Accurate transcriptome assembly by Nanopore RNA sequencing reveals novel functional transcripts in hepatocellular carcinoma.

The long reads of Nanopore sequencing permit accurate transcript assembly and ease in discovering novel transcripts with potentially important functions in cancers. The wide adoption of Nanopore sequencing for transcript quantification, however, is largely limited by high costs. To address this issue, we developed a bioinformatics software, NovelQuant, that can specifically quantify long-read-assembled novel transcripts with short-read sequencing data. Nanopore Direct RNA Sequencing was carried out on three hepatocellular carcinoma (HCC) patients' tumor, matched portal vein tumor thrombus, and peritumor to reconstruct the HCC transcriptome. Then, based on the reconstructed transcriptome, NovelQuant was applied on Illumina RNA sequencing data of 59 HCC patients' tumor and paired peritumor to quantify novel transcripts. Our further analysis revealed 361 novel transcripts dysregulated in HCC and that 101 of them were significantly associated with prognosis. There were 19 novel prognostic transcripts predicted to be long noncoding RNAs (lncRNAs), and some of them had regulatory targets that were reported to be associated with HCC. Additionally, 42 novel prognostic transcripts were predicted to be protein-coding mRNAs, and many of them could be involved in xenobiotic metabolism. Moreover, the tumor-suppressive roles of two representative novel prognostic transcripts, CDO1-novel (lncRNA) and CYP2A6-novel (protein-coding mRNA), were further functionally validated during HCC progression. Overall, the current study shows a possibility of combining long- and short-read sequencing to explore functionally important novel transcripts in HCC with accuracy and cost-efficiency, which expands the pool of molecular biomarkers that could enhance our understanding of the molecular mechanisms of HCC.

Barley long non-coding RNAs (lncRNA) responsive to excess boron.

Long non-coding RNA (lncRNA) has a misleading name, since although they do not encode proteins, they may encode small peptides. Such transcripts are emerging as regulatory molecules. With the advent of next-generation sequencing technologies and novel bioinformatics tools, a tremendous amount of lncRNAs have been identified in several plant species. Recent reports demonstrated roles of plant lncRNAs such as development and environmental response. Here, we reported a genome-wide discovery of ~8000 barley lncRNAs and measured their expression pattern upon excessive boron (B) treatment. According to the tissue-based comparison, leaves have a greater number of B-responsive differentially expressed lncRNAs than the root. Functional annotation of the coding transcripts, which were co-expressed with lncRNAs, revealed that molecular function of the ion transport, establishment of localization, and response to stimulus significantly enriched only in the leaf. On the other hand, 32 barley endogenous target mimics (eTM) as lncRNAs, which potentially decoy the transcriptional suppression activity of 18 miRNAs, were obtained. Also, six lncRNAs, differentially expressed upon B-treatment, were selected and quantitatively analyzed in both B-sensitive and B-tolerant cultivars treated by excess B-level. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed the B-responsive expressional changes obtained by RNA sequencing. Notably, some lncRNAs (i.e., TCONS_00045190 and TCONS_00056415) over-expressed only in B-tolerant cultivar upon excess B treatment. Presented data including identification, expression measurement, and functional characterization of barley lncRNAs suggest that B-stress response might also be regulated by lncRNA expression, via cooperative interaction of miRNA-eTM-coding target transcript modules.

Exploring the heat-responsive chaperones and microsatellite markers associated with terminal heat stress tolerance in developing wheat.

Global warming is a major threat for agriculture and food security, and in many cases the negative impacts are already apparent. Wheat is one of the most important staple food crops and is highly sensitive to the heat stress (HS) during reproductive and grain-filling stages. Here, whole transcriptome analysis of thermotolerant wheat cv. HD2985 was carried out at the post-anthesis stage under control (22 ± 3 °C) and HS-treated (42 °C, 2 h) conditions using Illumina Hiseq and Roche GS-FLX 454 platforms. We assembled ~24 million (control) and ~23 million (HS-treated) high-quality trimmed reads using different assemblers with optimal parameters. De novo assembly yielded 52,567 (control) and 59,658 (HS-treated) unigenes. We observed 785 transcripts to be upregulated and 431 transcripts to be downregulated under HS; 78 transcripts showed >10-fold upregulation such as HSPs, metabolic pathway-related genes, etc. Maximum number of upregulated genes was observed to be associated with processes such as HS-response, protein-folding, oxidation-reduction and photosynthesis. We identified 2008 and 2483 simple sequence repeats (SSRs) markers from control and HS-treated samples; 243 SSRs were observed to be overlying on stress-associated genes. Polymorphic study validated four SSRs to be heat-responsive in nature. Expression analysis of identified differentially expressed transcripts (DETs) showed very high fold increase in the expression of catalytic chaperones (HSP26, HSP17, and Rca) in contrasting wheat cvs. HD2985 and HD2329 under HS. We observed positive correlation between RNA-seq and qRT-PCR expression data. The present study culminated in greater understanding of the heat-response of tolerant genotype and has provided good candidate genes for the marker development and screening of wheat germplasm for thermotolerance.

