Massive Transcriptome Sequencing Research Articles

Databases for RNA Editing Collections.

A-to-I RNA editing in humans plays a relevant role since it can influence gene expression and increase proteome diversity. In addition, its deregulation has been linked to a variety of human diseases, including neurological disorders and cancer.In the last decade, massive transcriptome sequencing through the RNAseq technology has dramatically improved the investigation of RNA editing at single nucleotide resolution. Nowadays, different bioinformatics resources to discover and/or collect A-to-I events have been released. Hereafter, we initially provide an overview of the state-of-the-art RNA editing databases and, then, we focus on REDIportal, the largest collection of A-to-I events with more than 4.5 million sites from 2660 humans GTEx samples.

REDIportal: millions of novel A-to-I RNA editing events from thousands of RNAseq experiments

RNA editing is a relevant epitranscriptome phenomenon able to increase the transcriptome and proteome diversity of eukaryotic organisms. ADAR mediated RNA editing is widespread in humans in which millions of A-to-I changes modify thousands of primary transcripts. RNA editing has pivotal roles in the regulation of gene expression or modulation of the innate immune response or functioning of several neurotransmitter receptors. Massive transcriptome sequencing has fostered the research in this field. Nonetheless, different aspects of the RNA editing biology are still unknown and need to be elucidated. To support the study of A-to-I RNA editing we have updated our REDIportal catalogue raising its content to about 16 millions of events detected in 9642 human RNAseq samples from the GTEx project by using a dedicated pipeline based on the HPC version of the REDItools software. REDIportal now allows searches at sample level, provides overviews of RNA editing profiles per each RNAseq experiment, implements a Gene View module to look at individual events in their genic context and hosts the CLAIRE database. Starting from this novel version, REDIportal will start collecting non-human RNA editing changes for comparative genomics investigations. The database is freely available at http://srv00.recas.ba.infn.it/atlas/index.html.

Quantifying RNA Editing in Deep Transcriptome Datasets.

Massive transcriptome sequencing through the RNAseq technology has enabled quantitative transcriptome-wide investigation of co-/post-transcriptional mechanisms such as alternative splicing and RNA editing. The latter is abundant in human transcriptomes in which million adenosines are deaminated into inosines by the ADAR enzymes. RNA editing modulates the innate immune response and its deregulation has been associated with different human diseases including autoimmune and inflammatory pathologies, neurodegenerative and psychiatric disorders, and tumors. Accurate profiling of RNA editing using deep transcriptome data is still a challenge, and the results depend strongly on processing and alignment steps taken. Accurate calling of the inosinome repertoire, however, is required to reliably quantify RNA editing and, in turn, investigate its biological and functional role across multiple samples. Using real RNAseq data, we demonstrate the impact of different bioinformatics steps on RNA editing detection and describe the main metrics to quantify its level of activity.

Massive transcriptome sequencing of human spinal cord tissues provides new insights into motor neuron degeneration in ALS

ALS is a devastating and debilitating human disease characterized by the progressive death of upper and lower motor neurons. Although much effort has been made to elucidate molecular determinants underlying the onset and progression of the disorder, the causes of ALS remain largely unknown. In the present work, we have deeply sequenced whole transcriptome from spinal cord ventral horns of post-mortem ALS human donors affected by the sporadic form of the disease (which comprises ~90% of the cases but which is less investigated than the inherited form of the disease). We observe 1160 deregulated genes including 18 miRNAs and show that down regulated genes are mainly of neuronal derivation while up regulated genes have glial origin and tend to be involved in neuroinflammation or cell death. Remarkably, we find strong deregulation of SNAP25 and STX1B at both mRNA and protein levels suggesting impaired synaptic function through SNAP25 reduction as a possible cause of calcium elevation and glutamate excitotoxicity. We also note aberrant alternative splicing but not disrupted RNA editing.

Exploring the RNA editing potential of RNA-Seq data by ExpEdit.

Revealing the impact of A-to-I RNA editing in RNA-Seq experiments is relevant in humans because RNA editing can influence gene expression. In addition, its deregulation has been linked to a variety of human diseases. Exploiting the RNA editing potential in complete RNA-Seq datasets, however, is a challenging task. Indeed, no dedicated software is available, and sometimes deep computational skills and appropriate hardware resources are required. To explore the impact of known RNA editing events in massive transcriptome sequencing experiments, we developed the ExpEdit web service application. In the present work, we provide an overview of ExpEdit as well as methodologies to investigate known RNA editing in human RNA-Seq datasets.

