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Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs.

Background There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 μg of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated with DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation. Results Probe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3′ region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R > 0.9) than RNAse-H enzymatic depletion (R > 0.85). Conclusion In this study, our results showed that 1 μg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures.

Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG, TGA, and TAA) into three tumor suppressor genes, TP53, PTEN, and APC, in large white porcine fetal fibroblasts (PFFs). Among the isolated 290 single-cell colonies, 232 (80%) had premature stop codons in all the three genes. C−to−T conversion was found in 98.6%, 92.8%, and 87.2% of these cell colonies for TP53, PTEN, and APC, respectively. High frequencies of bystander C−to−T edits were observed within the editing window (positions 3–8), and there were nine (3.01%) clones with the designed simultaneous three-gene C−to−T conversion without bystander conversion. C−to−T conversion outside the editing window was found in 9.0%, 14.1%, and 26.2% of the 290 cell colonies for TP53, PTEN, and APC, respectively. Low-frequency C−to−G or C−to−A transversion occurred in APC. The mRNA levels of the three genes showed significant declines in triple-gene-mutant (Tri-Mut) cells as expected. No PTEN and a significantly lower (p < 0.05) APC protein expression were detected in Tri-Mut cells. Interestingly, the premature stop codon introduced into the TP53 gene did not eliminate the expression of its full-length protein in the Tri-Mut cells, suggesting that stop codon read-through occurred. Tri-Mut cells showed a significantly higher (p < 0.05) proliferation rate than WT cells. Furthermore, we identified 1418 differentially expressed genes (DEGs) between the Tri-Mut and WT groups, which were mainly involved in functions such as tumor progression, cell cycle, and DNA repair. This study indicates that hA3A-BE3-Y130F can be a powerful tool to create diverse knockout cell models without double-strand breaks (DSBs), with further possibilities to produce porcine models with various purposes.

Syftet med detta projekt var att undersöka hur vanligt använda cancerläkemedel påverkar mRNA-uttryck och kromatinorganisation i humana erytroleukemiceller. Som modell användes K562-celler från en patient i blastocystkris (2), för att utvärdera leukemicellernas svar på cancerläkemedel vinblastin och doxorubicin. Vinblastin och doxorubicin valdes på grund av deras distinkta mekanismer i cancercellen: medan doxorubicin interkaleras i DNA, hämmar topoisomeras II-aktivitet vilket orsakar celldöd, riktar vinblastin sig mot mikrotubuli för att stoppa mitotisk delning och proliferation. Uttryck av mRNA undersöktes i celler vid 0-timmar, 6-timmar och 24-timmar drogbehandling, samt efter en veckas återhämtning från 24-timmars drogbehandling. Kromatintillgänglighet med ATAC-seq undersöktes i K562-celler vid 0- timmar, 1-timmar, 6-timmar, 24-timmar och 24-timmar + en veckas återhämtning. Därefter utfördes DNA (ATAC-seq) och RNA (mRNA-seq) extraktion och biblioteksberedning på tre biologiska replikat, och öppna DNA-regioner samt mRNA expression undersöktes via sekvensering. Resultaten visade en stark korrelation mellan de biologiska replikaten, vilket indikerar att resultaten var upprepbara. Differentiellt uttryck av mRNA vid doxorubicin- och vinblastinbehandlingar utfördes genom att jämföra mRNA-nivåerna i läkemedelsbehandlade prover med obehandlade (0-timmar). Uppreglerade och nedreglerade gener identifierades och MA-grafer genererades för att visuellt analysera de differentiellt uttryckta generna vid olika tidpunkter efter läkemedelsbehandling och en veckas återhämtning. För att hitta anrikningar av funktionella genkategorier bland de läkemedelsinducerade eller -undertryckta generna, utfördes genontologianalyser. Slutligen användes verktyget Integrative Genomics Viewer (IGV) för att visuellt utforska mRNA-nivåerna och deras differentiella uttrycksmönster under läkemedelsbehandlingar. För ATAC-seq utfördes inte detaljerad dataanalys på grund av tidsbegränsning, men genomets öppenhet undersöktes visuellt genom IGV. Sammantaget inducerade doxorubicinbehandling en långsamt men långvarig förändring av genuttrycket, vilket involverade flera olika biologiska processer. Doxorubicinbehandlade K562-celler ändrade genuttryck att stöda kemoresistens snarare än att inducera apoptos eller celldöd. Behandlingen hade en långvarig inverkan på mRNA-nivåer som sträckte över återhämtningsveckan. Den totala uttrycksförändringen i återhämtningsproverna var förknippad med återhämtning av tumörigena egenskaper och återställning av mekanismener som stöder cellernas tillväxt. Vinblastine förorsakade snabb ökning av mRNA involverade i cytoskelettet. Vid 24-timmars vinblastinbehandling upplevde tumörcellerna stress på grund av grovt elongerad struktur, och de inducerade gener som stöder tumörbildning. En ökning av totala mRNA-nivåer detekterades i vinblastinbehandlade K562-leukemiceller, vilket var särskilt tydligt under återhämtningen. Resultaten visade att cellerna som överlevde vinblastinbehandling fokuserade på att återställa sin strukturella form. Sammantaget visade resultaten att monoterapi inte fungerar effektivt mot leukemiceller eftersom K562-leukemiceller inte bara överlevde läkemedelsbehandlingarna utan också inducerade mRNA som är involverade i resistens mot läkemedelsbehandlingar. The primary objective of this project is to investigate how commonly used cancer drugs affect mRNA expression and chromatin organization in human erythroleukemia cells. As a model, K562 cells derived from a patient in blastocyst crisis (2) were utilized, evaluating the leukemia cells’ cellular responses to cancer medicines vinblastine and doxorubicin. Vinblastine and doxorubicin were chosen due to the distinct pathways they target in the cell: while doxorubicin intercalates into DNA and inhibits topoisomerase II activity, which eventually cause cell death, vinblastine targets microtubules to stops mitotic division and excessive proliferation. Expression of mRNA was investigated in cells harvested at 0h, 6h, 24h and 24h + one week recovery. Chromatin accessibility with ATAC-seq was investigated in K562 cells harvested at 0h, 1h, 6h, 24h and 24h + one week recovery. Then DNA (ATAC-seq) and RNA (mRNA-seq) extraction and library preparation were performed on three replicates, and the genome-wide results was investigated via sequencing. The results showed a strong correlation between the biological replicates, indicating that the experimental conditions were sustained in these biological variables. Differential Expression of mRNA upon doxorubicin and vinblastine treatments was performed by comparing the mRNA levels in drug-treated samples to non-treated (0h) upregulated and down regulated genes were identified and MA plots generated to visually analyze the differentially expressed genes at different time points after drug treatment and one week recovery. To find enrichments of functional gene categories among the drug-induced or -repressed genes, gene ontology analyses were performed. Finally, the Integrative genomics viewer (IGV) tool was used to visually explore the mRNA levels and their differential expression pattern during drug treatments. For ATAC-seq, detailed data analysis was not performed due to limitation of time, and data was only visually explored through IGV. Taken together, doxorubicin treatment showed slow initial response within 6h followed by an extensive change in gene expression in 24h, involving several different biological processes. The response was more inclined towards chemoresistance rather than inducing apoptosis or cell death. There was a sustained increase in mRNA levels of doxorubicin treated leukemia cells during recovery week. The overall expression change in the recovery samples was majorly linked with not only gaining back the tumourigenic properties and restoring the mechanism which were affected by doxorubicin action but, based on changes in mRNA expression, it looks like doxorubicin treatment made the tumour cells more aggressive. The initial, 6h, response to vinblastine increases mRNAs involved in cytoskeleton. Upon 24h vinblastine treatment the tumour cells experienced stress due to shear force and structural deformity, and they induced genes supporting tumourigenesis. An increase in total mRNA levels was detected in vinblastine-treated K562 leukemia cells, which was particularly evident during recovery. The results indicated that the cells that survived vinblastine treatment focused on recovering its structural form. Overall, the results indicated that monotherapy does not effectively work against leukemia cells as K562 leukemia cells not only survived the drug treatments but also induced mRNAs involved in resistance against drug treatment.

