Abstract
Third-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcript isoforms identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection causes differential expression of host transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic transcriptomes in any organisms.
Highlights
Third-generation sequencing is able to read full-length transcripts and to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms
We applied the following techniques for the analysis of bovine transcriptome: (1) direct cDNA sequencing based on oligo(dT)-primed reverse transcription (RT), (2) amplified cDNA sequencing based on random-oligonucleotide-primed RT using nanopore sequencing on Oxford Nanopore Technologies (ONT) MinION platform, as well as (3) synthetic long-read sequencing (LoopSeq) on Illumina platform
All of the three techniques were used for bovine transcript annotation, whereas dcDNA-Seq was used for the time-varying analysis of the effect of Bovine alphaherpesvirus type 1 (BoHV-1) on host cell gene expression
Summary
Third-generation sequencing is able to read full-length transcripts and to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. We report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. The transcriptome analysis was performed using MinION sequencing from Oxford Nanopore Technologies (ONT) and Illumina-based LoopSeq from Loop Genomics
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