@article{Parker01042021,
author = {Parker, Matthew D. and Lindsey, Benjamin B. and Leary, Shay and Gaudieri, Silvana and Chopra, Abha and Wyles, Matthew and Angyal, Adrienn and Green, Luke R. and Parsons, Paul and Tucker, Rachel M. and Brown, Rebecca and Groves, Danielle and Johnson, Katie and Carrilero, Laura and Heffer, Joe and Partridge, David G. and Evans, Cariad and Raza, Mohammad and Keeley, Alexander J. and Smith, Nikki and Filipe, Ana Da Silva and Shepherd, James G. and Davis, Chris and Bennett, Sahan and Sreenu, Vattipally B. and Kohl, Alain and Aranday-Cortes, Elihu and Tong, Lily and Nichols, Jenna and Thomson, Emma C. and The COVID-19 Genomics UK (COG-UK) Consortium and Wang, Dennis and Mallal, Simon and de Silva, Thushan I.}, 
title = {Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data},
volume = {31}, 
number = {4}, 
pages = {645-658}, 
year = {2021}, 
doi = {10.1101/gr.268110.120}, 
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.}, 
URL = {http://genome.cshlp.org/content/31/4/645.abstract}, 
eprint = {http://genome.cshlp.org/content/31/4/645.full.pdf+html}, 
journal = {Genome Research} 
}