Parkinson’s disease (PD) is the second most common age-related neurodegenerative disease. It is presently only accurately diagnosed at an advanced stage by a series of motor deficits, which are predated by a litany of non-motor symptoms manifesting over years or decades. Aberrant epigenetic modifications exist across a range of diseases and are non-invasively detectable in blood as potential markers of disease. We performed comparative analyses of the methylome and transcriptome in blood from PD patients and matched controls. Our aim was to characterize DNA methylation and gene expression patterns in whole blood from PD patients as a foundational step toward the future goal of identifying molecular markers that could predict, accurately diagnose, or track the progression of PD. We found that differentially expressed genes (DEGs) were involved in the processes of transcription and mitochondrial function and that PD methylation profiles were readily distinguishable from healthy controls, even in whole-blood DNA samples. Differentially methylated regions (DMRs) were functionally varied, including near transcription factor nuclear transcription factor Y subunit alpha (NFYA), receptor tyrosine kinase DDR1, RING finger ubiquitin ligase (RNF5), acetyltransferase AGPAT1, and vault RNA VTRNA2-1. Expression quantitative trait methylation sites were found at long non-coding RNA PAX8-AS1 and transcription regulator ZFP57 among others. Functional epigenetic modules were highlighted by IL18R1, PTPRC, and ITGB2. We identified patterns of altered disease-specific DNA methylation and associated gene expression in whole blood. Our combined analyses extended what we learned from the DEG or DMR results alone. These studies provide a foundation to support the characterization of larger sample cohorts, with the goal of building a thorough, accurate, and non-invasive molecular PD biomarker.

Intellectual disability is closely related to impaired GABA neurotransmission. Brpf1 was specifically expressed in medial ganglionic eminence (MGE), a developmental niche of GABAergic interneurons, and patients with BRPF1 mutations showed intellectual disability. To test its role in the development and function of MGE-derived GABAergic interneurons, we performed immunofluorescence staining, whole-cell patch-clamp, MGE transplantation, and mRNA-Seq to understand its effect on neuronal differentiation, dendritic morphology, electrophysiology, migration, and gene regulation, using mouse MGE-derived GABAergic interneurons infected with AAV-shBrpf1. The results showed that Brpf1 knockdown had a decreasing trend, although not significant, on the differentiation of GABAergic interneurons into parvalbumin+ interneurons. Moreover, increased firing threshold, decreased number of evoked action potentials, and a reduced amplitude of miniature inhibitory postsynaptic currents were observed before any significant change of MAP2+ dendritic morphology and in vivo migration ability appeared. Finally, mRNA-Seq analysis revealed that genes related to neurodevelopment and synaptic transmission such as Map2k7 were dysregulated. Our results demonstrated a key role of Brpf1 in inhibitory neurotransmission and related gene expression of GABAergic interneurons.

