub-genomic RNA to date.

  • Abbreviations

    TRS
    Transcription regulation sequence (ACGAAC)
    ORF
    Open reading frame
    sgRNA
    sub-genomic RNA
    gRNA
    genomic RNA
    sgRPHT
    sub-genomic RNA reads per 100,000 mapped reads
    sgRPTg
    sub-genomic RNA reads per 1,000 genomic RNA reads
    gRPHT
    genomic RNA reads per 100,000 mapped reads
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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    periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data
    Matthew D Parker, Benjamin B Lindsey, Shay Leary, Silvana Gaudieri, Abha Chopra, Matthew Wyles, Adrienn Angyal, Luke R Green, Paul Parsons, Rachel M Tucker, Rebecca Brown, Danielle Groves, Katie Johnson, Laura Carrilero, Joe Heffer, David Partridge, Cariad Evans, Mohammad Raza, Alexander J Keeley, Nikki Smith, Dennis Wang, Simon Mallal, Thushan I de Silva
    bioRxiv 2020.07.01.181867; doi: https://doi.org/10.1101/2020.07.01.181867
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    periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data
    Matthew D Parker, Benjamin B Lindsey, Shay Leary, Silvana Gaudieri, Abha Chopra, Matthew Wyles, Adrienn Angyal, Luke R Green, Paul Parsons, Rachel M Tucker, Rebecca Brown, Danielle Groves, Katie Johnson, Laura Carrilero, Joe Heffer, David Partridge, Cariad Evans, Mohammad Raza, Alexander J Keeley, Nikki Smith, Dennis Wang, Simon Mallal, Thushan I de Silva
    bioRxiv 2020.07.01.181867; doi: https://doi.org/10.1101/2020.07.01.181867

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