Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data

Parker, M. D. et al. (2021) Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data. Genome Research, 31, pp. 645-658. (doi: 10.1101/gr.268110.120) (PMID:33722935) (PMCID:PMC8015849)

Abstract

We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed “subgenomic RNAs.” sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5′ UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5′ end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/− cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Aranday-Cortes, Dr Elihu and Tong, Dr Lily and Davis, Dr Chris and Shepherd, Dr James and Da Silva Filipe, Dr Ana and Nichols, Mrs Jenna and Thomson, Professor Emma and Bennett, Mr Sahan and Kohl, Professor Alain
• Authors: Parker, M. D., Lindsey, B. B., Leary, S., Gaudieri, S., Chopra, A., Wyles, M., Angyal, A., Green, L. R., Parsons, P., Tucker, R. M., Brown, R., Groves, D., Johnson, K., Carrilero, L., Heffer, J., Partridge, D. G., Evans, C., Raza, M., Keeley, A. J., Smith, N., Da Silva Filipe, A., Shepherd, J., Davis, C., Bennett, S., Sreenu, V. B., Kohl, A., Aranday-Cortes, E., Tong, L., Nichols, J., Thomson, E., Wang, D., Mallal, S., and de Silva, T. I.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity; College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: Genome Research
• Publisher: Cold Spring Harbor Laboratory Press
• ISSN: 1088-9051
• ISSN (Online): 1549-5469
• Published Online: 15 March 2021
• Copyright Holders: Copyright © 2021 Parker et al.
• First Published: First published in Genome Research 31:645-658
• Publisher Policy: Reproduced under a Creative Commons licence

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
				Medical Research Council (MRC)	MC_UU_12014/ 8
				Medical Research Council (MRC)	MC_UU_12014/ 12