The queen‐worker caste system of eusocial insects represents a prime example of developmental polyphenism (environmentally‐induced phenotypic polymorphism) and is intrinsic to the evolution of advanced eusociality. However, the comparative molecular basis of larval caste determination and subsequent differentiation in the eusocial Hymenoptera remains poorly known. To address this issue within bees, we profiled caste‐associated gene expression in female larvae of the intermediately eusocial bumblebee Bombus terrestris. In B. terrestris, female larvae experience a queen‐dependent period during which their caste fate as adults is determined followed by a nutrition‐sensitive period also potentially affecting caste fate but for which the evidence is weaker. We used mRNA‐seq and qRT‐PCR validation to isolate genes differentially expressed between each caste pathway in larvae at developmental stages before and after each of these periods. We show that differences in gene expression between caste pathways are small in totipotent larvae, then peak after the queen‐dependent period. Relatively few novel (i.e., taxonomically‐restricted) genes were differentially expressed between castes, though novel genes were significantly enriched in late‐instar larvae in the worker pathway. We compared sets of caste‐associated genes in B. terrestris with those reported from the advanced eusocial honeybee, Apis mellifera, and found significant but relatively low levels of overlap of gene lists between the two species. These results suggest both the existence of low numbers of shared toolkit genes and substantial divergence in caste‐associated genes between Bombus and the advanced eusocial Apis since their last common eusocial ancestor.

Background Biological adhesion (bioadhesion), enables organisms to attach to surfaces as well as to a range of other targets. Bioadhesion evolved numerous times independently and is ubiquitous throughout the kingdoms of life. To date, investigations have focussed on various taxa of animals, plants and bacteria, but the fundamental processes underlying bioadhesion and the degree of conservation in different biological systems remain poorly understood. This study had two aims: 1) To characterise tissue-specific gene regulation in the pedal disc of the model cnidarian Exaiptasia pallida, and 2) to elucidate putative genes involved in pedal disc adhesion. Results Five hundred and forty-seven genes were differentially expressed in the pedal disc compared to the rest of the animal. Four hundred and twenty-seven genes were significantly upregulated and 120 genes were significantly downregulated. Forty-one condensed gene ontology terms and 19 protein superfamily classifications were enriched in the pedal disc. Eight condensed gene ontology terms and 11 protein superfamily classifications were depleted. Enriched superfamilies were consistent with classifications identified previously as important for the bioadhesion of unrelated marine invertebrates. A host of genes involved in regulation of extracellular matrix generation and degradation were identified, as well as others related to development and immunity. Ab initio prediction identified 173 upregulated genes that putatively code for extracellularly secreted proteins. Conclusion The analytical workflow facilitated identification of genes putatively involved in adhesion, immunity, defence and development of the E. pallida pedal disc. When defence, immunity and development-related genes were identified, those remaining corresponded most closely to formation of the extracellular matrix (ECM), implicating ECM in the adhesion of anemones to surfaces. This study therefore provides a valuable high-throughput resource for the bioadhesion community and lays a foundation for further targeted research to elucidate bioadhesion in the Cnidaria. Electronic supplementary material The online version of this article (10.1186/s12864-019-5917-5) contains supplementary material, which is available to authorized users.

Background Feline injection-site sarcoma (FISS), an aggressive iatrogenic subcutaneous malignancy, is challenging to manage clinically and little is known about the molecular basis of its pathogenesis. Tumor transcriptome profiling has proved valuable for gaining insights into the molecular basis of cancers and for identifying new therapeutic targets. Here, we report the first study of the FISS transcriptome and the first cross-species comparison of the FISS transcriptome with those of anatomically similar soft-tissue sarcomas in dogs and humans. Methods Using high-throughput short-read paired-end sequencing, we comparatively profiled FISS tumors vs. normal tissue samples as well as cultured FISS-derived cell lines vs. skin-derived fibroblasts. We analyzed the mRNA-seq data to compare cancer/normal gene expression level, identify biological processes and molecular pathways that are associated with the pathogenesis of FISS, and identify multimegabase genomic regions of potential somatic copy number alteration (SCNA) in FISS. We additionally conducted cross-species analyses to compare the transcriptome of FISS to those of soft-tissue sarcomas in dogs and humans, at the level of cancer/normal gene expression ratios. Results We found: (1) substantial differential expression biases in feline orthologs of human oncogenes and tumor suppressor genes suggesting conserved functions in FISS; (2) a genomic region with recurrent SCNA in human sarcomas that is syntenic to a feline genomic region of probable SCNA in FISS; and (3) significant overlap of the pattern of transcriptional alterations in FISS with the patterns of transcriptional alterations in soft-tissue sarcomas in humans and in dogs. We demonstrated that a protein, BarH-like homeobox 1 (BARX1), has increased expression in FISS cells at the protein level. We identified 11 drugs and four target proteins as potential new therapies for FISS, and validated that one of them (GSK-1059615) inhibits growth of FISS-derived cells in vitro. Conclusions (1) Window-based analysis of mRNA-seq data can uncover SCNAs. (2) The transcriptome of FISS-derived cells is highly consistent with that of FISS tumors. (3) FISS is highly similar to soft-tissue sarcomas in dogs and humans, at the level of gene expression. This work underscores the potential utility of comparative oncology in improving understanding and treatment of FISS. Electronic supplementary material The online version of this article (10.1186/s12885-019-5501-z) contains supplementary material, which is available to authorized users.