Uncovering RNA Editing Sites in Long Non-Coding RNAs

RNA editing is an important co/post-transcriptional molecular process able to modify RNAs by nucleotide insertions/deletions or substitutions. In human, the most common RNA editing event involves the deamination of adenosine (A) into inosine (I) through the adenosine deaminase acting on RNA proteins. Although A-to-I editing can occur in both coding and non-coding RNAs, recent findings, based on RNA-seq experiments, have clearly demonstrated that a large fraction of RNA editing events alter non-coding RNAs sequences including untranslated regions of mRNAs, introns, long non-coding RNAs (lncRNAs), and low molecular weight RNAs (tRNA, miRNAs, and others). An accurate detection of A-to-I events occurring in non-coding RNAs is of utmost importance to clarify yet unknown functional roles of RNA editing in the context of gene expression regulation and maintenance of cell homeostasis. In the last few years, massive transcriptome sequencing has been employed to identify putative RNA editing changes at genome scale. Despite several efforts, the computational prediction of A-to-I sites in complete eukaryotic genomes is yet a challenging task. We have recently developed a software package, called REDItools, in order to simplify the detection of RNA editing events from deep sequencing data. In the present work, we show the potential of our tools in recovering A-to-I candidates from RNA-Seq experiments as well as guidelines to improve the RNA editing detection in non-coding RNAs, with specific attention to the lncRNAs.

Transcriptome survey of the lipid metabolic pathways involved in energy production and ecdysteroid synthesis in the salmon louse Caligus rogercresseyi (Crustacea: Copepoda).

The goal of this study was to identify and analyze the lipid metabolic pathways involved in energy production and ecdysteroid synthesis in the ectoparasite copepod Caligus rogercresseyi. Massive transcriptome sequencing analysis was performed during the infectious copepodid larval stage, during the attached chalimus larval stage, and also in female and male adults. Thirty genes were selected for describing the pathways, and these were annotated for proteins or enzymes involved in lipid digestion, absorption, and transport; fatty acid degradation; the synthesis and degradation of ketone bodies; and steroid and ecdysteroid syntheses. Differential expression of these genes was analyzed by ontogenic stage and discussed considering each stage's feeding habits and energetic needs. Copepodids showed a low expression of fatty acid digestion genes, reflected by a non-feeding behavior, and the upregulation of genes involved in steroid biosynthesis, which was consistent with a pathway for cholesterol synthesis during ecdysis. The chalimus stage showed an upregulation of genes related to fatty acid digestion, absorption, and transport, as well as to fatty acid degradation and the synthesis of ketone bodies, therefore suggesting that lipids ingested from the mucus and skin of the host fish are metabolized as important sources of energy. Adult females also showed a pattern of high lipid metabolism for energy supply and mobilization in relation to reproduction and vitellogenesis. Adult females and males revealed different lipid metabolism patterns that reflected different energetic needs. This study reports for the first time the probable lipid metabolic pathways involved in the energy production and ecdysteroid synthesis of C. rogercresseyi.

Reditools: Efficient RNA editing detection by RNA-seq data

The identification of RNA editing events from massive transcriptome sequencing data is yet a challenging task. Although few methodologies have been proposed no comprehensive software resource has been released up to now. To fill this gap we developed REDItools, a suite of python scripts to facilitate the study of RNA editing in the next generation sequencing era. REDItools work on whatever machine running unix/linux and are freely available at http://code.google.com/p/reditools/.

SNP discovery and High Resolution Melting Analysis from massive transcriptome sequencing in the California red abalone Haliotis rufescens

The California red abalone, Haliotis rufescens that belongs to the Haliotidae family, is the largest species of abalone in the world that has sustained the major fishery and aquaculture production in the USA and Mexico. This native mollusk has not been evaluated or assigned a conservation category even though in the last few decades it was heavily exploited until it disappeared in some areas along the California coast. In Chile, the red abalone was introduced in the 1970s from California wild abalone stocks for the purposes of aquaculture. Considering the number of years that the red abalone has been cultivated in Chile crucial genetic information is scarce and critical issues remain unresolved. This study reports and validates novel single nucleotide polymorphisms (SNP) markers for the red abalone H. rufescens using cDNA pyrosequencing. A total of 622 high quality SNPs were identified in 146 sequences with an estimated frequency of 1 SNP each 1000bp. Forty-five SNPs markers with functional information for gene ontology were selected. Of these, 8 were polymorphic among the individuals screened: Heat shock protein 70 (HSP70), vitellogenin (VTG), lysin, alginate lyase enzyme (AL), Glucose-regulated protein 94 (GRP94), fructose-bisphosphate aldolase (FBA), sulfatase 1A precursor (S1AP) and ornithine decarboxylase antizyme (ODC). Two additional sequences were also identified with polymorphisms but no similarities with known proteins were achieved. To validate the putative SNP markers, High Resolution Melting Analysis (HRMA) was conducted in a wild and hatchery-bred population. Additionally, SNP cross-amplifications were tested in two further native abalone species, Haliotis fulgens and Haliotis corrugata. This study provides novel candidate genes that could be used to evaluate loss of genetic diversity due to hatchery selection or inbreeding effects.

