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.
AbstractWe 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.
Objective-The molecular diagnosis of adult-onset autosomal recessive cerebellar ataxias remains challenging because of genetic heterogeneity. However, recently developed molecular genetic techniques will potentially revolutionise the diagnostic approach. Here we set out to define the genetic basis of the ataxia in two brothers with no molecular diagnosis.Methods-Clinical evaluation followed by whole exome second generation sequencing and comparative genomic hybridisation.Results-Whole exome sequencing identified a hemizygous novel spastic ataxia of CharlevoixSaguenay (SACS) stop-codon mutation in both brothers (c.13048G>T, p.E4350X) which was present in the mother, but not the father. Comparative genomic hybridisation revealed a 0.7Mb deletion on chromosome 13q12.12 in both brothers, which included SACs and was heterozygous in the asymptomatic father.Conclusions-The milder phenotype, caused by a whole gene deletion and a stop codon mutation in SACS, indicates a loss-of-function mechanism in late-onset autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), and illustrates the importance of chromosomal rearrangements in the investigation of adult-onset ataxia.
The complete genome sequences of RNA1 and RNA2 of the oca strain of the potato virus arracacha virus B were determined using next-generation sequencing. The RNA1 molecule is predicted to encode a 259-kDa polyprotein with homology to proteins of the cheraviruses apple latent spherical virus (ALSV) and cherry rasp leaf virus (CRLV). The RNA2 molecule is predicted to encode a 102-kDa polyprotein which also has homology to the corresponding protein of ALSV and, to a lesser degree, CRLV (30 % for RNA1, 24 % for RNA2). Detailed analysis of the genome sequence confirms that AVB is a distinct member of the genus Cheravirus.
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