Experimental designs such as matched-pair or longitudinal studies yield mRNA sequencing (mRNA-Seq) counts that are correlated across samples. Most of the approaches for the analysis of correlated mRNA-Seq data are restricted to a specific design and/or balanced data only (with the same number of samples in each group). We propose a model that is applicable to the analysis of correlated mRNA-Seq data of different types: paired, clustered, longitudinal, or others. Any combination of explanatory variables, as well as unbalanced data, can be processed within the proposed modeling framework. The model assumes that exon counts of a particular gene of an individual sample jointly follow a multivariate negative-binomial distribution. Additional correlation between exon counts obtained for, for example, individual samples within the same pair or cluster, is taken into account by including into the model a cluster-level normally distributed random effect. An interesting feature of the model is that it provides explicit expression for marginal correlation between exon counts at different levels. The performance of the model is evaluated by using a simulation study and an analysis of two real-life data sets: a paired mRNA-Seq experiment for 24 patients with clear-cell renal-cell carcinoma and a longitudinal mRNA-Seq experiment for 29 patients with Lyme disease.

MicroRNAs (miRNAs) are endogenously expressed small noncoding RNAs. The genomic locations of animal miRNAs are significantly clustered in discrete loci. We found duplication and de novo formation were important mechanisms to create miRNA clusters and the clustered miRNAs tend to be evolutionarily conserved. We proposed a "functional co-adaptation" model to explain how clustering helps newly emerged miRNAs survive and develop functions. We presented evidence that abundance of miRNAs in the same clusters were highly correlated and those miRNAs exerted cooperative repressive effects on target genes in human tissues. By transfecting miRNAs into human and fly cells and extensively profiling the transcriptome alteration with deep-sequencing, we further demonstrated the functional co-adaptation between new and old miRNAs in the miR-17-92 cluster. Our population genomic analysis suggest that positive Darwinian selection might be the driving force underlying the formation and evolution of miRNA clustering. Our model provided novel insights into mechanisms and evolutionary significance of miRNA clustering.

Acquired drug resistance is the major reason why patients fail to respond to cancer therapies. It is a challenging task to determine the tipping point of endocrine resistance and detect the associated molecules. Derived from new systems biology theory, the dynamic network biomarker (DNB) method is designed to quantitatively identify the tipping point of a drastic system transition and can theoretically identify DNB genes that play key roles in acquiring drug resistance. We analyzed time-course mRNA sequence data generated from the tamoxifen-treated estrogen receptor (ER)-positive MCF-7 cell line, and identified the tipping point of endocrine resistance with its leading molecules. The results show that there is interplay between gene mutations and DNB genes, in which the accumulated mutations eventually affect the DNB genes that subsequently cause the change of transcriptional landscape, enabling full-blown drug resistance. Survival analyses based on clinical datasets validated that the DNB genes were associated with the poor survival of breast cancer patients. The results provided the detection for the pre-resistance state or early signs of endocrine resistance. Our predictive method may greatly benefit the scheduling of treatments for complex diseases in which patients are exposed to considerably different drugs and may become drug resistant.

The development of techniques for measuring gene expression globally has greatly expanded our understanding of gene regulatory mechanisms in depth and scale. We can now quantify every intermediate and transition in the canonical pathway of gene expression-from DNA to mRNA to protein-genome-wide. Employing such measurements in parallel can produce rich datasets, but extracting the most information requires careful experimental design and analysis. Here, we argue for the value of genome-wide studies that measure multiple outputs of gene expression over many timepoints during the course of a natural developmental process. We discuss our findings from a highly parallel gene expression dataset of meiotic differentiation, and those of others, to illustrate how leveraging these features can provide new and surprising insight into fundamental mechanisms of gene regulation.

Huntington's and Parkinson's Diseases (HD and PD) are neurodegenerative disorders that share some pathological features but are disparate in others. For example, while both diseases are marked by aberrant protein aggregation in the brain, the specific proteins that aggregate and types of neurons affected differ. A better understanding of the molecular similarities and differences between these two diseases may lead to a more complete mechanistic picture of both the individual diseases and the neurodegenerative process in general. We sought to characterize the common transcriptional signature of HD and PD as well as genes uniquely implicated in each of these diseases using mRNA-Seq data from post mortem human brains in comparison to neuropathologically normal controls. The enriched biological pathways implicated by HD differentially expressed genes show remarkable consistency with those for PD differentially expressed genes and implicate the common biological processes of neuroinflammation, apoptosis, transcriptional dysregulation, and neuron-associated functions. Comparison of the differentially expressed (DE) genes highlights a set of consistently altered genes that span both diseases. In particular, processes involving nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and transcription factor cAMP response element-binding protein (CREB) are the most prominent among the genes common to HD and PD. When the combined HD and PD data are compared to controls, relatively few additional biological processes emerge as significantly enriched, suggesting that most pathways are independently seen within each disorder. Despite showing comparable numbers of DE genes, DE genes unique to HD are enriched in far more coherent biological processes than the DE genes unique to PD, suggesting that PD may represent a more heterogeneous disorder. The complexity of the biological processes implicated by this analysis provides impetus for the development of better experimental models to validate the results.

