Illumina mRNA-Seq Research Articles

Lizhong decoction ameliorates pulmonary infection secondary to severe traumatic brain injury in rats by regulating the intestinal physical barrier and immune response.

The pathogenesis of pulmonary infection secondary to severe traumatic brain injury (sTBI) is closely related to damage to the intestinal barrier. Lizhong decoction (LZD) is a prominent traditional Chinese medicine (TCM) that is widely used in clinical treatment to regulate gastrointestinal movement and enhance resistance. Nevertheless, the role and mechanism of LZD in lung infection secondary to sTBI have yet to be elucidated. Here, we evaluate the therapeutic effect of LZD on pulmonary infection secondary to sTBI in rats and discuss potential regulatory mechanisms. The chemical constituents of LZD were analyzed by ultra-high performance liquid chromatography-Q Exactive-tandem mass spectrometry(UPLC-QE-MS/MS). The efficacy of LZD on rats with lung infection secondary to sTBI was examined by changes in brain morphology, coma time, brain water content, mNSS score, colony counts, 16S rRNA/RNaseP/MRP30kDa(16S/RPP30), myeloperoxidase (MPO) content and pathology of lung tissue. The concentration of fluorescein isothiocyanate(FITC)-dextran in serum and the contents of secretory immunoglobulin A (SIgA) in colon tissue were detected by enzyme-linked immunosorbent assay (ELISA). Subsequently, Alcian Blue Periodic acid Schiff (AB-PAS) was used to detect colonic goblet cells. Immunofluorescence (IF) was used to detect the expression of tight junction proteins. The proportions of CD3+ cell, CD4+CD8+ T cells, CD45+ cell and CD103+ cells in the colon were analyzed by flow cytometry (FC). In addition, colon transcriptomics were analyzed by Illumina mRNA-Seq sequencing. Real-time quantitative polymerase chain reaction (qRT‒PCR) was used to verify the genes associated with LZD alleviation of intestinal barrier function. Twenty-nine chemical constituents of LZD were revealed with UPLC-QE-MS/MS analysis. Administration of LZD significantly reduced colony counts, 16S/RPP30 and MPO content in lung infection secondary to sTBI rats. In addition, LZD also reduced the serum FITC-glucan content and the SIgA content of the colon. Additionally, LZD significantly increased the number of colonic goblet cells and the expression of tight junction proteins. Furthermore, LZD significantly decreased the proportion of CD3+ cell, CD4+CD8+ T cells,CD45+ and CD103+ cells in colon tissue. Transcriptomic analysis identified 22 upregulated genes and 56 downregulated genes in sTBI compared to the sham group. The levels of seven genes were recovered after LZD treatment. qRT‒PCR successfully validated two genes (Jchain and IL-6) at the mRNA level. LZD can improves sTBI secondary lung infection by regulating the intestinal physical barrier and immune response. Thees results suggested that LZD may be a prospective treatment for pulmonary infection secondary to sTBI.

Transcriptomics and metabolomics reveal the cardioprotective effect of Compound Danshen tablet on isoproterenol-induced myocardial injury in high-fat-diet fed mice.

Compound Danshen tablet, an herbal preparation consisting of salviae miltiorrhizae, notoginseng and borneolum, is extensively employed clinically to treat angina pectoris, coronary arteriosclerosis and significantly improve microcirculation. To reveal the potential underlying cardioprotective mechanism(s) in isoproterenol-induced myocardial injury in high-fat-diet fed mice. Cardiac transcriptomics was analyzed by Illumina mRNA-Seq sequencing. The restored cardiovascular diseases (CVD)-related genes by Compound Danshen tablet were validated by quantitative real time polymerase chain reaction (qRT-PCR). Furthermore, Cardiac metabolomics were also performed using gas chromatography-mass spectrometry. From the transcriptomics study, we found the levels of 24 up-regulated and 44 down-regulated genes in the control compared to model groups. Among them, seven gene levels were restored by treatment of Compound Danshen tablet. Four CVD-related genes at the mRNA level (Sprr1a, Ppp1r3c, Bmp10 and Hspa1b) were validated successfully by qRT-PCR. From the metabolomics study, 37 differentially expressed metabolites were identified between the control and model groups. Among them, 21 metabolites were restored by treatment of Compound Danshen tablet. These altered metabolites are involved in glucose metabolism, fatty acid metabolism and amino acid metabolism. These genes and metabolites might provide clues for further molecular mechanistic study of Compound Danshen tablet.

