AtRTD - a comprehensive reference transcript dataset resource for accurate quantification of transcript-specific expression in Arabidopsis thaliana.
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ABSTRACT:
SUBMITTER: Zhang R
PROVIDER: S-EPMC4744958 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundRNA-Seq is revolutionizing the way transcript abundances are measured. A key challenge in transcript quantification from RNA-Seq data is the handling of reads that map to multiple genes or isoforms. This issue is particularly important for quantification with de novo transcriptome assemblies in the absence of sequenced genomes, as it is difficult to determine which transcripts are isoforms of the same gene. A second significant issue is the design of RNA-Seq experiments, in terms of the number of reads, read length, and whether reads come from one or both ends of cDNA fragments.ResultsWe present RSEM, an user-friendly software package for quantifying gene and isoform abundances from single-end or paired-end RNA-Seq data. RSEM outputs abundance estimates, 95% credibility intervals, and visualization files and can also simulate RNA-Seq data. In contrast to other existing tools, the software does not require a reference genome. Thus, in combination with a de novo transcriptome assembler, RSEM enables accurate transcript quantification for species without sequenced genomes. On simulated and real data sets, RSEM has superior or comparable performance to quantification methods that rely on a reference genome. Taking advantage of RSEM's ability to effectively use ambiguously-mapping reads, we show that accurate gene-level abundance estimates are best obtained with large numbers of short single-end reads. On the other hand, estimates of the relative frequencies of isoforms within single genes may be improved through the use of paired-end reads, depending on the number of possible splice forms for each gene.ConclusionsRSEM is an accurate and user-friendly software tool for quantifying transcript abundances from RNA-Seq data. As it does not rely on the existence of a reference genome, it is particularly useful for quantification with de novo transcriptome assemblies. In addition, RSEM has enabled valuable guidance for cost-efficient design of quantification experiments with RNA-Seq, which is currently relatively expensive.
| S-EPMC3163565 | biostudies-literature
Project description:BackgroundThe Complete Arabidopsis Transcript MicroArray (CATMA) initiative combines the efforts of laboratories in eight European countries 1 to deliver gene-specific sequence tags (GSTs) for the Arabidopsis research community. The CATMA initiative offers the power and flexibility to regularly update the GST collection according to evolving knowledge about the gene repertoire. These GST amplicons can easily be reamplified and shared, subsets can be picked at will to print dedicated arrays, and the GSTs can be cloned and used for other functional studies. This ongoing initiative has already produced approximately 24,000 GSTs that have been made publicly available for spotted microarray printing and RNA interference.ResultsGSTs from the CATMA version 2 repertoire (CATMAv2, created in 2002) were mapped onto the gene models from two independent Arabidopsis nuclear genome annotation efforts, TIGR5 and PSB-EuGène, to consolidate a list of genes that were targeted by previously designed CATMA tags. A total of 9,027 gene models were not tagged by any amplified CATMAv2 GST, and 2,533 amplified GSTs were no longer predicted to tag an updated gene model. To validate the efficacy of GST mapping criteria and design rules, the predicted and experimentally observed hybridization characteristics associated to GST features were correlated in transcript profiling datasets obtained with the CATMAv2 microarray, confirming the reliability of this platform. To complete the CATMA repertoire, all 9,027 gene models for which no GST had yet been designed were processed with an adjusted version of the Specific Primer and Amplicon Design Software (SPADS). A total of 5,756 novel GSTs were designed and amplified by PCR from genomic DNA. Together with the pre-existing GST collection, this new addition constitutes the CATMAv3 repertoire. It comprises 30,343 unique amplified sequences that tag 24,202 and 23,009 protein-encoding nuclear gene models in the TAIR6 and EuGène genome annotations, respectively. To cover the remaining untagged genes, we identified 543 additional GSTs using less stringent design criteria and designed 990 sequence tags matching multiple members of gene families (Gene Family Tags or GFTs) to cover any remaining untagged genes. These latter 1,533 features constitute the CATMAv4 addition.ConclusionTo update the CATMA GST repertoire, we designed 7,289 additional sequence tags, bringing the total number of tagged TAIR6-annotated Arabidopsis nuclear protein-coding genes to 26,173. This resource is used both for the production of spotted microarrays and the large-scale cloning of hairpin RNA silencing vectors. All information about the resulting updated CATMA repertoire is available through the CATMA database http://www.catma.org.