A Novel Computational Strategy to Identify A-to-I RNA Editing Sites by RNA-Seq Data: De Novo Detection in Human Spinal Cord Tissue

RNA editing is a post-transcriptional process occurring in a wide range of organisms. In human brain, the A-to-I RNA editing, in which individual adenosine (A) bases in pre-mRNA are modified to yield inosine (I), is the most frequent event. Modulating gene expression, RNA editing is essential for cellular homeostasis. Indeed, its deregulation has been linked to several neurological and neurodegenerative diseases. To date, many RNA editing sites have been identified by next generation sequencing technologies employing massive transcriptome sequencing together with whole genome or exome sequencing. While genome and transcriptome reads are not always available for single individuals, RNA-Seq data are widespread through public databases and represent a relevant source of yet unexplored RNA editing sites. In this context, we propose a simple computational strategy to identify genomic positions enriched in novel hypothetical RNA editing events by means of a new two-steps mapping procedure requiring only RNA-Seq data and no a priori knowledge of RNA editing characteristics and genomic reads. We assessed the suitability of our procedure by confirming A-to-I candidates using conventional Sanger sequencing and performing RNA-Seq as well as whole exome sequencing of human spinal cord tissue from a single individual.

Molecular Effects of Doxycycline Treatment on Pterygium as Revealed by Massive Transcriptome Sequencing

Pterygium is a lesion of the eye surface which involves cell proliferation, migration, angiogenesis, fibrosis, and extracellular matrix remodelling. Surgery is the only approved method to treat this disorder, but high recurrence rates are common. Recently, it has been shown in a mouse model that treatment with doxycycline resulted in reduction of the pterygium lesions. Here we study the mechanism(s) of action by which doxycycline achieves these results, using massive sequencing techniques. Surgically removed pterygia from 10 consecutive patients were set in short term culture and exposed to 0 (control), 50, 200, and 500 µg/ml doxycycline for 24 h, their mRNA was purified, reverse transcribed and sequenced through Illumina’s massive sequencing protocols. Acquired data were subjected to quantile normalization and analyzed using cytoscape plugin software to explore the pathways involved. False discovery rate (FDR) methods were used to identify 332 genes which modified their expression in a dose-dependent manner upon exposure to doxycycline. The more represented cellular pathways included all mitochondrial genes, the endoplasmic reticulum stress response, integrins and extracellular matrix components, and growth factors. A high correlation was obtained when comparing ultrasequencing data with qRT-PCR and ELISA results.Doxycycline significantly modified the expression of important cellular pathways in pterygium cells, in a way which is consistent with the observed efficacy of this antibiotic to reduce pterygium lesions in a mouse model. Clinical trials are under way to demonstrate whether there is a benefit for human patients.

De novo detection of A-To-I RNA editing sites in human mRNAs by massive transcriptome sequencing

Motivations. RNA editing is a widespread molecular phenomenon which modifies primary transcripts at specific positions [1]. It occurs in a variety of organisms including human and cooperates with alternative splicing in increasing both proteomic and transcriptomic complexity. RNA Editing can modulate gene expression and affect protein functionality. In human, such phenomenon is highly frequent in brain and its deregulation has been linked to a variety of neurological and neurodegenerative diseases [2]. Many editing events have been identified by next generation sequencing technologies employing massive transcriptome sequencing [3] together with whole genome or exome sequencing. Nowadays numerous RNA-Seq experiments are available through public databases and represent a relevant source of yet unexplored RNA editing sites. Hereafter we propose a simple computational strategy to identify de novo genomic positions enriched in novel potential RNA editing events through a new, two-step mapping procedure. Methods. To accurately predict RNA editing sites we developed a double mapping procedure in which millions of Illumina short reads were independently mapped onto the human transcriptome and the reference human genome tolerating at maximum two mismatches for each unique alignment. Only concordant alignments from the double mapping procedure were used for downstream analyses. All reads supporting each reference position were explored to calculate the empirical probability to observe a substitution. Such probabilities were then used to detect statistically significant base conversions by applying the Fisher's exact test by comparing the observed and expected occurrences in the aligned reads. Benjamini-Hochberg correction was finally employed to reduce the false discovery rate. A-to-I RNA editing candidates may be then selected according to P-value, coverage and editing extent for the experimental validation using the classical Sanger sequencing from RNA/DNA extracted from the same individual. Results. We initially tested our computational method on the SRA study SRA012427 involving high-throughput transcriptome sequencing of human brain tissues by Illumina technology. Over 22 millions 50 nt long paired-end reads were aligned onto the human reference genome (assembly hg18). Applying the above-described double mapping methodology and stringent filters we found 19 highly significant A-to-I conversions in known human coding regions. Interestingly, 11 of such changes have been already described in literature and 6 were experimentally confirmed. To further corroborate our strategy we carried out an RNA-Seq experiment on total RNA extracted from human spinal cord. More than 20 million of directional paired-end reads were analysed using the above mentioned procedure. At 0.05 significance level (FDR corrected) we obtained 15 RNA editing candidates covered by at least 30 independent reads and showing only A-to-G changes. In this case potential editing candidates were confirmed by whole exome sequencing performed on the individual and tissue in order to optimally exclude SNPs and somatic mutations. Notably, we were able to confirm 12 predicted candidates. Our results, therefore, indicate the feasibility and effectiveness of the above-described strategy to detect de novo A-to-I RNA editing events in human.

