Transcriptome Projects Research Articles

Growth differentiation factor 15 in erythroid health and disease

Growth differentiation factor 15 (GDF15) was identified as a hepcidin-suppression factor that is expressed at high levels in patients with ineffective erythropoiesis. This review addresses the regulation, expression and potential functions of GDF15 in the context of erythroid biology. GDF15 expression during late erythroid differentiation was discovered as part of an erythroblast transcriptome project. As GDF15 expression is associated with cellular stress or apoptosis, further investigation of the cytokine was focused upon its involvement in ineffective erythropoiesis. Remarkably high serum levels were detected in patients with thalassemia syndromes, congenital dyserythropoiesis and some acquired sideroblastic anemias. High-level GDF15 expression is not a feature of normal erythropoiesis, or erythroid recovery after bone-marrow transplantation. As GDF15 is a transforming growth factor-beta superfamily member, it was investigated as an effector of ineffective erythropoiesis that suppresses hepcidin expression despite iron overloading. In contrast to the low levels of GDF15 expressed during normal erythropoiesis, ineffective erythropoiesis causes high-level expression of GDF15. In patients with thalassemia and related anemias, GDF15 expression may contribute to iron overloading or other features of the disease phenotype.

Next generation transcriptomes for next generation genomes using est2assembly

BackgroundThe decreasing costs of capillary-based Sanger sequencing and next generation technologies, such as 454 pyrosequencing, have prompted an explosion of transcriptome projects in non-model species, where even shallow sequencing of transcriptomes can now be used to examine a range of research questions. This rapid growth in data has outstripped the ability of researchers working on non-model species to analyze and mine transcriptome data efficiently.ResultsHere we present a semi-automated platform 'est2assembly' that processes raw sequence data from Sanger or 454 sequencing into a hybrid de-novo assembly, annotates it and produces GMOD compatible output, including a SeqFeature database suitable for GBrowse. Users are able to parameterize assembler variables, judge assembly quality and determine the optimal assembly for their specific needs. We used est2assembly to process Drosophila and Bicyclus public Sanger EST data and then compared them to published 454 data as well as eight new insect transcriptome collections.ConclusionsAnalysis of such a wide variety of data allows us to understand how these new technologies can assist EST project design. We determine that assembler parameterization is as essential as standardized methods to judge the output of ESTs projects. Further, even shallow sequencing using 454 produces sufficient data to be of wide use to the community. est2assembly is an important tool to assist manual curation for gene models, an important resource in their own right but especially for species which are due to acquire a genome project using Next Generation Sequencing.

VIGS – genomics goes functional

Genome and EST sequencing projects generate a wealth of sequence information for agronomically and phylogenetically important plant species of which the majority is difficult to subject to functional genomics. Virus-induced gene silencing (VIGS) provides a powerful tool to enable gene functional analysis for species not amenable to stable genetic transformation. Recent approaches allow the use of VIGS as a high throughput method that will exploit the potential of genome and transcriptome projects further.

Global discovery of primate-specific genes in the human genome.

The genomic basis of primate phenotypic uniqueness remains obscure, despite increasing genome and transcriptome sequence data availability. Although factors such as segmental duplications and positive selection have received much attention as potential drivers of primate phenotypes, single-copy primate-specific genes are poorly characterized. To discover such genes genomewide, we screened a catalog of 38,037 human transcriptional units (TUs), compiled from EST and cDNA sequences in conjunction with the FANTOM3 transcriptome project. We identified 131 TUs from transcribed sequences residing within primate-specific insertions in 9-species sequence alignments and outside of segmental duplications. Exons of 120 (92%) of the TUs contained interspersed repeats, indicating that repeat insertions may have contributed to primate-specific gene genesis. Fifty-nine (46%) primate-specific TUs may encode proteins. Although primate-specific TU transcript lengths were comparable to known human gene mRNA lengths overall, 92 (70%) primate-specific TUs were single-exon. Thirty-two (24%) primate-specific TUs were localized to subtelomeric and pericentromeric regions. Forty (31%) of the TUs were nested in introns of known genes, indicating that primate-specific TUs may arise within older, protein-coding regions. Primate-specific TUs were preferentially expressed in reproductive organs and tissues (P < 0.011), consistent with the expectation that emergence of new, lineage-specific genes may accompany speciation or reproduction. Of the 33 primate-specific TUs with human Affymetrix microarray probe support, 21 were differentially expressed in human teratozoospermia. In addition to elucidating the likely functional relevance of primate-specific TUs to reproduction, we present a set of primate-specific genes for future functional studies, and we implicate nonduplicated pericentromeric and subtelomeric regions in gene genesis.

