Identification of herpesvirus transcripts from genomic regions around the replication origins

Long-read sequencing (LRS) techniques enable the identification of full-length RNA molecules in a single run eliminating the need for additional assembly steps. LRS research has exposed unanticipated transcriptomic complexity in various organisms, including viruses. Herpesviruses are known to produce a range of transcripts, either close to or overlapping replication origins (Oris) and neighboring genes related to transcription or replication, which possess confirmed or potential regulatory roles. In our research, we employed both new and previously published LRS and short-read sequencing datasets to uncover additional Ori-proximal transcripts in nine herpesviruses from all three subfamilies (alpha, beta and gamma). We discovered novel long non-coding RNAs, as well as splice and length isoforms of mRNAs. Moreover, our analysis uncovered an intricate network of transcriptional overlaps within the examined genomic regions. We demonstrated that herpesviruses display distinct patterns of transcriptional overlaps in the vicinity of or at the Oris. Our findings suggest the existence of a ‘super regulatory center’ in the genome of alphaherpesviruses that governs the initiation of both DNA replication and global transcription through multilayered interactions among the molecular machineries

Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

Abstract Background Epstein–Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read-sequencing and Pacific Biosciences RS II-based long-read sequencing technologies. Since the various sequencing methods have distinct strengths and limitations, the use of multiplatform approaches have proven to be valuable. The aim of this study is to provide a more complete picture on the transcriptomic architecture of EBV. Methods In this work, we apply the Oxford Nanopore Technologies MinION (long-read sequencing) platform for the generation of novel transcriptomic data, and integrate these with other’s data generated by another LRS approach, Pacific BioSciences RSII sequencing and Illumina CAGE-Seq and Poly(A)-Seq approaches. Both amplified and non-amplified cDNA sequencings were applied for the generation of sequencing reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. Results This study detected novel genes embedded into longer host genes containing 5′-truncated in-frame open reading frames, which potentially encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel replication-origin-associated transcripts. Additionally, novel mono- and multigenic transcripts were identified. An intricate meshwork of transcriptional overlaps was revealed. Conclusions An integrative approach applying multi-technique sequencing technologies is suitable for reliable identification of complex transcriptomes because each techniques has different advantages and limitations, and the they can be used for the validation of the results obtained by a particular approach. </jats:sec

Additional file 7 of Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

Additional file 7. The introns of EBV. The introns were detected using the LoRTIA software. Read counts per sample are shown. Splice junction coordinates are according to the genome of strain Akata (NCBI accession: KC207813.1)

Additional file 8 of Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

Additional file 8. Coding potential and BLAST analysis of alternatively spliced transcripts. For the analysis of the coding potential we used the CPAT tool

Additional file 4 of Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

Additional file 4. Transcripts of EBV. The transcripts were annotated using the LoRTIA software. The transcript coordinates are according to the genome of strain Akata (NCBI accession: KC207813.1). Transcript abundance in each sample is also shown

Additional file 3 of Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

Additional file 3. TESs of EBV. TESs were detected using the LoRTIA software. The -50–0 nt region upstream from the TES was analyzed for PASs. Distances from the TES are in nucleotides. The ±50 nts region of the TES was also retrieved for sequence analysis. The TES positions from previous PE-seq experiment was matched with our results. The sample number in which a given TES was detected is shown. TES coordinates are according to the genome of strain Akata (NCBI accession: KC207813.1)

Additional file 1 of Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

Additional file 1. Detailed descriptive statistics of the sequencing datasets. (A) Oxford Nanopore; B (Illumina)

Additional file 9 of Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

Additional file 9. Abundance of EBV transcripts. Color code. Light grey: low abundance: 1–10 reads, dark gray: medium abundance: 11–100 reads, black—high abundance: 101–3000 reads. See Additional file 4 for more details