Combined nanopore and single-molecule real-time sequencing survey of human betaherpesvirus 5 transcriptome

Abstract

Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing

“…The propensity of miRNAs to target 3 -UTRs, and recent findings that miRNAs were differentially regulated during natural OsHV-1 infection of Crassostrea gigas [49], might suggest an adaptive benefit of shorter 3 -UTRs in escaping host defenses. For HaHV-1, 27% of the genome was predicted to contain AS gene overlaps, similar to what was reported for cytomegalovirus [20]. Arguably, full-length RNA approaches (e.g., PacBio SMRT) minimized the AS overestimation due to the analysis of partial transcripts and allowed an accurate estimation of Natural Antisense Transcripts (NATs).…”

“…Arguably, full-length RNA approaches (e.g., PacBio SMRT) minimized the AS overestimation due to the analysis of partial transcripts and allowed an accurate estimation of Natural Antisense Transcripts (NATs). However, most of HaHV-1 and OsHV-1 head-to-head overlaps involved genes predicted to be coding, and should be considered as overlapping segments more than true NATs, as previously reported for cytomegalovirus using a similar approach [20]. In other herpesviruses the functions of single NATs have been often associated to the maintenance of latency and viral reactivation [50,51], but latency has not been shown for malacoherpesviruses thus far, which may suggest other functional roles for AS transcription.…”

“…Rather, polycistronism seems to be the result of viral transcript elongation in the stationary phase, mostly associated to 5 -RTT based on upstream TSSs and rarely impacting 3 -UTRs. Although AS transcription is often associated with 3'-UTR overlaps, an elongation of 5'-UTRs generating convergent overlaps was reported for cytomegalovirus [20]. This was the predominant situation in malacoherpesviruses, where 3 -UTRs appeared shorter compared to 5 -UTRs and generally not impacted by RTT.…”

“…The complex transcriptomic architectures typical of viruses, including nested isoforms and overlapping genes, can only be resolved with the recent introduction of long-read sequencing (LRS) technologies (single-molecule real-time (SMRT) and nanopore sequencing) [15][16][17]. Notably, this approach has revealed that as many as 2300 unique transcripts produced from the 79 genes of Herpes simplex virus type 1 (HSV-1) during in vitro infections [18,19] and has also been used to reveal variant transcripts and widespread antisense (AS) transcription in human cytomegalovirus [20]. In the dense-coding genomes of herpesviruses, AS transcription is quite frequent [21] and could play a functional role in controlling gene expression by RNA interference, chromatin remodeling, transcriptional interference, and RNA editing [22].…”

Application of second-generation sequencing (SGS) and third generation sequencing (TGS) in aquaculture breeding program

“…TGS platforms which are currently commercially available could sequence read lengths of up to15 kb for PacBio and > 30 kb for ONT, which exceed the read length of most transcripts (Kakuk et al, 2021). PacBio Iso-Seq® single-cell RNA sequencing method could capture fulllength transcripts of differentially expressed isoforms, homologous and superfamily genes in their entirety within a single sequencing read, without the need of downstream bioinformatic assembly (Rhoads and Au, 2015;Mehjabin et al, 2019).…”