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

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.

Generation of a novel SARS-CoV-2 sub-genomic RNA due to the R203K/G204R variant in nucleocapsid: homologous recombination has potential to change SARS-CoV-2 at both protein and RNA level

“…We hypothesized that the amount of sgRNA at each of the ORF TRS positions in the SARS- CoV-2 genome in ARTIC nanopore sequencing data could serve as a proxy for expression levels of each of the ORFs due to their positions in the amplicons (Fig 3). To test this hypothesis we developed a tool, periscope (19), which quantifies the number of sgRNA and genomic RNA reads at each ORF TRS position in ARTIC network nanopore sequencing data. We applied periscope to the 981 sequences in the Sheffield validation dataset.…”

“…We developed a tool, “periscope” (v0.0.0), to classify and quantify sub-genomic RNA in the Sheffield ARTIC network Nanopore dataset (10). The tool can be downloaded from git-hub at https://github.com/sheffield-bioinformatics-core/periscope.…”

Prediction of two novel overlapping ORFs in the genome of SARS-CoV-2

“…However, there is evidence for non-canonical subgenomic RNAs whose transcription starts immediately upstream the AUG initiation codon of ORF-Sh, located at nucleotide position 24051 (see Fig. 4A in Parker et al, 2021 ). As ORF-Sh is far from the 5’ end of the subgenomic RNA for S ( Fig.…”

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals

“…In the capture libraries, junction reads were identified in a million reads in all 17 samples in the N gene, followed by S gene and ORF7A in 14 samples, ORF3a, M, ORF6 and ORF8 in 13 samples, E gene in 11 samples, while in the rest of the samples, junction reads were either detected in just one sample (ORF1a) or none (ORF1ab, ORF7b and ORF10) in these samples. In one sample 192000072B, Periscope program identified a previously reported non-canonical sgRNA (N*) [ 34 ]. The average number of junction reads in million reads/sample was the highest for N gene (819.7), followed by ORF7a (201.2) ORF3a (129.3) and S gene (110.2).…”

“…Subgenomic RNAs in Illumina reads were analyzed using Periscope program ([ 34 ], downloaded on June 21, 2021) with “–technology illumina” option. From each sample, one million reads were selected using seqtk, version 1.3 ( https://github.com/lh3/seqtk ).…”