The human skeletal muscle transcriptome: sex differences, alternative splicing, and tissue homogeneity assessed with RNA sequencing.

Human skeletal muscle health is important for quality of life and several chronic diseases, including type II diabetes, heart disease, and cancer. Skeletal muscle is a tissue widely used to study mechanisms behind different diseases and adaptive effects of controlled interventions. For such mechanistic studies, knowledge about the gene expression profiles in different states is essential. Since the baseline transcriptome has not been analyzed systematically, the purpose of this study was to provide a deep reference profile of female and male skeletal muscle. RNA sequencing data were analyzed from a large set of 45 resting human muscle biopsies. We provide extensive information on the skeletal muscle transcriptome, including 5 previously unannotated protein-coding transcripts. Global transcriptional tissue homogeneity was strikingly high, within both a specific muscle and the contralateral leg. We identified >23,000 known isoforms and found >5000 isoforms that differ between the sexes. The female and male transcriptome was enriched for genes associated with oxidative metabolism and protein catabolic processes, respectively. The data demonstrate remarkably high tissue homogeneity and provide a deep and extensive baseline reference for the human skeletal muscle transcriptome, with regard to alternative splicing, novel transcripts, and sex differences in functional ontology.transcriptome: sex differences, alternative splicing, and tissue homogeneity assessed with RNA sequencing.

Mystifying Molecular Structure, Expression and Repertoire Diversity of IgM Heavy Chain Genes (Ighµ) in Clarias Catfish and Hybrids: Two Novel Transcripts in Vertebrates.

Two novel immunoglobulin heavy chain (Ighµ) transcripts encoding membrane-bound forms of IgM (mIgM) were discovered in bighead catfish, Clarias macrocephalus. The first transcript contains four constant and two transmembrane domains [Cµ1-Cµ2-Cµ3-Cµ4-TM1-TM2] that have never been reported in teleosts, and the second transcript is an unusual mIgM that has never been identified in any vertebrate [Cµ1-(Cδ2-Cδ3-Cδ4-Cδ5)-Cµ2-Cµ3-TM1-TM2]. Fluorescence in situ hybridization (FISH) in bighead catfish, North African catfish (C. gariepinus) and hybrid catfish revealed a single copy of Ighµ in individual parent catfish, while two gene copies were found in diploid hybrid catfish. Intensive sequence analysis demonstrated multiple distinct structural variabilities in the VH domain in Clarias, and hybrid catfish were defined and used to generate diversity with various mechanisms. Expression analysis of Ighµ in Aeromonas hydrophila infection of the head kidney, peripheral blood leukocytes and spleen revealed significantly higher levels in North African catfish and hybrid catfish than in bighead catfish.

Identification of a novel Sox5 transcript in mouse testis.

The transcription factor SOX5 is present in two distinct isoforms in both human and mouse, L-SOX5 and S-SOX5 (long and short isoforms of SOX5). Here, we identified and characterized a novel transcript of Sox5 (S-Sox5 variant) in mouse testis. eCLIP-based amplification of cDNA ends were performed to identify the potential Sox5 mRNA variant. This novel transcript shares a high similarity with the previously reported S-Sox5 in nucleotide sequence, but with a unique stretch of 5'UTR and an additional exon 9. Semi-quantitative PCR analysis revealed both S-Sox5 variant and S-Sox5 express specifically in mouse testis. Both transcripts increase significantly in mouse testis at postnatal day 21, when round spermatids appear. We further made a series of truncated Sox5 constructs and tagged them with eGFP in HeLa cells. In vitro transfection assay identified the N-terminus and the DNA-binding HMG domain are required for the nuclear localization of SOX5. Our results provides a basis for the future study to investigate the biological function of SOX5 in spermatogenesis.

High-throughput sequencing profile of laryngeal cancers: analysis of co-expression and competing endogenous RNA networks of circular RNAs, long non-coding RNAs, and messenger RNAs.

