Abstract
Long-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying polycistronic RNAs, transcript isoforms including splice and transcript end variants, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 2,300 previously undetected transcripts, including coding, and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we found previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5’ regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.
Highlights
Next-generation short-read sequencing (SRS) technology has revolutionized the research fields of genomics and transcriptomics due to its capacity of sequencing a large number of nucleic acid fragments simultaneously at a relatively low cost (Mortazavi et al, 2008; Wang et al, 2009; Djebali et al, 2012)
We report the application of two distinct Long-read sequencing (LRS) techniques, and multiple library approaches for the investigation of the herpes simplex virus type 1 (HSV-1) lytic transcriptome
2 sequencing platforms (PacBio Sequel and Oxford Nanopore Technologies (ONT) MinION) and 8 library preparation methods were applied for the investigation of the HSV-1 lytic transcriptome, including both poly(A)+ and poly(A)- RNAs
Summary
Next-generation short-read sequencing (SRS) technology has revolutionized the research fields of genomics and transcriptomics due to its capacity of sequencing a large number of nucleic acid fragments simultaneously at a relatively low cost (Mortazavi et al, 2008; Wang et al, 2009; Djebali et al, 2012). The PacBio approach is based on single-molecule real-time (SMRT) technology, while the ONT platform utilizes the nanopore sequencing concept Both techniques have already been applied for the structural and dynamic transcriptomic analysis of various organisms (Byrne et al, 2017; Chen et al, 2017; Cheng et al, 2017; Li et al, 2018; Nudelman et al, 2018; Wen et al, 2018; Zhang et al, 2018; Jiang et al, 2019; Zhao et al, 2019), including viruses (Boldogkői et al, 2019b), such as herpesviruses (Tombácz et al, 2015; O’Grady et al, 2016; Tombácz et al, 2016; Balázs et al, 2017a; Balázs et al, 2017b; Moldován et al, 2017b; Tombácz et al, 2017b; Tombácz et al, 2017a; Tombácz et al, 2018b; Depledge et al, 2019), poxviruses (Tombácz et al, 2018a), baculoviruses (Moldován et al, 2018b), retroviruses (Moldován et al, 2018a), coronaviruses (Viehweger et al, 2019), and circoviruses (Moldován et al, 2017a). The ONT technology is capable of sequencing DNA and RNA in its native form, allowing epigenetic and epitranscriptomic analysis (Wongsurawat et al, 2018; Liu et al, 2019; Shah et al, 2019)
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