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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.

Background The possibility of generating large RNA-sequencing datasets has led to development of various reference-based and de novo transcriptome assemblers with their own strengths and limitations. While reference-based tools are widely used in various transcriptomic studies, their application is limited to the organisms with finished and well-annotated genomes. De novo transcriptome reconstruction from short reads remains an open challenging problem, which is complicated by the varying expression levels across different genes, alternative splicing, and paralogous genes. Results Herein we describe the novel transcriptome assembler rnaSPAdes, which has been developed on top of the SPAdes genome assembler and explores computational parallels between assembly of transcriptomes and single-cell genomes. We also present quality assessment reports for rnaSPAdes assemblies, compare it with modern transcriptome assembly tools using several evaluation approaches on various RNA-sequencing datasets, and briefly highlight strong and weak points of different assemblers. Conclusions Based on the performed comparison between different assembly methods, we infer that it is not possible to detect the absolute leader according to all quality metrics and all used datasets. However, rnaSPAdes typically outperforms other assemblers by such important property as the number of assembled genes and isoforms, and at the same time has higher accuracy statistics on average comparing to the closest competitors.

Summary: Ability to generate large RNA-Seq datasets created a demand for both de novo and reference-based transcriptome assemblers. However, while many transcriptome assemblers are now available, there is still no unified quality assessment tool for RNA-Seq assemblies. We present rnaQUAST—a tool for evaluating RNA-Seq assembly quality and benchmarking transcriptome assemblers using reference genome and gene database. rnaQUAST calculates various metrics that demonstrate completeness and correctness levels of the assembled transcripts, and outputs them in a user-friendly report. Availability and Implementation: rnaQUAST is implemented in Python and is freely available at http://bioinf.spbau.ru/en/rnaquast. Contact: ap@bioinf.spbau.ru Supplementary information: Supplementary data are available at Bioinformatics online.