Sequencing and de novo assembly of a Dahlia hybrid cultivar transcriptome.
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ABSTRACT:
SUBMITTER: Lehnert EM
PROVIDER: S-EPMC4101353 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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Project description:MotivationIn the past years, the long read (LR) sequencing technologies, such as Pacific Biosciences and Oxford Nanopore Technologies, have been demonstrated to substantially improve the quality of genome assembly and transcriptome characterization. Compared to the high cost of genome assembly by LR sequencing, it is more affordable to generate LRs for transcriptome characterization. That is, when informative transcriptome LR data are available without a high-quality genome, a method for de novo transcriptome assembly and annotation is of high demand.ResultsWithout a reference genome, IDP-denovo performs de novo transcriptome assembly, isoform annotation and quantification by integrating the strengths of LRs and short reads. Using the GM12878 human data as a gold standard, we demonstrated that IDP-denovo had superior sensitivity of transcript assembly and high accuracy of isoform annotation. In addition, IDP-denovo outputs two abundance indices to provide a comprehensive expression profile of genes/isoforms. IDP-denovo represents a robust approach for transcriptome assembly, isoform annotation and quantification for non-model organism studies. Applying IDP-denovo to a non-model organism, Dendrobium officinale, we discovered a number of novel genes and novel isoforms that were not reported by the existing annotation library. These results reveal the high diversity of gene isoforms in D.officinale, which was not reported in the existing annotation library.Availability and implementationThe dataset of Dendrobium officinale used/analyzed during the current study has been deposited in SRA, with accession code SRP094520. IDP-denovo is available for download at www.healthcare.uiowa.edu/labs/au/IDP-denovo/.Supplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Sour cherry (Prunus cerasus) in the genus Prunus in the family Rosaceae is one of the most popular stone fruit trees worldwide. Of known sour cherry cultivars, the Schattenmorelle is a famous old sour cherry with a high amount of fruit production. The Schattenmorelle was selected before 1650 and described in the 1800s. This cultivar was named after gardens of the Chateau de Moreille in which the cultivar was initially found. In order to identify new genes and to develop genetic markers for sour cherry, we performed a transcriptome analysis of a sour cherry. We selected the cultivar Schattenmorelle, which is among commercially important cultivars in Europe and North America. We obtained 2.05 GB raw data from the Schattenmorelle (NCBI accession number: SRX1187170). De novo transcriptome assembly using Trinity identified 61,053 transcripts in which N50 was 611 bp. Next, we identified 25,585 protein coding sequences using TransDecoder. The identified proteins were blasted against NCBI's non-redundant database for annotation. Based on blast search, we taxonomically classified the obtained sequences. As a result, we provide the transcriptome of sour cherry cultivar Schattenmorelle using next generation sequencing.
| S-EPMC4664768 | biostudies-literature
Project description:UnlabelledPremise of the studyWe report the de novo assembly and characterization of the transcriptomes of Brachypodium sylvaticum (slender false-brome) accessions from native populations of Spain and Greece, and an invasive population west of Corvallis, Oregon, USA. •Methods and resultsMore than 350 million sequence reads from the mRNA libraries prepared from three B. sylvaticum genotypes were assembled into 120,091 (Corvallis), 104,950 (Spain), and 177,682 (Greece) transcript contigs. In comparison with the B. distachyon Bd21 reference genome and GenBank protein sequences, we estimate >90% exome coverage for B. sylvaticum. The transcripts were assigned Gene Ontology and InterPro annotations. Brachypodium sylvaticum sequence reads aligned against the Bd21 genome revealed 394,654 single-nucleotide polymorphisms (SNPs) and >20,000 simple sequence repeat (SSR) DNA sites. •ConclusionsTo our knowledge, this is the first report of transcriptome sequencing of invasive plant species with a closely related sequenced reference genome. The sequences and identified SNP variant and SSR sites will provide tools for developing novel genetic markers for use in genotyping and characterization of invasive behavior of B. sylvaticum.