Immune transcriptome reveals the mincle C-type lectin receptor acts as a partial replacement for TLR4 in lipopolysaccharide-mediated inflammatory response in barramundi (Lates calcarifer).

Fish represent the most diverse and abundant extant vertebrate infraclass. They are also one of the earliest divergent phyla with adaptive immunity based on antigen recognition by MHC and immunoglobulin. The aquaculture industry, which currently provides more than half of the fish for human consumption globally, has successfully exploited the adaptive immune system of fish through mass vaccination programs. However, vaccination against highly diverse antigens, mostly carbohydrates, such as capsular polysaccharides and lipopolysaccharide (LPS) is challenging. Fish have a subdued innate response to LPS, but adaptive response is generally high and type-specific. To better understand the link between initial innate response and early onset of adaptive immunity to carbohydrate antigens in the perciform barramundi (Lates calcarifer), an immune transcriptome was prepared from pronephros and spleen following vaccination with LPS and peptidoglycan. From 163,661 transcripts derived by Illumina mRNA-Seq, most grouped in neuronal, endocrine or immune system categories, suggesting a close relationship between the three systems. Moreover, digestive enzyme transcripts in spleen appeared to be highly inducible in barramundi. Most of the known TLRs were transcribed in the barramundi spleen and HK transcriptome, with the notable exception of TLR4, which is primarily responsible for LPS recognition in mammals. Several C-type lectin receptors were also identified, including CD209, CD205, and CLEC4E (Mincle). As Mincle has been shown to bind LPS and is abundant on dendritic cells, its role in response to LPS in barramundi was further investigated. A high dose of LPS induced TNF-alpha expression via Mincle. However, IL-6 regulation, whilst still regulated in response to LPS, did not depend upon the Mincle pathway, suggesting other routes of activation. This study thus suggests that Mincle acts as a partial substitute for TLR4 in barramundi in the processing of LPS.

Transcriptome analysis of genes involved in anthocyanins biosynthesis and transport in berries of black and white spine grapes (Vitis davidii).

BackgroundThe color of berry skin is an important economic trait for grape and is essentially determined by the components and content of anthocyanins. The fruit color of Chinese wild grapes is generally black, and the profile of anthocyanins in Chinese wild grapes is significantly different from that of Vitis vinifera. However, V. davidii is the only species that possesses white berry varieties among Chinese wild grape species. Thus, we performed a transcriptomic analysis to compare the difference of transcriptional level in black and white V. davidii, in order to find some key genes that are related to anthocyanins accumulation in V. davidii.ResultsThe results of anthocyanins detection revealed that 3,5-O-diglucoside anthocyanins is the predominant anthocyanins in V. davidii. It showed obvious differences from V. vinifera in the profile of the composition of anthocyanins. The transcriptome sequencing by Illumina mRNA-Seq technology generated an average of 57 million 100-base pair clean reads from each sample. Differential gene expression analysis revealed thousands of differential expression genes (DEGs) in the pairwise comparison of different fruit developmental stages between and within black and white V. davidii. After the analysis of functional category enrichment and differential expression patterns of DEGs, 46 genes were selected as the candidate genes. Some genes have been reported as being related to anthocyanins accumulation, and some genes were newly found in our study as probably being related to anthocyanins accumulation. We inferred that 3AT (VIT_03s0017g00870) played an important role in anthocyanin acylation, GST4 (VIT_04s0079g00690) and AM2 (VIT_16s0050g00910) played important roles in anthocyanins transport in V. davidii. The expression of some selected DEGs was further confirmed by quantitative real-time PCR (qRT-PCR).ConclusionsThe present study investigated the transcriptomic profiles of berry skin from black and white spine grapes at three fruit developmental stages by Illumina mRNA-Seq technology. It revealed the variety specificity of anthocyanins accumulation in V. davidi at the transcriptional level. The data reported here will provide a valuable resource for understanding anthocyanins accumulation in grapes, especially in V. davidii.Electronic supplementary materialThe online version of this article (doi:10.1186/s41065-016-0021-1) contains supplementary material, which is available to authorized users.