The environment has profound effects on the expression of many traits and reaction norms describe the expression dynamics of a trait across a broad range of environmental conditions. Here, we analyze gene expression in Drosophila melanogaster across four different developmental temperatures (13–29 °C). Gene expression is highly plastic with 83.3% of the genes being differentially expressed. We distinguished three components of plasticity: 1) Dynamics of gene expression intensity (sum of change), 2) direction of change, and 3) curvature of the reaction norm (linear vs. quadratic). Studying their regulatory architecture we found that all three plasticity components were most strongly affected by the number of different transcription factors (TFs) binding to the target gene. More TFs were found in genes with less expression changes across temperatures. Although the effect of microRNAs was weaker, we consistently noted a trend in the opposite direction. The most plastic genes were regulated by fewer TFs and more microRNAs than less plastic genes. Different patterns of plasticity were also reflected by their functional characterization based on gene ontology. Our results suggest that reaction norms provide an important key to understand the functional requirements of natural populations exposed to variable environmental conditions.

// David J. Duffy 1,7,* , Aleksandar Krstic 1,* , Melinda Halasz 1,* , Thomas Schwarzl 1,8,* , Dirk Fey 1 , Kristiina Iljin 6 , Jai Prakash Mehta 1 , Kate Killick 1 , Jenny Whilde 1 , Benedetta Turriziani 1 , Saija Haapa-Paananen 6 , Vidal Fey 6 , Matthias Fischer 5 , Frank Westermann 4 , Kai-Oliver Henrich 4 , Steffen Bannert 4 , Desmond G. Higgins 1,2,3 and Walter Kolch 1,2,3 1 Systems Biology Ireland, University College Dublin, Belfield, Dublin, Ireland 2 Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin, Ireland 3 School of Medicine and Medical Science, University College Dublin, Belfield, Dublin, Ireland 4 Division of NB Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Department of Paediatric Haematology and Oncology and Center for Molecular Medicine Cologne (CMMC), University Hospital Cologne, Cologne, Germany 6 VTT Technical Research Centre of Finland, Tietotie 2, Espoo, Finland 7 The Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, Florida, USA 8 European Molecular Biology Laboratory (EMBL), Meyerhofstrase, Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: David J. Duffy, email: // Keywords : MYC (c-MYC), neuroblastoma, transcriptional regulation, mRNA sequencing (mRNA-seq), 4sU-seq Received : November 15, 2015 Accepted : November 23, 2015 Published : December 11, 2015 Abstract Despite intensive study, many mysteries remain about the MYCN oncogene’s functions. Here we focus on MYCN’s role in neuroblastoma, the most common extracranial childhood cancer. MYCN gene amplification occurs in 20% of cases, but other recurrent somatic mutations are rare. This scarcity of tractable targets has hampered efforts to develop new therapeutic options. We employed a multi-level omics approach to examine MYCN functioning and identify novel therapeutic targets for this largely un-druggable oncogene. We used systems medicine based computational network reconstruction and analysis to integrate a range of omic techniques: sequencing-based transcriptomics, genome-wide chromatin immunoprecipitation, siRNA screening and interaction proteomics, revealing that MYCN controls highly connected networks, with MYCN primarily supressing the activity of network components. MYCN’s oncogenic functions are likely independent of its classical heterodimerisation partner, MAX. In particular, MYCN controls its own protein interaction network by transcriptionally regulating its binding partners. Our network-based approach identified vulnerable therapeutically targetable nodes that function as critical regulators or effectors of MYCN in neuroblastoma. These were validated by siRNA knockdown screens, functional studies and patient data. We identified β-estradiol and MAPK/ERK as having functional cross-talk with MYCN and being novel targetable vulnerabilities of MYCN-amplified neuroblastoma. These results reveal surprising differences between the functioning of endogenous, overexpressed and amplified MYCN, and rationalise how different MYCN dosages can orchestrate cell fate decisions and cancerous outcomes. Importantly, this work describes a systems-level approach to systematically uncovering network based vulnerabilities and therapeutic targets for multifactorial diseases by integrating disparate omic data types.

Ocean acidification is an emerging problem that is expected to impact ocean species to varying degrees. Currently, little is known about its effect on molecular mechanisms induced in fleshy macroalgae. To elucidate genome wide responses to acidification, a comparative transcriptome analysis was carried out between Sargassum vulgare populations growing under acidified conditions at volcanic CO2 vents and a control site. Several transcripts involved in a wide range of cellular and metabolic processes were differentially expressed. No drastic changes were observed in the carbon acquisition processes and RuBisCO level. Moreover, relatively few stress genes, including those for antioxidant enzymes and heat shock proteins, were affected. Instead, increased expression of transcripts involved in energy metabolism, photosynthetic processes, and ion homeostasis suggested that algae increased energy production to maintain ion-homeostasis and other cellular processes. Also, an increased allocation of carbon to cell wall and carbon storage was observed. A number of genes encoding proteins involved in cellular signaling, information storage and processing, and transposition were differentially expressed between the two conditions. The transcriptional changes of key enzymes were largely confirmed by enzymatic activity measurements. Altogether, the changes induced by acidification indicate an adaptation of growth and development of S. vulgare at the volcanic CO2 vents, suggesting that this fleshy alga exhibits a high plasticity to low pH and can adopt molecular strategies to grow also in future more acidified waters. This article is protected by copyright. All rights reserved.