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Project description:Pseudostellaria heterophylla (Miq.) Pax is a mild tonic herb widely cultivated in the Southern part of China. The tuberous roots of P. heterophylla accumulate high levels of secondary metabolism products of medicinal value such as saponins, flavonoids, and isoquinoline alkaloids. Despite numerous studies on the pharmacological importance and purification of these compounds in P. heterophylla, their biosynthesis is not well understood. In the present study, we used Illumina HiSeq 4000 sequencing platform to sequence the RNA from flowers, leaves, stem, root cortex and xylem tissues of P. heterophylla. We obtained 616,413,316 clean reads that we assembled into 127, 334 unique sequences with an N50 length of 951 bp. Among these unigenes, 53,184 unigenes (41.76%) were annotated in a public database and 39, 795 unigenes were assigned to 356 KEGG pathways; 23,714 unigenes (8.82%) had high homology with the genes from Beta vulgaris. We discovered 32, 095 DEGs in different tissues and performed GO and KEGG enrichment analysis. The most enriched KEGG pathway of secondary metabolism showed up-regulated expression in tuberous roots as compared with the ground parts of P. heterophylla. Moreover, we identified 72 candidate genes involved in triterpenoids saponins biosynthesis in P. heterophylla. The expression profiles of 11 candidate unigenes were analyzed by quantitative real-time PCR (RT-qPCR). Our study established a global transcriptome database of P. heterophylla for gene identification and regulation. We also identified the candidate unigenes involved in triterpenoids saponins biosynthesis. Our results provide an invaluable resource for the secondary metabolites and physiological processes in different tissues of P. heterophylla.
| S-EPMC5072632 | biostudies-literature
Project description:Raphanus sativus is an important Brassicaceae plant and also an edible vegetable with great economic value. However, currently there is not enough transcriptome information of R. sativus tissues, which impedes further functional genomics research on R. sativus. In this study, RNA-seq technology was employed to characterize the transcriptome of leaf tissues. Approximately 70 million clean pair-end reads were obtained and used for de novo assembly by Trinity program, which generated 68,086 unigenes with an average length of 576 bp. All the unigenes were annotated against GO and KEGG databases. In the meanwhile, we merged leaf sequencing data with existing root sequencing data and obtained better de novo assembly of R. sativus using Oases program. Accordingly, potential simple sequence repeats (SSRs), transcription factors (TFs) and enzyme codes were identified in R. sativus. Additionally, we detected a total of 3563 significantly differentially expressed genes (DEGs, P = 0.05) and tissue-specific biological processes between leaf and root tissues. Furthermore, a TFs-based regulation network was constructed using Cytoscape software. Taken together, these results not only provide a comprehensive genomic resource of R. sativus but also shed light on functional genomic and proteomic research on R. sativus in the future.
| S-EPMC4428447 | biostudies-literature
Project description:Transcriptome analysis has important applications in many biological fields. However, assembling a transcriptome without a known reference remains a challenging task requiring algorithmic improvements. We present two methods for substantially improving transcriptome de novo assembly. The first method relies on the observation that the use of a single k-mer length by current de novo assemblers is suboptimal to assemble transcriptomes where the sequence coverage of transcripts is highly heterogeneous. We present the Multiple-k method in which various k-mer lengths are used for de novo transcriptome assembly. We demonstrate its good performance by assembling de novo a published next-generation transcriptome sequence data set of Aedes aegypti, using the existing genome to check the accuracy of our method. The second method relies on the use of a reference proteome to improve the de novo assembly. We developed the Scaffolding using Translation Mapping (STM) method that uses mapping against the closest available reference proteome for scaffolding contigs that map onto the same protein. In a controlled experiment using simulated data, we show that the STM method considerably improves the assembly, with few errors. We applied these two methods to assemble the transcriptome of the non-model catfish Loricaria gr. cataphracta. Using the Multiple-k and STM methods, the assembly increases in contiguity and in gene identification, showing that our methods clearly improve quality and can be widely used. The new methods were used to assemble successfully the transcripts of the core set of genes regulating tooth development in vertebrates, while classic de novo assembly failed.
| S-EPMC2945192 | biostudies-literature
Project description:Single-molecule sequencing instruments can generate multikilobase sequences with the potential to greatly improve genome and transcriptome assembly. However, the error rates of single-molecule reads are high, which has limited their use thus far to resequencing bacteria. To address this limitation, we introduce a correction algorithm and assembly strategy that uses short, high-fidelity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on reads generated by a PacBio RS instrument from phage, prokaryotic and eukaryotic whole genomes, including the previously unsequenced genome of the parrot Melopsittacus undulatus, as well as for RNA-Seq reads of the corn (Zea mays) transcriptome. Our long-read correction achieves >99.9% base-call accuracy, leading to substantially better assemblies than current sequencing strategies: in the best example, the median contig size was quintupled relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly.