Transcriptome and proteome analyses provide insight into laticifer's defense of Euphorbia tirucalli against pests

The cytoplasm of laticifers, which are plant cells specialized for rubber production and defense against microbes and herbivores, is a latex. Although laticifers share common functions, the protein constituents of latexes are highly variable among plant species and even among organs. In this study, transcriptomic and proteomic analyses of Euphorbia tirucalli's (Euphorbiaceae) latex were conducted to determine the molecular basis of the laticifer's functions in this plant. The hybrid de novo assembly of Illumina mRNA-seq and expressed sequence tags obtained by Sanger's sequencing revealed 26,447 unigenes. A unigene similar to Arabidopsis embryo-specific protein 3 (AT5G62200), which is a PLAT domain-containing protein, and rubber elongation factor showed the highest expression levels. The proteome analysis, studied by liquid chromatography–mass spectrometry with the de novo assembled unigenes as the database, revealed 161 proteins in the latex, 107 of which were not detected in the stem. A gene ontology analysis indicated that the laticifer's proteome was enriched with proteins related to proteolysis, phosphatase, defense against various environmental stresses and lipid metabolisms. D-mannose-binding lectin, ricin (which lacked the N-terminal conserved ribosome-inactivating protein domain), chitinase and peroxidase were highly accumulated, as confirmed by two-dimensional polyacrylamide gel electrophoresis. Thus, the lectins and chitinase may be the major defensive proteins against pests, and the other defense-related proteins and transcripts detected in latex may work in coordination with them. Highly expressing unigenes with unknown functions are candidate novel defense- or rubber production-related genes.

Patient-Specific Mutation-Derived Tumor Antigens As Targets for Cancer Immunotherapy in Multiple Myeloma