CD24 is an anchored cell surface marker that is highly expressed in cancer cells (Lee et al., 2009) and its expression is associated with poorer outcome of cancer patients (Kristiansen et al., 2003). Phenotype comparison between two subpopulations derived from the Mvt1 cell line, CD24(-) cells (with no CD24 cell surface expression) and the CD24(+) cells, identified high tumorigenic capacity for the CD24(+) cells. In order to reveal the transcripts that support the CD24(+) aggressive and invasive phenotype we compared the gene profiles of these two subpopulations. mRNA profiles of CD24(-) and CD24(+) cells were generated by deep sequencing, in triplicate, using an Illumina HiSeq 2500. Here we provide a detailed description of the mRNA-seq analysis from our recent study (Rostoker et al., 2015). The mRNA-seq data have been deposited in the NCBI GEO database (accession number GSE68746).

Despite its eradication over 30 years ago, smallpox (as well as other orthopox viruses) remains a pathogen of interest both in terms of biodefense and for its use as a vector for vaccines and immunotherapies. Here we describe the application of mRNA-Seq transcriptome profiling to understanding immune responses in smallpox vaccine recipients. Contrary to other studies examining gene expression in virally infected cell lines, we utilized a mixed population of peripheral blood mononuclear cells in order to capture the essential intercellular interactions that occur in vivo, and would otherwise be lost, using single cell lines or isolated primary cell subsets. In this mixed cell population we were able to detect expression of all annotated vaccinia genes. On the host side, a number of genes encoding cytokines, chemokines, complement factors and intracellular signaling molecules were downregulated upon viral infection, whereas genes encoding histone proteins and the interferon response were upregulated. We also identified a small number of genes that exhibited significantly different expression profiles in subjects with robust humoral immunity compared with those with weaker humoral responses. Our results provide evidence that differential gene regulation patterns may be at work in individuals with robust humoral immunity compared with those with weaker humoral immune responses.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate. © 2013 Federation of European Biochemical Societies.

Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink, and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a. España , Ministerio de Ciencia e Innovación BES-2010-031073 España , Ministerio de Ciencia e Innovación CGL2009-08257 España , Ministerio de Ciencia e Innovación CGL2012-37646

Background Fish reared under intensive conditions are repeatedly exposed to stress, which negatively impacts growth. Although most fish follow a conserved pattern of stress response, with increased concentrations of cortisol, each species presents specificities in the cell response and stress tolerance. Therefore, culturing new species requires a detailed knowledge of these specific responses. The red cusk-eel (Genypterus chilensis) is a new economically important marine species for the Chilean aquaculture industry. However, there is no information on the stress- and cortisol-induced mechanisms that decrease skeletal muscle growth in this teleost. Results Using Illumina RNA-seq technology, skeletal muscle sequence reads for G. chilensis were generated under control and handling stress conditions. Reads were mapped onto a reference transcriptome, resulting in the in silico identification of 785 up-regulated and 167 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant up-regulation of catabolic genes associated with skeletal muscle atrophy. These results were validated by RT-qPCR analysis for ten candidates genes involved in ubiquitin-mediated proteolysis, autophagy and skeletal muscle growth. Additionally, using a primary culture of fish skeletal muscle cells, the effect of cortisol was evaluated in relation to red cusk-eel skeletal muscle atrophy. Conclusions The present data demonstrated that handling stress promotes skeletal muscle atrophy in the marine teleost G. chilensis through the expression of components of the ubiquitin-proteasome and autophagy-lysosome systems. Furthermore, cortisol was a powerful inductor of skeletal muscle atrophy in fish myotubes. This study is an important step towards understanding the atrophy system in non-model teleost species and provides novel insights on the cellular and molecular mechanisms that control skeletal muscle growth in early vertebrates. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2232-7) contains supplementary material, which is available to authorized users.

Pre-mRNA splicing is regulated through the combinatorial activity of RNA motifs, including splice sites and splicing regulatory elements. Here we show that the activity of the G-run (polyguanine sequence) class of splicing enhancer elements is approx4-fold higher when adjacent to intermediate strength 5' splice sites (ss) than when adjacent to weak 5' ss, and approx1.3-fold higher relative to strong 5' ss. We observed this dependence on 5' ss strength in both splicing reporters and in global microarray and mRNA-Seq analyses of splicing changes following RNA interference against heterogeneous nuclear ribonucleoprotein (hnRNP) H, which cross-linked to G-runs adjacent to many regulated exons. An exon's responsiveness to changes in hnRNP H levels therefore depends in a complex way on G-run abundance and 5' ss strength. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5' ss mutations, augmenting both the frequency of 5' ss polymorphism and the evolution of new splicing patterns. Certain other splicing factors may function similarly., American Heart Association, Human Frontier Science Program (Strasbourg, France), National Institutes of Health (U.S.), National Science Foundation (U.S.) (equipment grant DBI-0821391)

Background Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. Results We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. Conclusions We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth. peerReviewed

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0729-7) contains supplementary material, which is available to authorized users.