| S-EPMC3707490 | biostudies-literature
Project description:BACKGROUND:Poinsettia is a popular and important ornamental crop, mostly during the Christmas season. Its bract coloration ranges from pink/red to creamy/white shades. Despite its ornamental value, there is a lack of knowledge about the genetics and molecular biology of poinsettia, especially on the mechanisms of color formation. We performed an RNA-Seq analysis in order to shed light on the transcriptome of poinsettia bracts. Moreover, we analyzed the transcriptome differences of red- and white-bracted poinsettia varieties during bract development and coloration. For the assembly of a bract transcriptome, two paired-end cDNA libraries from a red and white poinsettia pair were sequenced with the Illumina technology, and one library from a red-bracted variety was used for PacBio sequencing. Both short and long reads were assembled using a hybrid de novo strategy. Samples of red- and white-bracted poinsettias were sequenced and comparatively analyzed in three color developmental stages in order to understand the mechanisms of color formation and accumulation in the species. RESULTS:The final transcriptome contains 288,524 contigs, with 33% showing confident protein annotation against the TAIR10 database. The BUSCO pipeline, which is based on near-universal orthologous gene groups, was applied to assess the transcriptome completeness. From a total of 1440 BUSCO groups searched, 77% were categorized as complete (41% as single-copy and 36% as duplicated), 10% as fragmented and 13% as missing BUSCOs. The gene expression comparison between red and white varieties of poinsettia showed a differential regulation of the flavonoid biosynthesis pathway only at particular stages of bract development. An initial impairment of the flavonoid pathway early in the color accumulation process for the white poinsettia variety was observed, but these differences were no longer present in the subsequent stages of bract development. Nonetheless, GSTF11 and UGT79B10 showed a lower expression in the last stage of bract development for the white variety and, therefore, are potential candidates for further studies on poinsettia coloration. CONCLUSIONS:In summary, this transcriptome analysis provides a valuable foundation for further studies on poinsettia, such as plant breeding and genetics, and highlights crucial information on the molecular mechanism of color formation.
| S-EPMC6882326 | biostudies-literature
Project description:BackgroundIsatis indigotica, the source of the traditional Chinese medicine Radix isatidis (Ban-Lan-Gen), is an extremely important economical crop in China. To facilitate biological, biochemical and molecular research on the medicinal chemicals in I. indigotica, here we report the first I. indigotica transcriptome generated by RNA sequencing (RNA-seq).ResultsRNA-seq library was created using RNA extracted from a mixed sample including leaf and root. A total of 33,238 unigenes were assembled from more than 28 million of high quality short reads. The quality of the assembly was experimentally examined by cDNA sequencing of seven randomly selected unigenes. Based on blast search 28,184 unigenes had a hit in at least one of the protein and nucleotide databases used in this study, and 8 unigenes were found to be associated with biosynthesis of indole and its derivatives. According to Gene Ontology classification, 22,365 unigenes were categorized into 48 functional groups. Furthermore, Clusters of Orthologous Group and Swiss-Port annotation were assigned for 7,707 and 18,679 unigenes, respectively. Analysis of repeat motifs identified 6,400 simple sequence repeat markers in 4,509 unigenes.ConclusionOur data provide a comprehensive sequence resource for molecular study of I. indigotica. Our results will facilitate studies on the functions of genes involved in the indole alkaloid biosynthesis pathway and on metabolism of nitrogen and indole alkaloids in I. indigotica and its related species.
| S-EPMC4178013 | biostudies-literature
Project description:Contact with poison ivy plants is widely dreaded because they produce a natural product called urushiol that is responsible for allergenic contact delayed-dermatitis symptoms lasting for weeks. For this reason, the catchphrase most associated with poison ivy is "leaves of three, let it be", which serves the purpose of both identification and an appeal for avoidance. Ironically, despite this notoriety, there is a dearth of specific knowledge about nearly all other aspects of poison ivy physiology and ecology. As a means of gaining a more molecular-oriented understanding of poison ivy physiology and ecology, Next Generation DNA sequencing technology was used to develop poison ivy root and leaf RNA-seq transcriptome resources. De novo assembled transcriptomes were analyzed to generate a core set of high quality expressed transcripts present in poison ivy tissue. The predicted protein sequences were evaluated for similarity to SwissProt homologs and InterProScan domains, as well as assigned both GO terms and KEGG annotations. Over 23,000 simple sequence repeats were identified in the transcriptome, and corresponding oligo nucleotide primer pairs were designed. A pan-transcriptome analysis of existing Anacardiaceae transcriptomes revealed conserved and unique transcripts among these species.
| S-EPMC5704230 | biostudies-literature