| S-EPMC2147040 | biostudies-literature
Project description:BACKGROUND:The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. RESULTS:A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts - BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427-433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20-28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. CONCLUSION:A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.
| S-EPMC6907147 | biostudies-literature
Project description:BACKGROUND: Arabidopsis thaliana transcript profiles indicate effects of abiotic and biotic stresses and tissue-specific and cell-specific gene expression. Organizing these datasets could reveal the structure and mechanisms of responses and crosstalk between pathways, and in which cells the plants perceive, signal, respond to, and integrate environmental inputs. RESULTS: We clustered Arabidopsis transcript profiles for various treatments, including abiotic, biotic, and chemical stresses. Ubiquitous stress responses in Arabidopsis, similar to those of fungi and animals, employ genes in pathways related to mitogen-activated protein kinases, Snf1-related kinases, vesicle transport, mitochondrial functions, and the transcription machinery. Induced responses to stresses are attributed to genes whose promoters are characterized by a small number of regulatory motifs, although secondary motifs were also apparent. Most genes that are downregulated by stresses exhibited distinct tissue-specific expression patterns and appear to be under developmental regulation. The abscisic acid-dependent transcriptome is delineated in the cluster structure, whereas functions that are dependent on reactive oxygen species are widely distributed, indicating that evolutionary pressures confer distinct responses to different stresses in time and space. Cell lineages in roots express stress-responsive genes at different levels. Intersections of stress-responsive and cell-specific profiles identified cell lineages affected by abiotic stress. CONCLUSION: By analyzing the stress-dependent expression profile, we define a common stress transcriptome that apparently represents universal cell-level stress responses. Combining stress-dependent and tissue-specific and cell-specific expression profiles, and Arabidopsis 5'-regulatory DNA sequences, we confirm known stress-related 5' cis-elements on a genome-wide scale, identify secondary motifs, and place the stress response within the context of tissues and cell lineages in the Arabidopsis root.
| S-EPMC1896000 | biostudies-literature
Project description:Gene expression maps for model organisms, including Arabidopsis thaliana, have typically been created using gene-centric expression arrays. Here, we describe a comprehensive expression atlas, Arabidopsis thaliana Tiling Array Express (At-TAX), which is based on whole-genome tiling arrays. We demonstrate that tiling arrays are accurate tools for gene expression analysis and identified more than 1,000 unannotated transcribed regions. Visualizations of gene expression estimates, transcribed regions, and tiling probe measurements are accessible online at the At-TAX homepage.
| S-EPMC2530869 | biostudies-other
Project description:Plant viruses often harm their hosts, which have developed mechanisms to prevent or minimize the effects of virus infection. Resistance and tolerance are the two main plant defences to pathogens. Although resistance to plant viruses has been studied extensively, tolerance has received much less attention. Theory predicts that tolerance to low-virulent parasites would be achieved through resource reallocation from growth to reproduction, whereas tolerance to high-virulent parasites would be attained through shortening of the pre-reproductive period. We have shown previously that the tolerance of Arabidopsis thaliana to Cucumber mosaic virus (CMV), a relatively low-virulent virus in this host, accords to these predictions. However, whether other viruses trigger the same response, and how A. thaliana copes with highly virulent virus infections remains unexplored. To address these questions, we challenged six A. thaliana wild genotypes with five viruses with different genomic structures, life histories and transmission modes. In these plants, we quantified virus multiplication, virulence, and the effects of infection on plant growth and reproduction, and on the developmental schedule. Our results indicate that virus multiplication varies according to the virus × host genotype interaction. Conversely, effective tolerance is observed only on CMV infection, and is associated with resource reallocation from growth to reproduction. Tolerance to the other viruses is observed only in specific host-virus combinations and, at odds with theoretical predictions, is linked to longer pre-reproductive periods. These findings only partially agree with theoretical predictions, and contribute to a better understanding of pathogenic processes in plant-virus interactions.