Flipping NextGen: using biological systems to characterize NextGen sequencing technologies

Finally, de-novo sequencing (i.e. without a reference) would require a minimum of 1/2 of the sequence length to be unique in order to allow sufficient contig extension in the assembly process. For example, 40–50 bp reads are necessary for de-novo characterization of these systems uniquely defined by 20–25 bp reads. As of 2009, short read NextGen sequencing technologies have moved to 50 bp and beyond, ushering in what is expected to be the start of a revolution in genomics. Conclusion These results establish a lower bound on sequence length (x) required to sufficiently conduct re-sequencing, transcriptome, and de-novo sequencing projects. The asymptotic nature of the results also provides a guide for what percentage of the total space (y) we might expect to define in genomes/transcriptomes of similar size and complexity. from UT-ORNL-KBRIN Bioinformatics Summit 2009 Pikeville, TN, USA. 20–22 March 2009

A multi-Fc-species system for recombinant antibody production

BackgroundGenomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies.ResultsWe developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use. This series enables the fusion of single chain Fv antibodies with human, mouse or rabbit Fc so that a given antibody is no longer restricted to a particular species. This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies. We also show that this multi-Fc species production system can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies and we converted the widely used 9E10 mouse anti-Myc-tag antibody into a human and a rabbit antibody.ConclusionAltogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.

The long and short of RNAs

The known world of RNA is expanding faster than that of any other cellular building block. The latest additions are types of long and short non-coding RNAs formed by bidirectional transcription and unusual processing. The transcriptomes of eukaryotic cells are unexpectedly complex, with virtually the entire non-repeat portions of many genomes being transcribed. This paper from the Affymetrix/Cold Spring Harbor Laboratory ENCODE Transcriptome Project reports a deep sequencing study revealing that a remarkable breadth of RNA species is produced both from within annotated genes and from unannotated intergenic regions in human cells. Importantly, many of these small RNAs possess cap structures and appear to be processed from mature mRNAs resulting in populations of long and short RNAs with capped 5 ends that coincide. Transfecting synthetic PASRs (promoter-associated short RNAs) corresponding to the c-MYC transcriptional start site reduced MYC mRNA abundance, demonstrating a biological impact for at least one class of these small RNAs.

Identification of putative peptide paracrines/hormones in the water flea Daphnia pulex (Crustacea; Branchiopoda; Cladocera) using transcriptomics and immunohistochemistry

The cladoceran crustacean Daphnia pulex has emerged as a model species for many biological fields, in particular environmental toxicology and toxicogenomics. Recently, this species has been the subject of an extensive transcriptome project, resulting in the generation and public deposition of over 150,000 expressed sequence tags (ESTs). This resource makes D. pulex an excellent model for protein discovery using bioinformatics. Here, in silico searches of the D. pulex EST database were conducted to identify transcripts encoding putative peptide precursors. Moreover, the mature peptides contained within the deduced prepro-hormones were predicted using online peptide processing programs and homology to known arthropod isoforms. In total, 63 putative peptide-encoding ESTs were identified encompassing 14 distinct peptide families/subfamilies: A-type allatostatin, B-type allatostatin, C-type allatostatin, bursicon (both α and β subunit peptides), crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone (CHH)/ion transport peptide (both CHH- and moult-inhibiting hormone-like subfamilies), diuretic hormone (calcitonin-like), ecdysis-triggering hormone (ETH), FMRFamide (both neuropeptide F and short neuropeptide F subfamilies), orcokinin and pigment dispersing hormone. From these transcripts, the structures of 76 full-length/partial peptides were predicted, which included the first C-type allatostatin-like peptide identified from a crustacean, the first crustacean calcitonin-like diuretic hormone, an undescribed CCAP isoform, two hitherto unknown ETH variants, and two new orcokinins. Neuronal localization of several of the identified peptide families was confirmed using immunohistochemitry ( i.e. A-type allatostatin, CCAP, FMRFamide and PDH). In addition, immunohistochemical analyses identified other putative neuropeptides for which no ESTs had been found ( i.e. corazonin, insect kinin, proctolin, red pigment concentrating hormone, SIFamide, sulfakinin and tachykinin-related peptide). Collectively, the data presented here not only catalog an extensive array of putative D. pulex peptide paracrines/hormones, but also provide a strong foundation for future investigations of the effects of environmental/anthropogenic stressors on peptidergic control in this model organism.