Background Seedless grapes are greatly appreciated for fresh and dry fruit consumption. Parthenocarpy and stenospermocarpy have been described as the main phenomena responsible for seedlessness in Vitis vinifera. However, the key genes underpinning molecular and cellular processes that play a significant role in seed development are not well characterized. To identify important regulators and mechanisms that may be altered in the seedless phenotype, we performed a comprehensive transcriptional analysis to compare the transcriptomes of a popular seeded wine cultivar (wild-type) and its seedless somatic variant (mutant) at three key developmental stages. Results The transcriptomes revealed by Illumina mRNA-Seq technology had approximately 98% of grapevine annotated transcripts and about 80% of them were commonly expressed in the two lines. Differential gene expression analysis revealed a total of 1075 differentially expressed genes (DE) in the pairwise comparison of developmental stages, which included DE genes specific to the wild-type background, DE genes specific to the mutant background and DE genes commonly shared in both backgrounds. The analysis of differential expression patterns and functional category enrichment of wild-type and mutant DE genes highlighted significant coordination and enrichment of pollen and ovule developmental pathways. The expression of some selected DE genes was further confirmed by real-time RT-PCR analysis. Conclusions This study represents the most comprehensive attempt to characterize the genetic bases of seed formation in grapevine. With a high throughput method, we have shown that a seeded wine grape and its seedless somatic variant are similar in several biological processes. Nevertheless, we could identify an inventory of genes with altered expression in the mutant compared to the wild-type, which may be responsible for the seedless phenotype. The genes located within known genomic regions regulating seed content may be used for the development of molecular tools to assist table grape breeding. Therefore the data reported here have provided a rich genomic resource for practical use and functional characterization of the genes that potentially underpin seedlessness in grapevine. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1030) contains supplementary material, which is available to authorized users.

Background Neurodegenerative diseases are progressive and irreversible and they can be initiated by mutations in specific genes. Spalt-like genes (Sall) encode transcription factors expressed in the central nervous system. In humans, SALL mutations are associated with hereditary syndromes characterized by mental retardation, sensorineural deafness and motoneuron problems, among others. Drosophila sall mutants exhibit severe neurodegeneration of the central nervous system at embryonic stage 16, which surprisingly reverts later in development at embryonic stage 17, suggesting a potential to recover from neurodegeneration. We hypothesize that this recovery is mediated by a reorganization of the transcriptome counteracting SALL lost. To identify genes associated to neurodegeneration and neuroprotection, we used mRNA-Seq to compare the transcriptome of Drosophila sall mutant and wild type embryos from neurodegeneration and reversal stages. Results Neurodegeneration stage is associated with transcriptional changes in 220 genes, of which only 5% were already described as relevant for neurodegeneration. Genes related to the groups of Redox, Lifespan/Aging and Mitochondrial diseases are significantly represented at this stage. By contrast, neurodegeneration reversal stage is associated with significant changes in 480 genes, including 424 not previously associated with neuroprotection. Immune response and Salt stress are the most represented groups at this stage. Conclusions We identify new genes associated to neurodegeneration and neuroprotection by using an mRNA-Seq approach. The strong homology between Drosophila and human genes raises the possibility to unveil novel genes involved in neurodegeneration and neuroprotection also in humans.

Background The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life-threatening infections in immuno-compromised patients. Recently developed high-throughput transcriptome and proteome technologies, such as microarrays, RNA deep-sequencing, and LC-MS/MS of peptide mixtures, are of enormous value for systematically investigating pathogenic organisms. In the field of infection biology, one of the priorities is to collect and standardise data, in order to generate datasets that can be used to investigate and compare pathways and gene responses involved in pathogenicity. The “omics” era provides a multitude of inputs that need to be integrated and assessed. We therefore evaluated the potential of paired-end mRNA-Seq for investigating the regulatory role of the central mitogen activated protein kinase (MpkA). This kinase is involved in the cell wall integrity signalling pathway of A. fumigatus and essential for maintaining an intact cell wall in response to stress. Results The comparison of the transcriptome and proteome of an A. fumigatus wild-type strain with an mpkA null mutant strain revealed that 70.4% of the genome was found to be expressed and that MpkA plays a significant role in the regulation of many genes involved in cell wall remodelling, oxidative stress and iron starvation response, and secondary metabolite biosynthesis. Moreover, absence of the mpkA gene also strongly affects the expression of genes involved in primary metabolism. The data were further processed to evaluate the potential of the mRNA-Seq technique. We comprehensively matched up our data to published transcriptome studies and were able to show an improved data comparability of mRNA-Seq experiments independently of the technique used. Analysis of transcriptome and proteome data revealed only a weak correlation between mRNA and protein abundance. Conclusions High-throughput analysis of MpkA-dependent gene expression confirmed many previous findings that this kinase is important for regulating many genes involved in metabolic pathways. Our analysis showed more than 2000 differentially regulated genes. RNA deep-sequencing is less error-prone than established microarray-based technologies. It also provides additional information in A. fumigatus studies and as a result is more suitable for the creation of extensive datasets.

