Abstract
ObjectiveIn this study, we applied two long-read sequencing (LRS) approaches, including single-molecule real-time and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of host gene expression as a response to Vaccinia virus infection. Transcriptomes determined using short-read sequencing approaches are incomplete because these platforms are inefficient or fail to distinguish between polycistronic RNAs, transcript isoforms, transcriptional start sites, as well as transcriptional readthroughs and overlaps. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases.ResultsIn this work, we identified a number of novel transcripts and transcript isoforms of Chlorocebus sabaeus. Additionally, analysis of the most abundant 768 host transcripts revealed a significant overrepresentation of the class of genes in the “regulation of signaling receptor activity” Gene Ontology annotation as a result of viral infection.
Highlights
Vaccinia virus (VACV) the prototypic member of poxviruses, contains a large, double-stranded DNA molecule encoding more than 200 protein-coding genes.Long-read sequencing (LRS) has become a mainstream approach in transcriptome studies
The Guppy basecaller was run: we found almost perfect match between the results generated by the two toolkits [15]
The obtained datasets were used to determine the VACV transcripts [15] using the LoRTIA pipeline that was developed by our group [19]
Summary
The time-varying transcriptome of CV-1 cells were profiled applying LRS datasets. ‘Static’ host transcriptome Using the LoRTIA toolkit, we annotated a total of 478 transcription start sites (TSSs), 2011 transcription end sites (TESs), and 24,574 splice junctions, each represented by at least 10 reads (Additional files 6, 7: Tables S2, S3). Transcripts represented by less than ten reads were excluded These assessments revealed a total of 758 transcript isoforms, 207 with TSSs and TESs in ± 10-nt intervals of previously annotated transcripts, 692 length isoforms, and 66 alternatively spliced isoforms. 239 mRNA length isoforms differed from previously annotated transcripts in their TSS positions [including those with TSSs downstream of respective translation initiation sites (TISs)], 19 in their TES positions, and 56 in both. 31 transcript isoforms were found with TSSs downstream of previously annotated TISs, and contained curtailed forms of open reading frames (ORFs) These RNAs might code for N-terminally truncated forms of canonical proteins. Most of the present clusters were not significantly enriched in genes of specific
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