Expression patterns of transcribed human endogenous retrovirus HERV-K(HML-2) loci in human tissues and the need for a HERV Transcriptome Project

BackgroundA significant proportion of the human genome is comprised of human endogenous retroviruses (HERVs). HERV transcripts are found in every human tissue. Expression of proviruses of the HERV-K(HML-2) family has been associated with development of human tumors, in particular germ cell tumors (GCT). Very little is known about transcriptional activity of individual HML-2 loci in human tissues, though.ResultsBy employing private nucleotide differences between loci, we assigned ~1500 HML-2 cDNAs to individual HML-2 loci, identifying, in total, 23 transcriptionally active HML-2 proviruses. Several loci are active in various human tissue types. Transcription levels of some HML-2 loci appear higher than those of other loci. Several HML-2 Rec-encoding loci are expressed in GCT and non-GCT tissues. A provirus on chromosome 22q11.21 appears strongly upregulated in pathologic GCT tissues and may explain high HML-2 Gag protein levels in GCTs. Presence of Gag and Env antibodies in GCT patients is not correlated with activation of individual loci. HML-2 proviruses previously reported capable of forming an infectious HML-2 variant are transcriptionally active in germ cell tissue. Our study furthermore shows that Expressed Sequence Tag (EST) data are insufficient to describe transcriptional activity of HML-2 and other HERV loci in tissues of interest.ConclusionOur, to date, largest-scale study reveals in greater detail expression patterns of individual HML-2 loci in human tissues of clinical interest. Moreover, large-scale, specialized studies are indicated to better comprehend transcriptional activity and regulation of HERVs. We thus emphasize the need for a specialized HERV Transcriptome Project.

Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica

The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis.

Structural differences of orthologous genes: Insights from human–primate comparisons

The genomic basis of phenotypic distinctions between humans and nonhuman primates remains insufficiently explained. We hypothesized that interspecies structural differences of orthologous genes can cause such distinctions and searched protein-coding genes conserved between humans and nonhuman primates for species-specific initial and terminal exons. We inferred gene structure differences from genomic locations where portions of primate transcripts aligned with the human genome outside of any human exons. Of 22,466 high-confidence FANTOM3 human transcriptional units, 7424 (33%) had nonhuman primate full-length cDNA support. One hundred eighty-three of the loci contained 68,424 bp of sequence exonic in nonhuman primates but not humans. Fifty-four of 183 included species-specific portions of protein-coding regions. Six genes had evidence of intergenic splicing in a nonhuman primate but not in human. It is imperative that primate transcriptome projects be accelerated on par with genome projects to understand better interspecies gene structure distinctions.

Recent advances in crustacean genomics

Crustaceans are a diverse and ancient group of arthropods that have long been studied as interesting model systems in biology, especially for understanding animal evolution and physiology and for environmentally relevant studies. Like many model systems, advances in DNA-sequencing methodologies have led to a large amount of genomics-related projects. The purpose of this article is to highlight the genome projects and functional genomics (transcriptomics) projects that are currently underway in crustacean biology. Specifically, we have surveyed the amount of publicly available DNA sequence data (both genomic and EST data) across all crustacean taxa for which a significant number of DNA sequences have been generated. Several ongoing projects are presented including the ecology of invasive species, thermal physiology, ion and water balance, ecology and evolutionary biology, and developmental biology.

Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

BackgroundTartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics.FindingsA new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine.ConlusionThe gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism.

Low Frequency of KIT Gene Mutation in Pediatric Acute Myeloid Leukemia with inv(16)(p13q22): A Study of the Japanese Childhood AML Cooperative Study Group

aDepartment of Hematology/Oncology, Gunma Children’s Medical Center, Gunma, Japan; bCancer Transcriptome Project, National Cancer Center Research Institute, Tokyo, Japan; cDepartment of Molecular Laboratory Medicine, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan; dDepartment of Pediatrics, Hamamatsu University School of Medicine, Shizuoka, Japan; eDepartment of Pediatric Hematology/Oncology, Osaka City General Hospital, Osaka, Japan; fDepartment of Pediatrics, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan; gDepartment of Pediatrics, National Hospital Organization Osaka National Hospital, Osaka, Japan; hFirst Department of Pediatrics, Toho University School of Medicine, Tokyo, Japan

Bioinformatics for the Citrus EST Project (CitEST)

In this work we describe all the computational environments, pipelines, and web services developed for the CitEST transcriptome project, on which all the annotation researchers relied. We also present a complete list of CitEST libraries and, for each of them, the general features after the in silico processing, showing some quantitative information.

Functional genome of the human pathogenic fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation process that this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanisms involved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This review focuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimorphism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium and yeast phases.

The Power of an Integrated Informatic and Molecular Approach to Type 1 Diabetes Research

Recent years have witnessed an explosive growth in available biological data. This includes a tremendous quantity of sequence data (e.g., biological structures, genetic and physical maps, pathways) generated by genome and transcriptome projects focused on humans, mice, and a multitude of other species. Diabetes research stands to greatly benefit from this data, which is distributed across public and private databases and the scientific literature. The increasing quantity and complexity of this biological data necessitates use of novel bioinformatics strategies for its efficient retrieval, analysis, and interpretation. Bioinformatic capability is becoming increasingly indispensable for fast and comprehensive analysis of biological data by diabetes researchers. There is great potential for diabetes scientists and clinicians to take advantage of recent bioinformatics and knowledge discovery developments to radically transform and advance this field of research. This paper will review advances in the field of bioinformatics relevant to diabetes research and preview a new specialty diabetes database, Diabetaeta, that we are creating to serve as a central bioinformatic portal for type 1 diabetes research, as well as serving as a public repository for beta cell gene and protein expression data.

Systematic genome‐wide approach to positional candidate cloning for identification of novel human disease genes

Recent large-scale genome projects afford a unique opportunity to identify many novel disease genes and thereby better understand the genetic basis of human disease. Functional Annotation of Mouse (FANTOM) 2, the largest mouse transcriptome project yet, provides a wealth of data on novel genes, splice variants and non-coding RNA, and provides a unique opportunity to identify novel human disease genes. To demonstrate the power of combining the FANTOM 2 cDNA dataset with a positional candidate approach and bioinformatics analysis to identify genes underlying human genetic disease. By mapping all FANTOM 2 cDNA to the human genome, we were able to identify mouse clones that co-localised on the human genome with mapped but uncloned human disease loci. By this method we identified mouse and corresponding human genes mapping within the loci of 100 different human genetic diseases (mapped interval of <5 cM). Of particular interest was the elucidation through FANTOM 2 novel mouse gene data of candidate human genes for the following: (i) developmental -disorders: neural tube defect, Meckel syndrome, Wolf--Hirschhorn syndrome and keratosis follicularis spinulosa decalvans cum ophiasi; (ii) neurological disorders: benign familial infantile convulsions 3, early-onset cerebellar ataxia with retained tendon reflexes, infantile-onset spinocerebellar ataxia and vacuolar neuro-myopathy and (iii) cancer-related syndromes: tylosis with oesophageal cancer and low-grade B-cell chronic lymphatic leukaemia. The FANTOM 2 data will dramatically accelerate efforts to identify genes underlying human disease. It will also facilitate the creation of transgenic mouse models to help elucidate the function of potential human disease genes.

