Long-Read Isoform Sequencing Reveals a Hidden Complexity of the Transcriptional Landscape of Herpes Simplex Virus Type 1

Abstract

In this study, we used the amplified isoform sequencing technique from Pacific Biosciences to characterize the poly(A)+ fraction of the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). Our analysis detected 34 formerly unidentified protein-coding genes, 10 non-coding RNAs, as well as 17 polycistronic and complex transcripts. This work also led us to identify many transcript isoforms, including 13 splice and 68 transcript end variants, as well as several transcript overlaps. Additionally, we determined previously unascertained transcriptional start and polyadenylation sites. We analyzed the transcriptional activity from the complementary DNA strand in five convergent HSV gene pairs with quantitative RT-PCR and detected antisense RNAs in each gene. This part of the study revealed an inverse correlation between the expressions of convergent partners. Our work adds new insights for understanding the complexity of the pervasive transcriptional overlaps by suggesting that there is a crosstalk between adjacent and distal genes through interaction between their transcription apparatuses. We also identified transcripts overlapping the HSV replication origins, which may indicate an interplay between the transcription and replication machineries. The relative abundance of HSV-1 transcripts has also been established by using a novel method based on the calculation of sequencing reads for the analysis.

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

“…Recently, nanopore sequencing has been used for metagenomic forays into the virosphere 29 and studies focusing on transmission routes 30,31 . Furthermore, viral transcriptomes have been investigated using nanopore sequencing of cDNA [32][33][34][35] , being subject to bias from reverse transcription and amplification. Other studies used DRS to study the human poly(A) transcriptome 36 and the transcriptome of DNA viruses such as HSV 37 .…”

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

“…Recently, nanopore sequencing has been used for metagenomic forays into the virosphere (Warwick-Dugdale et al 2019) and studies focusing on transmission routes Faria et al 2017). Furthermore, viral transcriptomes have been investigated using nanopore sequencing of cDNA (Moldován et al , 2018aTombácz et al 2017), being subject to bias from reverse transcription and amplification. Other studies used DRS to study the human poly(A) transcriptome (Workman et al 2018) and the transcriptome of DNA viruses such as HSV (Depledge et al 2018).…”

Demand for Multiplatform and Meta-analytic Approaches in Transcriptome Profiling

“…The LoRTIA tool was used to annotate introns and TSSs, and TESs from the LRS data, whereas we used the STAR software was used to detect introns from the SRS samples. The previously published introns (Tang et al 8 , Wishnant et al 10 , and Tombácz et al 11,13 ) were compared with each other, reanalyzed, and validated by using the datasets from all of the aforementioned publications.…”

“…The datasets generated by Depledge et al 7 and five other datasets (Tombácz et al 11,13 ; Tang et al 8 ; Rutkowski et al 9 , and Whisnant et al 10 ) were reanalyzed in order to define the complete HSV transcriptome.…”