Abstract
Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of VACV gene expression.
Highlights
Poxviruses infect a wide variety of vertebrate and invertebrate species [1]
The time-varying transcriptome of vaccinia virus (VACV) was profiled using the Pacific Biosciences (PacBio) isoform sequencing (Iso-Seq) template preparation protocol for Sequel platform, and the Oxford Nanopore Technologies (ONT) sequencing 1D-Seq library preparation protocol for MinION platform
We ran the Guppy sequencing pipeline for this purpose. These programs are based on the translation of electrical signals generated by the DNA strand passing through the nanopore into nucleotides
Summary
Poxviruses infect a wide variety of vertebrate and invertebrate species [1]. Among these, variola virus is the causative agent of smallpox [2] which was declared eradicated in 1980 by a vaccination program using the closely related vaccinia virus (VACV) as a live vaccine [3]. The variola genome exhibits roughly 90% sequence homology with VACV, which is the prototypic member of poxviruses. The VACV contains a 195 kbp doublestranded DNA molecule encoding more than 200 protein-coding genes. The viral genome is able to code for transcription factors and enzymes for DNA and RNA syntheses, and for capping [4] and polyadenylation [5] of RNAs, which allows VACV to replicate in the cytoplasm. A unique feature of poxviruses is that the entire transcription apparatus is packed into the virion, and E genes are expressed instantly following the entry to the host cells [10]. The step is the onset of DNA replication in conjunction with the expression of first the I and the L kinetic classes of genes.
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