Third-generation Sequencing Reveals Extensive Polycistronism and Transcriptional Overlapping in a Baculovirus

Abstract

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an insect-pathogen baculovirus. In this study, we applied the Oxford Nanopore Technologies platform for the analysis of the polyadenylated fraction of the viral transcriptome using both cDNA and direct RNA sequencing methods. We identified and annotated altogether 132 novel transcripts and transcript isoforms, including 4 coding and 4 non-coding RNA molecules, 47 length variants, 5 splice isoforms, as well as 23 polycistronic and 49 complex transcripts. All of the identified novel protein-coding genes were 5′-truncated forms of longer host genes. In this work, we demonstrated that in the case of transcript start site isoforms, the promoters and the initiator sequence of the longer and shorter variants belong to the same kinetic class. Long-read sequencing also revealed a complex meshwork of transcriptional overlaps, the function of which needs to be clarified. Additionally, we developed bioinformatics methods to improve the transcript annotation and to eliminate the non-specific transcription reads generated by template switching and false priming.

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).…”

Protein composition of the occlusion bodies of Epinotia aporema granulovirus

“…On the other hand, Epap126 orthogroup is present in group II Baculovirus genomes are densely populated with coding sequences (overlapping in several cases) and contain short intergenic regions [2]. A recent study has described the transcriptional landscape of baculovirus infection, demonstrating the existence of several polycistronic and overlapping viral transcripts [42]. Together with other technologies, proteogenomic mapping is a valuable tool to improve the annotation of these complex coding regions.…”