BACKGROUND: Circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) have been recently identified as new classes of non-coding RNAs which participate in carcinogenesis and tumor progression. However, the functions of these non-coding RNAs and gene expression patterns are largely unknown. METHODS: We carried out high-throughput sequencing to analyze the differential expression of RNAs in 5 coupled laryngeal cancer (LC) and corresponding adjacent noncancerous tissues. Bioinformatics analyses were performed to predict the functions of these non-coding RNAs via co-expression, competing endogenous RNA networks and pathway enrichment analysis. The differential expression of the selected RNAs were confirmed using RT-qPCR. The CCK8, EDU, Transwell, and wound healing assays were conducted to validate the biological functions of SNHG29 in LC. Western blot assay was performed to identify the effects of SNHG29 having on the epithelial to mesenchymal transition process. Kaplan-Meier analysis was used to investigate whether the expression level of SNHG29 correlated with survival in LC patients. One-way ANOVA was used to analyze the correlation between the expression of SNHG29 and clinicopathological parameters of the included patients. RESULTS: Compared to normal laryngeal tissues, 31,763 non-coding RNAs were upregulated and 11,557 non-coding RNAs were downregulated in cancer tissues. SNHG29 expression was low in the LC cell lines and tissues predicting a better clinical prognosis. SNHG29 was also found to inhibit the proliferation, migration, and invasion ability of LC, exerting a suppressive role in the epithelial to mesenchymal transition process as well. SNHG29 downregulation was significantly correlated with differentiation (P=0.026), T-stage (P=0.041), lymphatic metastasis (P=0.044), and clinical stage (P=0.037). We found that the biological functions of differentially expressed transcripts included cell adhesion, biological adhesion, and migration and invasion related to adherens junction pathways. CONCLUSIONS: Our study was the first to describe the non-coding RNA profile of LC, and suggested that dysregulated non-coding RNAs could be involved in LC tumorigenesis. SNHG29 was demonstrated to play crucial roles in inhibiting the pathogenesis and progression of LC. Our findings provide a new approach for further analyses of pathogenetic mechanisms, the detection of novel transcripts, and the identification of valuable biomarkers for this tumor.

Discovery and Annotation of Plant Endogenous Target Mimicry Sequences from Public Transcriptome Libraries: A Case Study of Prunus persica.

Novel transcript discovery through RNA sequencing has substantially improved our understanding of the transcriptome dynamics of biological systems. Endogenous target mimicry (eTM) transcripts, a novel class of regulatory molecules, bind to their target microRNAs (miRNAs) by base pairing and block their biological activity. The objective of this study was to provide a computational analysis framework for the prediction of putative eTM sequences in plants, and as an example, to discover previously un-annotated eTMs in Prunus persica (peach) transcriptome. Therefore, two public peach transcriptome libraries downloaded from Sequence Read Archive (SRA) and a previously published set of long non-coding RNAs (lncRNAs) were investigated with multi-step analysis pipeline, and 44 putative eTMs were found. Additionally, an eTM-miRNA-mRNA regulatory network module associated with peach fruit organ development was built via integration of the miRNA target information and predicted eTM-miRNA interactions. My findings suggest that one of the most widely expressed miRNA families among diverse plant species, miR156, might be potentially sponged by seven putative eTMs. Besides, the study indicates eTMs potentially play roles in the regulation of development processes in peach fruit via targeting specific miRNAs. In conclusion, by following the step-by step instructions provided in this study, novel eTMs can be identified and annotated effectively in public plant transcriptome libraries.

Application of RNA-Seq Technology in Cancer Chemoprevention.

RNA-sequencing is a revolutionary tool to follow differential expression after treatment with cancer chemopreventive agents. It allows a real genome-wide screening independent of prior assumptions and is well suited for analyzing coding but also long noncoding RNAs. It still consents the discovery of new genes and isoforms and increased our knowledge of antisense and other noncoding RNAs in a tremendous manner. Moreover, it permits to detect low-abundance and biologically critical isoforms and reveals genetic variants and gene fusions in one single assay. Here, we provide a detailed protocol for stranded RNA-sequencing.

Re-analysis of RNA-seq transcriptome data reveals new aspects of gene activity in Arabidopsis root hairs.

Root hairs, tubular-shaped outgrowths from root epidermal cells, play important roles in the acquisition of nutrients and water, interaction with microbe, and in plant anchorage. As a specialized cell type, root hairs, especially in Arabidopsis, provide a pragmatic research system for various aspects of studies. Here, we re-analyzed the RNA-seq transcriptome profile of Arabidopsis root hair cells by Tophat software and used Cufflinks program to mine the differentially expressed genes. Results showed that ERD14, RIN4, AT5G64401 were among the most abundant genes in the root hair cells; while ATGSTU2, AT5G54940, AT4G30530 were highly expressed in non-root hair tissues. In total, 5409 genes, with a fold change greater than two-fold (FDR adjusted P < 0.05), showed differential expression between root hair cells and non-root hair tissues. Of which, 61 were expressed only in root hair cells. One hundred and thirty-six out of 5409 genes have been reported to be "core" root epidermal genes, which could be grouped into nine clusters according to expression patterns. Gene ontology (GO) analysis of the 5409 genes showed that processes of "response to salt stress," "ribosome biogenesis," "protein phosphorylation," and "response to water deprivation" were enriched. Whereas only process of "intracellular signal transduction" was enriched in the subset of 61 genes expressed only in the root hair cells. One hundred and twenty-one unannotated transcripts were identified and 14 of which were shown to be differentially expressed between root hair cells and non-root hair tissues, with transcripts XLOC_000763, XLOC_031361, and XLOC_005665 being highly expressed in the root hair cells. The comprehensive transcriptomic analysis provides new information on root hair gene activity and sets the stage for follow-up experiments to certify the biological functions of the newly identified genes and novel transcripts in root hair cell morphogenesis.