A Guide to the Mammalian Genome: Figure 1

Sequencing of a Transcriptome The rapid completion and public release of the genome sequences of mouse and human has led to a downgrading of the number of “genes” predicted in the mammalian genome to the region of 30,000 (Mouse Genome Sequencing Consortium, Waterston et al. 2002). In simpler organisms such as yeast, the estimate of gene number is comparatively straightforward, because the majority of the genome clearly encodes proteins, and individual genes generally have a welldefined start and finish and a single mRNA output. In mammals, the task is muchmore complex. Only a small proportion of the genome encodes mRNAs that in turn encode protein, and protein-coding sequence is interspersed with large introns or intergenic regions. Even protein coding genes have proven difficult to annotate reliably (Kawai et al. 2001), and non–protein coding genes are essentially impossible to annotate a priori. The key to reliable annotation of a mammalian genome is the comprehensive characterization of the transcriptional output, the transcriptome. There are two approaches to this problem. The most common is highthroughput sequencing of cDNA ends (ESTs). In mouse and human, and to a lesser extent in many other mammals, there are millions of EST sequences in various repositories. EST sequences can be computationally assembled into clusters, as in the UniGene projects (http : / /www.ncbi .nlm.nih.gov/ UniGene). There are many drawbacks with this approach, both from the cDNA cloning and sequence quality and from computational perspectives, but the most compelling is that the sequences are generated in silico and are not necessarily supported by a physical clone. It is also rather inefficient, because even with the best subtraction and normalization, abundant transcripts have been sequenced thousands of times, whereas many rare transcripts are absent from EST databases. EST assemblies are particularly difficult to interpret when there are multigene families or complex alternative splicing. The alternative approach is to systematically isolate and sequence fulllength cDNAs. The logistics of this approach are daunting, and it is actually far more challenging than is genomic sequencing, especially using shotgun approaches because of the difficulties in the collection of the samples. Nevertheless, the RIKEN Mouse Gene Encyclopedia Project has taken this approach. In the process, the RIKEN team has provided a model for eukaryotic transcriptome projects. The task required a range of new technologies and approaches. In outline, the RIKEN team developed new approaches to production of full-length cDNAs (Carninci et al. 2003) that required (1) a novel reverse transcriptase reaction (to enable effective complete firststrand synthesis), (2) novel 5 end capture technology, and (3) novel approaches to normalization and subtraction of cDNA libraries. Starting with their first libraries, the RIKEN team sequenced 3 ends (and later 5 ends) in a Phase 1 sequencing pipeline and, for each individual clone, determined whether the sequence had been sequenced previously or could be ascribed to a new cluster. In the second phase, individual representatives of EST clusters were selected and fully sequenced to produce a full-length cDNA sequence representing the sequence of an individual physical clone. At a number of stages in the project, the RIKEN team assembled a set of cDNAs that had previously been sequenced and used them to subtract successive libraries. The success of the approach is outlined in detail in Carninci et al. (2003). The output of this pipeline was analyzed in the FANTOM2 meeting (April 29 to May 5, 2002, Yokohama, Japan), which is the basis of this special issue of Genome Research.

Distribution of DNA repair-related ESTs in sugarcane

DNA repair pathways are necessary to maintain the proper genomic stability and ensure the survival of the organism, protecting it against the damaging effects of endogenous and exogenous agents. In this work, we made an analysis of the expression patterns of DNA repair-related genes in sugarcane, by determining the EST (expressed sequence tags) distribution in the different cDNA libraries of the SUCEST transcriptome project. Three different pathways - photoreactivation, base excision repair and nucleotide excision repair - were investigated by employing known DNA repair proteins as probes to identify homologous ESTs in sugarcane, by means of computer similarity search. The results showed that DNA repair genes may have differential expressions in tissues, depending on the pathway studied. These in silico data provide important clues on the potential variation of gene expression, to be confirmed by direct biochemical analysis.