Leveraging multiple transcriptome assembly methods for improved gene structure annotation.
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ABSTRACT:
SUBMITTER: Venturini L
PROVIDER: S-EPMC6105091 | biostudies-literature | 2018 Aug
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:The investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection. In contrast, long-reads sequencing techniques revealed their potential to generate complete transcript assemblies even when a reference genome is lacking. Cynara cardunculus var. altilis (DC) (cultivated cardoon) is a perennial hardy crop adapted to dry environments with many industrial and nutraceutical applications due to the richness of secondary metabolites mostly produced in flower heads. The investigation of this species benefited from the recent release of a draft genome, but the transcriptome profile during the capitula formation still remains unexplored. In the present study we show a transcriptome analysis of vegetative and inflorescence organs of cultivated cardoon through a novel hybrid RNA-seq assembly approach utilizing both long and short RNA-seq reads. RESULTS:The inclusion of a single Nanopore flow-cell output in a hybrid sequencing approach determined an increase of 15% complete assembled genes and 18% transcript isoforms respect to short reads alone. Among 25,463 assembled unigenes, we identified 578 new genes and updated 13,039 gene models, 11,169 of which were alternatively spliced isoforms. During capitulum development, 3424 genes were differentially expressed and approximately two-thirds were identified as transcription factors including bHLH, MYB, NAC, C2H2 and MADS-box which were highly expressed especially after capitulum opening. We also show the expression dynamics of key genes involved in the production of valuable secondary metabolites of which capitulum is rich such as phenylpropanoids, flavonoids and sesquiterpene lactones. Most of their biosynthetic genes were strongly transcribed in the flower heads with alternative isoforms exhibiting differentially expression levels across the tissues. CONCLUSIONS:This novel hybrid sequencing approach allowed to improve the transcriptome assembly, to update more than half of annotated genes and to identify many novel genes and different alternatively spliced isoforms. This study provides new insights on the flowering cycle in an Asteraceae plant, a valuable resource for plant biology and breeding in Cynara and an effective method for improving gene annotation.
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Project description:Annotation of gene models and transcripts is a fundamental step in genome sequencing projects. Often this is performed with automated prediction pipelines, which can miss complex and atypical genes or transcripts. RNA sequencing (RNA-seq) data can aid the annotation with empirical data. Here we present de novo transcriptome assemblies generated from RNA-seq data in four Dictyostelid species: D. discoideum, P. pallidum, D. fasciculatum and D. lacteum. The assemblies were incorporated with existing gene models to determine corrections and improvement on a whole-genome scale. This is the first time this has been performed in these eukaryotic species.An initial de novo transcriptome assembly was generated by Trinity for each species and then refined with Program to Assemble Spliced Alignments (PASA). The completeness and quality were assessed with the Benchmarking Universal Single-Copy Orthologs (BUSCO) and Transrate tools at each stage of the assemblies. The final datasets of 11,315-12,849 transcripts contained 5,610-7,712 updates and corrections to >50% of existing gene models including changes to hundreds or thousands of protein products. Putative novel genes are also identified and alternative splice isoforms were observed for the first time in P. pallidum, D. lacteum and D. fasciculatum.In taking a whole transcriptome approach to genome annotation with empirical data we have been able to enrich the annotations of four existing genome sequencing projects. In doing so we have identified updates to the majority of the gene annotations across all four species under study and found putative novel genes and transcripts which could be worthy for follow-up. The new transcriptome data we present here will be a valuable resource for genome curators in the Dictyostelia and we propose this effective methodology for use in other genome annotation projects.
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Project description:BACKGROUND:Physalis peruviana commonly known as Cape gooseberry is a member of the Solanaceae family that has an increasing popularity due to its nutritional and medicinal values. A broad range of genomic tools is available for other Solanaceae, including tomato and potato. However, limited genomic resources are currently available for Cape gooseberry. RESULTS:We report the generation of a total of 652,614 P. peruviana Expressed Sequence Tags (ESTs), using 454 GS FLX Titanium technology. ESTs, with an average length of 371?bp, were obtained from a normalized leaf cDNA library prepared using a Colombian commercial variety. De novo assembling was performed to generate a collection of 24,014 isotigs and 110,921 singletons, with an average length of 1,638?bp and 354?bp, respectively. Functional annotation was performed using NCBI's BLAST tools and Blast2GO, which identified putative functions for 21,191 assembled sequences, including gene families involved in all the major biological processes and molecular functions as well as defense response and amino acid metabolism pathways. Gene model predictions in P. peruviana were obtained by using the genomes of Solanum lycopersicum (tomato) and Solanum tuberosum (potato). We predict 9,436 P. peruviana sequences with multiple-exon models and conserved intron positions with respect to the potato and tomato genomes. Additionally, to study species diversity we developed 5,971 SSR markers from assembled ESTs. CONCLUSIONS:We present the first comprehensive analysis of the Physalis peruviana leaf transcriptome, which will provide valuable resources for development of genetic tools in the species. Assembled transcripts with gene models could serve as potential candidates for marker discovery with a variety of applications including: functional diversity, conservation and improvement to increase productivity and fruit quality. P. peruviana was estimated to be phylogenetically branched out before the divergence of five other Solanaceae family members, S. lycopersicum, S. tuberosum, Capsicum spp, S. melongena and Petunia spp.