mRNA-Seq is a precise and highly reproducible technique for measurement of transcripts levels and yields sequence information of a transcriptome at a single nucleotide base-level thus enabling us to determine splice junctions and alternative splicing events with high confidence. Often analysis of mRNA-Seq data does not attempt to quantify the expressions at isoform level. In this paper our objective would be use the mRNA-Seq data to infer expression at isoform level, where splicing patterns of a gene is assumed to be known. A Bayesian latent variable based modeling framework is proposed here, where the parameterization enables us to infer at various levels. For example, expression variability of an isoform across different conditions; the model parameterization also allows us to carry out two-sample comparisons, e.g., using a Bayesian t-test, in addition simple presence or absence of an isoform can also be estimated by the use of the latent variables present in the model. In this paper we would carry out inference on isoform expression under different normalization techniques, since it has been recently shown that one of the most prominent sources of variation in differential call using mRNA-Seq data is the normalization method used. The statistical framework is developed for multiple isoforms and easily extends to reads mapping to multiple genes. This could be achieved by slight conceptual modifications in definitions of what we consider as a gene and what as an exon. Additionally proposed framework can be extended by appropriate modeling of the design matrix to infer about yet unknown novel transcripts. However such attempts should be made judiciously since the input date used in the proposed model does not use reads from splice junctions.

Background Transcriptome analysis is increasingly being used to study the evolutionary origins and ecology of non-model plants. One issue for both transcriptome assembly and differential gene expression analyses is the common occurrence in plants of hybridisation and whole genome duplication (WGD) and hybridization resulting in allopolyploidy. The divergence of duplicated genes following WGD creates near identical homeologues that can be problematic for de novo assembly and also reference based assembly protocols that use short reads (35 - 100 bp). Results Here we report a successful strategy for the assembly of two transcriptomes made using 75 bp Illumina reads from Pachycladon fastigiatum and Pachycladon cheesemanii. Both are allopolyploid plant species (2n = 20) that originated in the New Zealand Alps about 0.8 million years ago. In a systematic analysis of 19 different coverage cutoffs and 20 different k-mer sizes we showed that i) none of the genes could be assembled across all of the parameter space ii) assembly of each gene required an optimal set of parameter values and iii) these parameter values could be explained in part by different gene expression levels and different degrees of similarity between genes. Conclusions To obtain optimal transcriptome assemblies for allopolyploid plants, k-mer size and k-mer coverage need to be considered simultaneously across a broad parameter space. This is important for assembling a maximum number of full length ESTs and for avoiding chimeric assemblies of homeologous and paralogous gene copies.

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina's genome analyzer performs sequencing of a large number (10(7)) of relatively short sequence reads (150 bp).The "paired end" approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly. One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described(1, 2) but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published(3, 4) but require microgram amounts of starting total RNA. Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing(5), where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined "T7LA" (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome(6) and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples(7).

There is controversy as to whether gene expression is silenced in the functional centromere. The complete genomic sequences of the centromeric regions in higher eukaryotes have not been fully elucidated, because the presence of highly repetitive sequences complicates many aspects of genomic sequencing. We performed resequencing, assembly, and sequence finishing of two P1-derived artificial chromosome clones in the centromeric region of rice (Oryza sativa L.) chromosome 5 (Cen5). The pericentromeric region, where meiotic recombination is silenced, is located at the center of chromosome 5, and is 2.14 Mb in length; a total of six restriction-fragment-length polymorphism markers (R448, C1388, S20487S, E3103S, C53260S, and R2059) genetically mapped at 54.6 cM were located in this region. In the pericentromeric region, 28 genes were annotated on the short arm and 45 genes on the long arm. To quantify all transcripts in this region, we performed massive parallel sequencing of mRNA. Transcriptional density (total length of transcribed region/length of the genomic region) and expression level (number of uniquely mapped reads/length of transcribed region) were calculated on the basis of the mapped reads on the rice genome. Transcriptional density and expression level were significantly lower in Cen5 than in the average of the other chromosomal regions. Moreover, transcriptional density in Cen5 was significantly lower on the short arm than on the long arm; the distribution of transcriptional density was asymmetric. The genomic sequence of Cen5 has been integrated into the most updated reference rice genome sequence constructed by the International Rice Genome Sequencing Project.

To compare the two RNA-sequencing protocols, ribo-minus RNA-sequencing (rmRNA-seq) and polyA-selected RNA-sequencing (mRNA-seq), we acquired transcriptomic data–52 and 32 million alignable reads of 35 bases in length–from the mouse cerebrum, respectively. We found that a higher proportion, 44% and 25%, of the uniquely alignable rmRNA-seq reads, is in intergenic and intronic regions, respectively, as compared to 23% and 15% from the mRNA-seq dataset. Further analysis made an additional discovery of transcripts of protein-coding genes (such as Histone, Heg1, and Dux), ncRNAs, snoRNAs, snRNAs, and novel ncRNAs as well as repeat elements in rmRNA-seq dataset. This result suggests that rmRNA-seq method should detect more polyA- or bimorphic transcripts. Finally, through comparative analyses of gene expression profiles among multiple datasets, we demonstrated that different RNA sample preparations may result in significant variations in gene expression profiles.