| S-EPMC6638070 | biostudies-literature
Project description:Using an integrative genome annotation pipeline (iGAP) for proteome-wide protein structure and functional domain assignment, we analyzed all the proteins of Arabidopsis thaliana. Three-dimensional structures at the level of the domain are assigned by fold recognition and threading based on a novel fold library that extends common domain classifications. iGAP is being applied to proteins from all available proteomes as part of a comparative proteomics resource. The database is accessible from the web.
| S-EPMC193643 | biostudies-literature
Project description:Genetic differences between Arabidopsis thaliana accessions underlie the plant's extensive phenotypic variation, and until now these have been interpreted largely in the context of the annotated reference accession Col-0. Here we report the sequencing, assembly and annotation of the genomes of 18 natural A. thaliana accessions, and their transcriptomes. When assessed on the basis of the reference annotation, one-third of protein-coding genes are predicted to be disrupted in at least one accession. However, re-annotation of each genome revealed that alternative gene models often restore coding potential. Gene expression in seedlings differed for nearly half of expressed genes and was frequently associated with cis variants within 5 kilobases, as were intron retention alternative splicing events. Sequence and expression variation is most pronounced in genes that respond to the biotic environment. Our data further promote evolutionary and functional studies in A. thaliana, especially the MAGIC genetic reference population descended from these accessions.
| S-EPMC4856438 | biostudies-literature
Project description:Quantitative real-time polymerase chain reaction (qPCR) is a widely used method to analyse the gene expression pattern in the reproductive tissues along with detecting gene levels in mutant backgrounds. This technique requires stable reference genes to normalise the expression level of target genes. Nonetheless, a considerable number of publications continue to present qPCR results normalised to a single reference gene and, to our knowledge, no comparative evaluation of multiple reference genes has been carried out in specific reproductive tissues of Arabidopsis thaliana. Herein, we assessed the expression stability levels of ten candidate reference genes (UBC9, ACT7, GAPC-2, RCE1, PP2AA3, TUA2, SAC52, YLS8, SAMDC and HIS3.3) in two conditional sets: one across flower development and the other using inflorescences from different genotypes. The stability analysis was performed using the RefFinder tool, which combines four statistical algorithms (geNorm, NormFinder, BestKeeper and the comparative ΔCt method). Our results showed that RCE1, SAC52 and TUA2 had the most stable expression in different flower developmental stages while YLS8, HIS3.3 and ACT7 were the top-ranking reference genes for normalisation in mutant studies. Furthermore, we validated our results by analysing the expression pattern of genes involved in reproduction and examining the expression of these genes in published mutant backgrounds. Overall, we provided a pool of appropriate reference genes for expression studies in reproductive tissues of A. thaliana, which will facilitate further gene expression studies in this context. More importantly, we presented a framework that will promote a consistent and accurate analysis of gene expression in any scientific field. Simultaneously, we highlighted the relevance of clearly defining and describing the experimental conditions associated with qPCR to improve scientific reproducibility.
| S-EPMC10046263 | biostudies-literature
Project description:We present whole-genome assemblies of four divergent Arabidopsis thaliana strains that complement the 125-Mb reference genome sequence released a decade ago. Using a newly developed reference-guided approach, we assembled large contigs from 9 to 42 Gb of Illumina short-read data from the Landsberg erecta (Ler-1), C24, Bur-0, and Kro-0 strains, which have been sequenced as part of the 1,001 Genomes Project for this species. Using alignments against the reference sequence, we first reduced the complexity of the de novo assembly and later integrated reads without similarity to the reference sequence. As an example, half of the noncentromeric C24 genome was covered by scaffolds that are longer than 260 kb, with a maximum of 2.2 Mb. Moreover, over 96% of the reference genome was covered by the reference-guided assembly, compared with only 87% with a complete de novo assembly. Comparisons with 2 Mb of dideoxy sequence reveal that the per-base error rate of the reference-guided assemblies was below 1 in 10,000. Our assemblies provide a detailed, genomewide picture of large-scale differences between A. thaliana individuals, most of which are difficult to access with alignment-consensus methods only. We demonstrate their practical relevance in studying the expression differences of polymorphic genes and show how the analysis of sRNA sequencing data can lead to erroneous conclusions if aligned against the reference genome alone. Genome assemblies, raw reads, and further information are accessible through http://1001genomes.org/projects/assemblies.html.
| S-EPMC3121819 | biostudies-other