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Project description:The common toad Rhinella arenarum is widely distributed in Argentina, where it is utilised as an autochthonous model in ecotoxicological research and environmental toxicology. However, the lack of a reference genome makes molecular assays and gene expression studies difficult to carry out on this non-model species. To address this issue, we performed a genome-wide transcriptome analysis on R. arenarum larvae through massive RNA sequencing, followed by de novo assembly, annotation, and gene prediction. We obtained 57,407 well-annotated transcripts representing 99.4% of transcriptome completeness (available at http://rhinella.uncoma.edu.ar). We also defined a set of 52,800 high-confidence lncRNA transcripts and demonstrated the reliability of the transcriptome data to perform phylogenetic analysis. Our comprehensive transcriptome analysis of R. arenarum represents a valuable resource to perform functional genomic studies and to identify potential molecular biomarkers in ecotoxicological research.
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Project description:Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complemented by transcriptome information that will enhance genome assembly and annotation. Previously, transcriptome reference sequences were reported using data from different sources. Although the previous work added a great wealth of sequences, a complete and well-annotated transcriptome is still needed. In addition, gene expression in different tissues was not completely addressed in the previous studies. In this study, non-normalized cDNA libraries were sequenced from 13 different tissues of a single doubled haploid rainbow trout from the same source used for the rainbow trout genome sequence. A total of ~1.167 billion paired-end reads were de novo assembled using the Trinity RNA-Seq assembler yielding 474,524 contigs > 500 base-pairs. Of them, 287,593 had homologies to the NCBI non-redundant protein database. The longest contig of each cluster was selected as a reference, yielding 44,990 representative contigs. A total of 4,146 contigs (9.2%), including 710 full-length sequences, did not match any mRNA sequences in the current rainbow trout genome reference. Mapping reads to the reference genome identified an additional 11,843 transcripts not annotated in the genome. A digital gene expression atlas revealed 7,678 housekeeping and 4,021 tissue-specific genes. Expression of about 16,000-32,000 genes (35-71% of the identified genes) accounted for basic and specialized functions of each tissue. White muscle and stomach had the least complex transcriptomes, with high percentages of their total mRNA contributed by a small number of genes. Brain, testis and intestine, in contrast, had complex transcriptomes, with a large numbers of genes involved in their expression patterns. This study provides comprehensive de novo transcriptome information that is suitable for functional and comparative genomics studies in rainbow trout, including annotation of the genome.
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Project description:The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage.We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource.We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3' and 5' UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis regulatory motifs). The P. tetraurelia improved transcriptome resource, gene annotations for P. tetraurelia, P. biaurelia, P. sexaurelia and P. caudatum, and Paramecium-trained EuGene configuration are available through ParameciumDB ( http://paramecium.i2bc.paris-saclay.fr ). TrUC software is freely distributed under a GNU GPL v3 licence ( https://github.com/oarnaiz/TrUC ).
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Project description:BackgroundsMacaca fascicularis (M. fascicularis) is a primate model organism that played important role in studying human health. It is vital to better understand the similarity and differences of gene regulation between M. fascicularis and human. Current comparative study of gene regulation between the two species are limited by low quality of gene annotation and lack of regulatory element data on M. fascicularis genome.ResultsIn this study, we improved the M. fascicularis gene annotation with 57 gene expression data from multiple tissues and, more importantly, a manual curation procedure. The new annotation enabled us to map gene expression and identify gene location more accurately.ConclusionsComparing with human gene expression data from the same cell types, we characterized the evolution of expression patterns of homologous genes.
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Project description:We present a new transcriptome assembly of the Pacific whiteleg shrimp (Litopenaeus vannamei), the species most farmed for human consumption. Its functional annotation, a substantial improvement over previous ones, is provided freely. RNA-Seq with Illumina HiSeq technology was used to analyze samples extracted from shrimp abdominal muscle, hepatopancreas, gills and pleopods. We used the Trinity and Trinotate software suites for transcriptome assembly and annotation, respectively. The quality of this assembly and the affiliated targeted homology searches greatly enrich the curated transcripts currently available in public databases for this species. Comparison with the model arthropod Daphnia allows some insights into defining characteristics of decapod crustaceans. This large-scale gene discovery gives the broadest depth yet to the annotated transcriptome of this important species and should be of value to ongoing genomics and immunogenetic resistance studies in this shrimp of paramount global economic importance.
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Project description:Genomes sequenced using short-read, next-generation sequencing technologies can have many errors and may be fragmented into thousands of small contigs. These incomplete and fragmented assemblies lead to errors in gene identification, such that single genes spread across multiple contigs are annotated as separate gene models. Such biases can confound inferences about the number and identity of genes within species, as well as gene gain and loss between species.We present AGOUTI (Annotated Genome Optimization Using Transcriptome Information), a tool that uses RNA sequencing data to simultaneously combine contigs into scaffolds and fragmented gene models into single models. We show that AGOUTI improves both the contiguity of genome assemblies and the accuracy of gene annotation, providing updated versions of each as output. Running AGOUTI on both simulated and real datasets, we show that it is highly accurate and that it achieves greater accuracy and contiguity when compared with other existing methods.AGOUTI is a powerful and effective scaffolder and, unlike most scaffolders, is expected to be more effective in larger genomes because of the commensurate increase in intron length. AGOUTI is able to scaffold thousands of contigs while simultaneously reducing the number of gene models by hundreds or thousands. The software is available free of charge under the MIT license.
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