Abstract
Characterization of global transcriptomes using conventional short-read sequencing is challenging due to the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps. Long-read sequencing (LRS) can overcome these limitations by reading full-length transcripts. Employment of these technologies has led to the redefinition of transcriptional complexities in reported organisms. In this study, we applied LRS platforms from Pacific Biosciences and Oxford Nanopore Technologies to profile the vaccinia virus (VACV) transcriptome. We performed cDNA and direct RNA sequencing analyses and revealed an extremely complex transcriptional landscape of this virus. In particular, VACV genes produce large numbers of transcript isoforms that vary in their start and termination sites. A significant fraction of VACV transcripts start or end within coding regions of neighbouring genes. This study provides new insights into the transcriptomic profile of this viral pathogen.
Highlights
Characterization of global transcriptomes using conventional short-read sequencing is challenging due to the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps
For Oxford Nanopore Technologies (ONT) sequencing, we used the company’s own library preparation approach (1D-Seq), or the Teloprime Cap-selection protocol from Lexogen, which was adapted for the MinION sequencer
We identified transcription start sites (TSSs) for 90% of the promoters described by Yang et al, (2010), with the exception of G2R, J6R, A18R, A20R, and five hypothetical genes located in the repeat region of the vaccinia virus (VACV) genome
Summary
Characterization of global transcriptomes using conventional short-read sequencing is challenging due to the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps. We used two long-read sequencing (LRS) platform Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)] to profile the transcriptomes of herpesviruses, including pseudorabies virus[27,28], herpes simplex virus type 129,30, varicella-zoster virus[31], and human cytomegalovirus[32,33]. These analyses identified polycistronic RNAs, transcript isoforms, and transcriptional overlaps, and facilitated the kinetic characterization of viral t ranscripts[34,35]
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