Multiple myeloma (MM) is an incurable plasma cell malignancy accounting for more than 10,000 deaths in the US each year. Novel therapeutic approaches for relapsed MM are urgently needed. Tumor-specific mutations are ideal targets for cancer immunotherapy as they can be potentially recognized as neo-antigens by mature T-cells. Targeting tumor-specific antigens harboring somatic mutations presented on major histocompatibility complex class I molecules (MHC-I) with peptides could personalize the therapeutic approach for relapsed patients. To test this possibility, we examined 6 relapsed MM tumor samples from Mount Sinai, NY to predict in silico patient-specific tumor mutations that may activate the patient's immune systems. This is the first study to utilize Whole-Exome Sequence data (WES) from relapsed MM patients to show the feasibility of using exome sequencing to identify mutation derived neo-antigens that are patient-specific.DNA and RNA from six MM patients were extracted from sorted CD138+ cells from bone marrow aspirates. At the time of sample collection all patients had relapsed following at least five lines of therapy including Autologous Stem Cell Transplantation. The exome capture for DNA sequencing was carried out using the Agilent human whole-exome SureSelect assay. RNA-seq libraries were prepared using Illumina mRNA-seq protocol. All libraries were sequenced on an Illumina HiSeq2500 to generate 100 nucleotide reads. RNA reads were aligned to human reference genome (hg19) and assembled into transcripts using Bowtie-TopHat-Cufflinks. WES data was mapped to reference genome by BWA and then processed by MuTect to detect somatic mutations. Patient-specific alleles were determined using Seq2HLA. The identified mutations lead to candidate antigenic peptides that were filtered by tumor expression level (FPKM >2) using RNA sequence data. Candidate peptides of 8-11 character long were then ranked based on peptide-MHC binding affinity prediction (IC50nM) performed in silico using NetMHCpan.We identified a total of 340 tumor-specific nonsynonymous somatic mutations expressed in the context of patient specific HLA type. 263 (77%) genes were strong binders (IC50<150nM) and 77 (23%) genes were moderate/weak binders (IC50150-500nM). The number of mutated genes that were immunogenic per patient ranged from a minimum of 6 genes to 147 genes. Further, Database for Annotation, Visualization and Integrated Discovery (DAVID) tool was used to identify potentially enriched biological processes among the 340 genes using Gene Ontology (GO) terms. Enrichment analysis of 263 genes showed that they are mainly involved in myeloid cell activation during immune response (eg.LAT2, MYO1F), cell cycle process (CDK1, CHEK2, DNM2, EP300, SETD8), cellular response to stress (RAD21, HDAC2, MAT2B), chromatin silencing (SIRT2, SMARCA4), cell apoptosis and signal transduction (KRAS, NLRP1, ING4, IGF2R). Similarly enrichment analysis of 77 genes revealed their involvement mainly in B cell activation and leukocyte differentiation (LRRC8A, CD3E, PRKDC). Examples of some of these significantly mutated genes with binding affinity and predicted peptides are shown in Table 1.In this study, we show for the first time a correlation between tumor mutations and the epitope landscape by in silico data, suggesting that somatic mutations in MM are immunogenic and could potentially confer antitumor vaccine activity. Our results support an approach in creating cancer vaccines that use tumor-specific immunogenic mutations for the development of personalized vaccines for MM patients.Table 1Immunogenic mutations in Multiple Myeloma#Patient Specific allelesPeptidesIC50Mutated GenesEffectPatient#11HLA-C*14:02CYGHTMVAF57.75LZTR1p.R284C2HLA-C*14:02LYFFGMHVQEY29.75EP300p.C1372Y3HLA-C*14:02TFNEPSSEYF114.21SMARCA4p.G1146SPatient#24HLA-B*15:01MSLHNLGTVF26.2BCRp.A1160G5HLA-A*31:01SIISDSPR149.38FcRL5p.V269I6HLA-A*31:01HYFMHLLK37.16SIRT2p.R116HPatient#37HLA-C*05:01ITDFGHSEIL25.18CHEK2p.K344EPatient#48HLA-A*11:01VVGARGVGK121.47KRASp.G12R9HLA-B*41:01LEIDQLFRI132.84CDK1p.S208LPatient#510HLA-A*31:01AVGCGFRRARR106.68MAT2Bp.P65RPatient#611HLA-A*30:01HQRVLYIEI93.61HDAC2p.D145E12HLA-A*30:01FTRCLTPLL63.19RAD21p.V397L DisclosuresChari:Array Biopharma: Consultancy, Other: Institutional Research Funding, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Biotest: Other: Institutional Research Funding; Novartis: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Jagannath:BMS: Honoraria; Janssen: Honoraria; MERCK: Honoraria; Novartis Pharmaceuticals Corporation: Honoraria; Celgene: Honoraria. Dudley:NuMedii, Inc: Patents & Royalties; Janssen Pharmaceuticals: Consultancy; GlaxoSmithKline: Consultancy; Personalis: Patents & Royalties; Ayasdi, Inc: Other: Equity; Ecoeos, Inc: Other: Equity. Hammerbacher:Cloudera: Membership on an entity's Board of Directors or advisory committees; Bay Sensors: Other: Equity; Cambrian Genomics: Other: Equity; Genome Compiler Corporation: Other: Equity; Science Exchange: Other: Equity; Transcriptic: Other: Equity; Pymetrics: Other: Equity. Schadt:Pacific Biosciences: Consultancy; Berg Pharma: Other: Scientific Advisory Board; GNS Healthcare: Other: Scientific Advisory Board; Clinical Gene Networks AB: Other: Equity. Bhardwaj:Dynavax Technologies Corporation: Consultancy; Crucell: Other: Equity; Dendreon Corporation: Other: Scientific Advisory Board; Merck & Co., Inc.: Other: Scientific Advisory Board; Neostem, Inc.: Other: Equity.

Transcriptomic Analysis of the Mouse Mammary Gland Reveals New Insights for the Role of Serotonin in Lactation.

Serotonin regulates numerous processes in the mammary gland. Our objective was to discover novel genes, pathways and functions which serotonin modulates during lactation. The rate limiting enzyme in the synthesis of non-neuronal serotonin is tryptophan-hydroxylase (TPH1). Therefore, we used TPH1 deficient dams (KO; serotonin deficient, n = 4) and compared them to wild-type (WT; n = 4) and rescue (RC; KO + 100 mg/kg 5-hydroxytryptophan injected daily, n = 4) dams. Mammary tissues were collected on day 10 of lactation. Total RNA extraction, amplification, library preparation and sequencing were performed following the Illumina mRNA-Seq. Overall, 97 and 204 genes (false discovery rate, FDR ≤ 0.01) exhibited a minimum of a 2-fold expression difference between WT vs. KO and WT vs. RC dams, respectively. Most differentially expressed genes were related to calcium homeostasis, apoptosis regulation, cell cycle, cell differentiation and proliferation, and the immune response. Additionally, gene set enrichment analysis using Gene Ontology and Medical Subject Headings databases revealed the alteration of several biological processes (FDR ≤ 0.01) including fat cell differentiation and lipid metabolism, regulation of extracellular signal-related kinase and mitogen-activated kinase cascades, insulin resistance, nuclear transport, membrane potential regulation, and calcium release from the endoplasmic reticulum into the cytosol. The majority of the biological processes and pathways altered in the KO dams are central for mammary gland homeostasis. Increasing peripheral serotonin in the RC dams affects specific pathways that favor lactation. Our data confirms the importance of serotonin during lactation in the mammary gland.

