Global transcript structure resolution of high gene density genomes through multi-platform data integration

Abstract

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome.

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis

“…This enriched pool of EBV transcripts was then oligo-dT primed to generate long cDNAs that were sequenced using a PacBio RS II analyzer. We were able to enrich the Wp/Cp driven EBV transcripts by ~7,000-fold over SMRT sequencing previously published from total LCL mRNA (O’Grady et al, 2016; Tilgner et al, 2014). EBV transcripts make up a low percentage of total LCL transcripts, as seen in our RNA-Seq meta-analysis (EBV transcripts were only ~0.70% of total reads).…”

“…4C). Previous SMRT sequencing of LCLs did not include any enrichment for Cp/Wp-driven transcripts, and so failed to detect any splicing between the Y exons and the HF exon in latent transcripts (O’Grady et al, 2016). We have therefore identified a novel, yet common, splicing pattern for EBNA-LP transcripts that allows the miR-BHRF1-2 and 1-3 pri-miRNA stem-loops to downregulate EBNA-LP expression in cis .…”

Current Progress in EBV-Associated B-Cell Lymphomas

“…About 300 novel EBV transcripts have been predicted by combining multiple platform data from PacBio SMART Iso-Seq, RNA-Seq, and deep-CAGE, which illustrates the complex regulation of viral gene transcription during EBV infection [63]. Studies on miRNA targetome show that EBV miRNAs mainly target cellular transcription factors to manipulate the microenvironment during latent infection, suggesting the importance of EBV-expressed miRNAs in contributing to viral-mediated oncogenesis [64].…”

Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells

“…In addition, the transcript annotations for these herpesviruses and the derived GFF sequence feature files are frequently inadequate to fully characterize the viral transcriptome. New approaches to resolve the transcript structure of high density genomes are critical to understanding virus biology and pathology [14]. …”