Abstract 5664: Molecular effects of doxycycline treatment on pterygium as revealed by massive transcriptome sequencing

Abstract Pterygium is a common benign tumor growth of the ocular surface attributed to chronic ultraviolet light exposure, representing a tremendous burden, both human and financial, in many countries. At the cellular level, pterygium is characterized by proliferation of limbal cells, inflammatory infiltrates, fibrosis, angiogenesis, and extracellular matrix breakdown. Pterygia also display tumor-like features, such as propensity to invade normal tissue and high recurrence rates following resection. Currently, the only approved treatment for pterygium is surgery which, in some cases, results in high recurrence rates. In a recent article, our group has shown that doxycycline, a common oral antibiotic, was able to dramatically reduce pterygium-like lesions in a mouse model and we are nowadays conducting a clinical trial to test this ability in humans. In the present study we investigated the molecular effects of doxycycline on short-term primary cultures of pterygium cells, using ultrasequencing techniques, to better understand all the pathways affected by the antibiotic in pterygium. Use of pterygium specimens was approved by our Institutional Review Board (CEICLAR). For each patient, cells were subjected to 4 treatments: 0 (control), 50, 200, and 500 μg/mL doxycycline for 24 h. Total RNA was isolated and a library was prepared for ultrasequencing following Illumina's protocols. Gene products whose expression was significantly changed upon application of doxycycline were chosen for further confirmation by quantitative real time PCR using Taqman probes. Some secreted proteins were quantified in the supernatant of the cultures by commercially available ELISA kits Our results showed that the exposure of pterygium cells to doxycycline induced dose-dependent changes in mitochondrial-related genes as well as ER stress-related genes, VEGF signalling, and integrin related genes in pterygium. Twenty two of the differentially-induced genes identified by ultrasequencing were selected for confirmation by qRT-PCR. All of them showed a similar dose dependence on doxycycline treatment. Correlation analysis between ultrasequencing data and qRT-PCR showed a coefficient of correlation (r2) of 0.7. In addition, several cytokines were analysed by ELISA confirming the validity of the technique. The doxycycline-activated pathways identified by ultrasequencing techniques provide a plausible mechanism by which doxycycline exerts its anti-proliferative effects on pterygia and unveiled new therapeutic targets for the treatment of the disease. In addition, the high correlation between ultrasequencing and qRT-PCR data supports the use of Massive Transcriptome Sequencing as a useful and powerful tool to study the mechanism underlining complex diseases. Supported by a Grant from Spain's Ministry of Health, Social Policy, and Equality, No. DIN-021. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5664. doi:1538-7445.AM2012-5664

Transformation and regeneration of the holoparasitic plant Phelipanche aegyptiaca

BackgroundTransformation and subsequent regeneration of holoparasitic plants has never been reported, in part due to challenges in developing transformation protocols, but also because regeneration of obligate parasites is difficult since their survival depends completely on successful haustorium penetration of a host and the formation of vascular connections. The recent completion of a massive transcriptome sequencing project (the Parasitic Plant Genome Project) will fuel the use of genomic tools for studies on parasitic plants. A reliable system for holoparasite transformation is needed to realize the full value of this resource for reverse genetics and functional genomics studies.ResultsHere we demonstrate that transformation of Phelipanche aegyptiaca is achieved by infection of 3 month-old in vitro grown P. aegyptiaca calli with Agrobacterium rhizogenes harboring the yellow fluorescent protein (YFP). Four months later, YFP-positive regenerated calli were inoculated onto tomato plants growing in a minirhizotron system. Eight days after inoculation, transgenic parasite tissue formed lateral haustoria that penetrated the host and could be visualized under UV illumination through intact host root tissue. YFP-positive shoot buds were observed one month after inoculation.ConclusionsThis work constitutes a breakthrough in holoparasitic plant research methods. The method described here is a robust system for transformation and regeneration of a holoparasitic plant and will facilitate research on unique parasitic plant capabilities such as host plant recognition, haustorial formation, penetration and vascular connection.

Analysis of transcript and protein overlap in a human osteosarcoma cell line

BackgroundAn interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq) to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC) and immunofluorescence microscopy (IF).ResultsA large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2%) were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies.ConclusionThis suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.