Genome distribution of differential homoeologue contributions to leaf gene expression in bread wheat

SummaryUsing a combination of de novo transcriptome assembly, a newly developed 9495‐marker transcriptome SNP genetic linkage map and comparative genomics approaches, we developed an ordered set of nonredundant transcripts for each of the subgenomes of hexaploid wheat: A (47 160 unigenes), B (59 663 unigenes) and D (40 588 unigenes). We used these as reference sequences against which to map Illumina mRNA‐Seq reads derived from young leaf tissue. Transcript abundance was quantified for each unigene. Using a three‐way reciprocal BLAST approach, 15 527 triplet sets of homoeologues (one from each genome) were identified. Differential expression (P < 0.05) was identified for 5248 unigenes, with 2906 represented at greater abundance than their two homoeologues and 2342 represented at lower abundance than their two homoeologues. Analysis of gene ontology terms revealed no biases between homoeologues. There was no evidence of genomewide dominance effects, rather the more highly transcribed individual genes were distributed throughout all three genomes. Transcriptome display tile plot, a visualization approach based on CMYK colour space, was developed and used to assess the genome for regions of skewed homoeologue transcript abundance. Extensive striation was revealed, indicative of many small regions of genome dominance (transcripts of homoeologues from one genome more abundant than the others) and many larger regions of genome repression (transcripts of homoeologues from one genome less abundant than the others).

Transcriptome analysis during berry development provides insights into co-regulated and altered gene expression between a seeded wine grape variety and its seedless somatic variant.

BackgroundSeedless 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.ResultsThe 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.ConclusionsThis 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 materialThe online version of this article (doi:10.1186/1471-2164-15-1030) contains supplementary material, which is available to authorized users.

RNA-seq Profiles of Immune Related Genes in the Staghorn Coral Acropora cervicornis Infected with White Band Disease

Coral diseases are among the most serious threats to coral reefs worldwide, yet most coral diseases remain poorly understood. How the coral host responds to pathogen infection is an area where very little is known. Here we used next-generation RNA-sequencing (RNA-seq) to produce a transcriptome-wide profile of the immune response of the Staghorn coral Acropora cervicornis to White Band Disease (WBD) by comparing infected versus healthy (asymptomatic) coral tissues. The transcriptome of A. cervicornis was assembled de novo from A-tail selected Illumina mRNA-seq data from whole coral tissues, and parsed bioinformatically into coral and non-coral transcripts using existing Acropora genomes in order to identify putative coral transcripts. Differentially expressed transcripts were identified in the coral and non-coral datasets to identify genes that were up- and down-regulated due to disease infection. RNA-seq analyses indicate that infected corals exhibited significant changes in gene expression across 4% (1,805 out of 47,748 transcripts) of the coral transcriptome. The primary response to infection included transcripts involved in macrophage-mediated pathogen recognition and ROS production, two hallmarks of phagocytosis, as well as key mediators of apoptosis and calcium homeostasis. The strong up-regulation of the enzyme allene oxide synthase-lipoxygenase suggests a key role of the allene oxide pathway in coral immunity. Interestingly, none of the three primary innate immune pathways - Toll-like receptors (TLR), Complement, and prophenoloxydase pathways, were strongly associated with the response of A. cervicornis to infection. Five-hundred and fifty differentially expressed non-coral transcripts were classified as metazoan (n = 84), algal or plant (n = 52), fungi (n = 24) and protozoans (n = 13). None of the 52 putative Symbiodinium or algal transcript had any clear immune functions indicating that the immune response is driven by the coral host, and not its algal symbionts.

Simultaneous transcriptome analysis of Sorghum and Bipolaris sorghicola by using RNA-seq in combination with de novo transcriptome assembly.

