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In this study we identified two 3'-coterminal RNA molecules in the pseudorabies virus. The highly abundant short transcript (CTO-S) proved to be encoded between the ul21 and ul22 genes in close vicinity of the replication origin (OriL) of the virus. The less abundant long RNA molecule (CTO-L) is a transcriptional readthrough product of the ul21 gene and overlaps OriL. These polyadenylated RNAs were characterized by ascertaining their nucleotide sequences with the Illumina HiScanSQ and Pacific Biosciences Real-Time (PacBio RSII) sequencing platforms and by analyzing their transcription kinetics through use of multi-time-point Real-Time RT-PCR and the PacBio RSII system. It emerged that transcription of the CTOs is fully dependent on the viral transactivator protein IE180 and CTO-S is not a microRNA precursor. We propose an interaction between the transcription and replication machineries at this genomic location, which might play an important role in the regulation of DNA synthesis.

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.

Herpesvirus gene expression is co-ordinately regulated and sequentially ordered during productive infection. The viral genes can be classified into three distinct kinetic groups: immediate-early, early, and late classes. In this study, a massively parallel sequencing technique that is based on PacBio Single Molecule Real-time sequencing platform, was used for quantifying the poly(A) fraction of the lytic transcriptome of pseudorabies virus (PRV) throughout a 12-hour interval of productive infection on PK-15 cells. Other approaches, including microarray, real-time RT-PCR and Illumina sequencing are capable of detecting only the aggregate transcriptional activity of particular genomic regions, but not individual herpesvirus transcripts. However, SMRT sequencing allows for a distinction between transcript isoforms, including length- and splice variants, as well as between overlapping polycistronic RNA molecules. The non-amplified Isoform Sequencing (Iso-Seq) method was used to analyse the kinetic properties of the lytic PRV transcripts and to then classify them accordingly. Additionally, the present study demonstrates the general utility of long-read sequencing for the time-course analysis of global gene expression in practically any organism.

SummaryPersonalized medicine is expected to benefit from combining genomic information with regular monitoring of physiological states by multiple high-throughput methods. Here, we present an integrative personal omics profile (iPOP), an analysis that combines genomic, transcriptomic, proteomic, metabolomic, and autoantibody profiles from a single individual over a 14 month period. Our iPOP analysis revealed various medical risks, including type 2 diabetes. It also uncovered extensive, dynamic changes in diverse molecular components and biological pathways across healthy and diseased conditions. Extremely high-coverage genomic and transcriptomic data, which provide the basis of our iPOP, revealed extensive heteroallelic changes during healthy and diseased states and an unexpected RNA editing mechanism. This study demonstrates that longitudinal iPOP can be used to interpret healthy and diseased states by connecting genomic information with additional dynamic omics activity.

Pseudorabies virus (PRV) is a neurotropic herpesvirus that causes Aujeszky's disease in pigs. PRV strains are widely used as transsynaptic tracers for mapping neural circuits. We present here the complete and fully annotated genome sequence of strain Kaplan of PRV, determined by Pacific Biosciences RSII long-read sequencing technology.

Personal transcriptomes in which all of an individual's genetic variants (e.g., single nucleotide variants) and transcript isoforms (transcription start sites, splice sites, and polyA sites) are defined and quantified for full-length transcripts are expected to be important for understanding individual biology and disease, but have not been described previously. To obtain such transcriptomes, we sequenced the lymphoblastoid transcriptomes of three family members (GM12878 and the parents GM12891 and GM12892) by using a Pacific Biosciences long-read approach complemented with Illumina 101-bp sequencing and made the following observations. First, we found that reads representing all splice sites of a transcript are evident for most sufficiently expressed genes ≤3 kb and often for genes longer than that. Second, we added and quantified previously unidentified splicing isoforms to an existing annotation, thus creating the first personalized annotation to our knowledge. Third, we determined SNVs in a de novo manner and connected them to RNA haplotypes, including HLA haplotypes, thereby assigning single full-length RNA molecules to their transcribed allele, and demonstrated Mendelian inheritance of RNA molecules. Fourth, we show how RNA molecules can be linked to personal variants on a one-by-one basis, which allows us to assess differential allelic expression (DAE) and differential allelic isoforms (DAI) from the phased full-length isoform reads. The DAI method is largely independent of the distance between exon and SNV--in contrast to fragmentation-based methods. Overall, in addition to improving eukaryotic transcriptome annotation, these results describe, to our knowledge, the first large-scale and full-length personal transcriptome.

Global RNA studies have become central to understanding biological processes, but methods such as microarrays and short-read sequencing are unable to describe an entire RNA molecule from 5' to 3' end. Here we use single-molecule long-read sequencing technology from Pacific Biosciences to sequence the polyadenylated RNA complement of a pooled set of 20 human organs and tissues without the need for fragmentation or amplification. We show that full-length RNA molecules of up to 1.5 kb can readily be monitored with little sequence loss at the 5' ends. For longer RNA molecules more 5' nucleotides are missing, but complete intron structures are often preserved. In total, we identify ∼14,000 spliced GENCODE genes. High-confidence mappings are consistent with GENCODE annotations, but >10% of the alignments represent intron structures that were not previously annotated. As a group, transcripts mapping to unannotated regions have features of long, noncoding RNAs. Our results show the feasibility of deep sequencing full-length RNA from complex eukaryotic transcriptomes on a single-molecule level.

Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A+) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono- and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.

Protein kinases are key regulators of cellular processes. In spite of considerable effort, a full understanding of the pathways they participate in remains elusive. We globally investigated the proteins that interact with the majority of yeast protein kinases using protein microarrays. Eighty-five kinases were purified and used to probe yeast proteome microarrays. One-thousand-twenty-three interactions were identified, and the vast majority were novel. Coimmunoprecipitation experiments indicate that many of these interactions occurred in vivo. Many novel links of kinases to previously distinct cellular pathways were discovered. For example, the well-studied Kss1 filamentous pathway was found to bind components of diverse cellular pathways, such as those of the stress response pathway and the Ccr4–Not transcriptional/translational regulatory complex; genetic tests revealed that these different components operate in the filamentation pathway in vivo. Overall, our results indicate that kinases operate in a highly interconnected network that coordinates many activities of the proteome. Our results further demonstrate that protein microarrays uncover a diverse set of interactions not observed previously.