The recent development of RNA sequencing (RNA-seq) technology has enabled us to analyze the transcriptomes of plants and their pathogens simultaneously. However, RNA-seq often relies on aligning reads to a reference genome and is thus unsuitable for analyzing most plant pathogens, as their genomes have not been fully sequenced. Here, we analyzed the transcriptomes of Sorghum bicolor (L.) Moench and its pathogen Bipolaris sorghicola simultaneously by using RNA-seq in combination with de novo transcriptome assembly. We sequenced the mixed transcriptome of the disease-resistant sorghum cultivar SIL-05 and B. sorghicola in infected leaves in the early stages of infection (12 and 24 h post-inoculation) by using Illumina mRNA-Seq technology. Sorghum gene expression was quantified by aligning reads to the sorghum reference genome. For B. sorghicola, reads that could not be aligned to the sorghum reference genome were subjected to de novo transcriptome assembly. We identified genes of B. sorghicola for growth of this fungus in sorghum, as well as genes in sorghum for the defense response. The genes of B. sorghicola included those encoding Woronin body major protein, LysM domain-containing intracellular hyphae protein, transcriptional factors CpcA and HacA, and plant cell-wall degrading enzymes. The sorghum genes included those encoding two receptors of the simple eLRR domain protein family, transcription factors that are putative orthologs of OsWRKY45 and OsWRKY28 in rice, and a class III peroxidase that is a homolog involved in disease resistance in the Poaceae. These defense-related genes were particularly strongly induced among paralogs annotated in the sorghum genome. Thus, in the absence of genome sequences for the pathogen, simultaneous transcriptome analysis of plant and pathogen by using de novo assembly was useful for identifying putative key genes in the plant–pathogen interaction.

Expressed Sequences and Polymorphisms in Rohu Carp (Labeo rohita, Hamilton) Revealed by mRNA-seq

Expressed genes and polymorphisms were identified in lines of rohu Labeo rohita selected for resistance or susceptibility to Aeromonas hydrophila, an important bacterial pathogen causing aeromoniasis. All animals were grown in a common environment and RNA from ten individuals from each line pooled for Illumina mRNA-seq. De novo transcriptome assembly produced 137,629 contigs with 40× average coverage.Forty-four percent of the assembled sequences were annotated with gene names and ontology terms. Of these, 3,419 were assigned biological process terms related to "stress response" and 1,939 "immune system". Twenty-six contigs containing 38 single nucleotide polymorphisms (SNPs) were found to map to the Cyprinus carpio mitochondrial genome and over 26,000 putative SNPs and 1,700 microsatellite loci were detected. Seventeen percent of the 100 transcripts with coverage data most indicative of higher-fold expression(>5.6 fold) in the resistant line pool showed homology to major histocompatibility (MH), heat shock proteins (HSP)30, 70 and 90, glycoproteins or serum lectin genes with putative functions affecting immune response. Forty-one percent of these 100 transcripts showed no or low homology to known genes. Of the SNPs identified, 96 showing the highest allele frequency differences between susceptible and resistant line fish included transcripts with homology to MH class I and galactoside-binding soluble lectin, also with putative functions affecting innate and acquired immune response. A comprehensive sequence resource for L. rohita, including annotated microsatellites and SNPs from a mixture of A. hydrophila-susceptible and -resistant individuals, was created for subsequent experiments aiming to identify genes associated with A. hydrophila resistance.

Genetic dissection of transcript, metabolite, growth and wood property traits in an F2 pseudo-backcross pedigree of Eucalyptus grandis x E. urophylla

Background E. grandis is used extensively for the production of pulp and paper due to its rapid growth, good form and ease of vegetative propagation. E. urophylla exhibits tolerance to fungal diseases that limit the growth of E. grandis in tropical and subtropical regions. Interspecific hybrids of these two species are, therefore, commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions (e.g., South Africa, Congo and Brazil). These hybrids often exhibit superior growth and wood quality compared to the pure species, but the underlying genetic basis of the observed hybrid superiority remains unclear. Phenotypic variation observed in interspecific mapping populations has been used to identify QTLs in several genetic linkage studies in Eucalyptus. However, QTL intervals have generally been wide (20 to 30 cM) and may include several hundred genes. To bridge the gap between fine mapping and QTL validation studies, the expression levels of genes and metabolites in individuals from the segregating population can be treated as quantitative traits and used for eQTL and mQTL mapping, respectively. Co-localization of wood property, expression and metabolite QTLs will facilitate the identification of positional candidate genes and other components of regulatory networks underlying phenotypic variation. Methods To identify genetic factors controlling growth and wood property traits in Eucalyptus, an F2 pseudo-backcross mapping family derived from a cross between an F1 hybrid (GUSAP1, E. grandisxE. urophylla, Sappi Forest Research) and an E. urophylla parent (USAP1), consisting of 555 individuals, was used for genetic linkage map construction using microsatellite and DArT markers. Phenotypic trait assessment included physical measurements of tree diameter and wood density performed on 319 three-year-old individuals. Klason (acid-soluble & -insoluble) lignin and cell wall sugar content were determined for a selection of 100 backcross progeny and used for near-infrared analysis (NIRA) calibration. NIRA predictions for glucose, xylose, arabinose, cellulose and total lignin content, as well as pulp yield were made for all 315 individuals. Total lignin and S:G ratios were also separately measured for the 315 individuals. Immature xylem tissues, collected from 192 backcross progeny, were used for metabolite profiling (ORNL, Oak Ridge, TN) and Illumina mRNA-Seq (PE50, BGI Americas) quantification of transcript levels.

Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing.

Characterizing the Impact of Smoking and Lung Cancer on the Airway Transcriptome Using RNA-Seq

Cigarette smoke creates a molecular field of injury in epithelial cells that line the respiratory tract. We hypothesized that transcriptome sequencing (RNA-Seq) will enhance our understanding of the field of molecular injury in response to tobacco smoke exposure and lung cancer pathogenesis by identifying gene expression differences not interrogated or accurately measured by microarrays. We sequenced the high-molecular-weight fraction of total RNA (>200 nt) from pooled bronchial airway epithelial cell brushings (n = 3 patients per pool) obtained during bronchoscopy from healthy never smoker (NS) and current smoker (S) volunteers and smokers with (C) and without (NC) lung cancer undergoing lung nodule resection surgery. RNA-Seq libraries were prepared using 2 distinct approaches, one capable of capturing non-polyadenylated RNA (the prototype NuGEN Ovation RNA-Seq protocol) and the other designed to measure only polyadenylated RNA (the standard Illumina mRNA-Seq protocol) followed by sequencing generating approximately 29 million 36 nt reads per pool and approximately 22 million 75 nt paired-end reads per pool, respectively. The NuGEN protocol captured additional transcripts not detected by the Illumina protocol at the expense of reduced coverage of polyadenylated transcripts, while longer read lengths and a paired-end sequencing strategy significantly improved the number of reads that could be aligned to the genome. The aligned reads derived from the two complementary protocols were used to define the compendium of genes expressed in the airway epithelium (n = 20,573 genes). Pathways related to the metabolism of xenobiotics by cytochrome P450, retinol metabolism, and oxidoreductase activity were enriched among genes differentially expressed in smokers, whereas chemokine signaling pathways, cytokine-cytokine receptor interactions, and cell adhesion molecules were enriched among genes differentially expressed in smokers with lung cancer. There was a significant correlation between the RNA-Seq gene expression data and Affymetrix microarray data generated from the same samples (P < 0.001); however, the RNA-Seq data detected additional smoking- and cancer-related transcripts whose expression was were either not interrogated by or was not found to be significantly altered when using microarrays, including smoking-related changes in the inflammatory genes S100A8 and S100A9 and cancer-related changes in MUC5AC and secretoglobin (SCGB3A1). Quantitative real-time PCR confirmed differential expression of select genes and non-coding RNAs within individual samples. These results demonstrate that transcriptome sequencing has the potential to provide new insights into the biology of the airway field of injury associated with smoking and lung cancer. The measurement of both coding and non-coding transcripts by RNA-Seq has the potential to help elucidate mechanisms of response to tobacco smoke and to identify additional biomarkers of lung cancer risk and novel targets for chemoprevention.

De novo assembled expressed gene catalog of a fast-growing Eucalyptus tree produced by Illumina mRNA-Seq

BackgroundDe novo assembly of transcript sequences produced by short-read DNA sequencing technologies offers a rapid approach to obtain expressed gene catalogs for non-model organisms. A draft genome sequence will be produced in 2010 for a Eucalyptus tree species (E. grandis) representing the most important hardwood fibre crop in the world. Genome annotation of this valuable woody plant and genetic dissection of its superior growth and productivity will be greatly facilitated by the availability of a comprehensive collection of expressed gene sequences from multiple tissues and organs.ResultsWe present an extensive expressed gene catalog for a commercially grown E. grandis × E. urophylla hybrid clone constructed using only Illumina mRNA-Seq technology and de novo assembly. A total of 18,894 transcript-derived contigs, a large proportion of which represent full-length protein coding genes were assembled and annotated. Analysis of assembly quality, length and diversity show that this dataset represent the most comprehensive expressed gene catalog for any Eucalyptus tree. mRNA-Seq analysis furthermore allowed digital expression profiling of all of the assembled transcripts across diverse xylogenic and non-xylogenic tissues, which is invaluable for ascribing putative gene functions.ConclusionsDe novo assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in Eucalyptus and other non-model organisms. The transcriptome resource (Eucspresso, http://eucspresso.bi.up.ac.za/) generated by this study will be of value for genomic analysis of woody biomass production in Eucalyptus and for comparative genomic analysis of growth and development in woody and herbaceous plants.

Abstract 1987: RNA-seq of the bronchial airway transcriptome identifies novel gene and microRNA expression changes associated with smoking and lung cancer

Abstract Prior studies have shown that cigarette smoke creates a field of molecular injury in the airway epithelium. Using microarrays, our group has previously identified gene and microRNA expression differences in bronchial airway epithelial cells that are associated with smoking and lung cancer. We hypothesize that RNA-seq of the airway transcriptome will enhance our understanding of the response to tobacco smoke exposure and lung cancer pathogenesis by identifying mRNA splice variants, non-coding RNA, and novel RNA not interrogated by microarrays. We profiled pooled bronchial airway epithelial cell brushings (n=3 patients/pool) obtained during bronchoscopy from healthy never and current smoker volunteers and smokers with and without lung cancer undergoing surgery for suspicion of lung cancer. The high MW fraction (&amp;gt;200 bp) was amplified using a combination of oligo d(T) and random hexamers (NuGEN, San Carlos, CA) followed by library preparation with Illumina's mRNA Seq Library Prep Kit and sequencing using Illumina's Genome Analyzer generating ∼27-30 million 36 bp reads/sample. The low MW fraction (15-40 bp) was processed using ABI's Small RNA Library Sequencing Kit and sequenced using ABI's SOLiD system generating ∼50-90 million 35 bp reads/sample. The high MW reads were aligned to rRNA (5-13%), the human genome (33-40%), and to computationally generated splice junctions (1%) using BowTie. Differential gene and isoform expression was evaluated using Cufflinks and the R package Genominator. Low MW reads were aligned using both RNA2MAP and Geospiza to a filter containing rRNA, tRNA, and repeats (4-14%), to miRBase (4-10%), and the human genome minus miRBase sequences (4-8%). Expression values were calculated for microRNAs. We compared Affymetrix Exon 1.0 ST, HGU133A2, and Invitrogen miRNA microarrays to RNA-seq and found strong correlation for genes/miRNAs interrogated by microarrays, with most smoking- and cancer related changes in transcript expression being identified by sequencing and not by arrays. Genes found differentially expressed only by sequencing were validated by RT-PCR. RNA-seq also reveals differentially expressed isoforms, potential novel miRNAs, and several isoforms of known miRNAs which are being further investigated. RNA-seq provides a comprehensive and high-resolution view of the airway transcriptome and will provide insights into the molecular field of injury induced by smoking and the pathogenesis of smoking-related lung disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1987.

Into the unknown: expression profiling without genome sequence information in CHO by next generation sequencing

The arrival of next-generation sequencing (NGS) technologies has led to novel opportunities for expression profiling and genome analysis by utilizing vast amounts of short read sequence data. Here, we demonstrate that expression profiling in organisms lacking any genome or transcriptome sequence information is feasible by combining Illumina’s mRNA-seq technology with a novel bioinformatics pipeline that integrates assembled and annotated Chinese hamster ovary (CHO) sequences with information derived from related organisms. We applied this pipeline to the analysis of CHO cells which were chosen as a model system owing to its relevance in the production of therapeutic proteins. Specifically, we analysed CHO cells undergoing butyrate treatment which is known to affect cell cycle regulation and to increase the specific productivity of recombinant proteins. By this means, we identified sequences for >13 000 CHO genes which added sequence information of ∼5000 novel genes to the CHO model. More than 6000 transcript sequences are predicted to be complete, as they covered >95% of the corresponding mouse orthologs. Detailed analysis of selected biological functions such as DNA replication and cell cycle control, demonstrated the potential of NGS expression profiling in organisms without extended genome sequence to improve both data quantity and quality.