Hybrid transcriptome sequencing approach improved assembly and gene annotation in Cynara cardunculus (L.)

Abstract

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.

Growth and Meat Quality of Grass Carp (Ctenopharyngodon idellus) Responded to Dietary Protein (Soybean Meal) Level Through the Muscle Metabolism and Gene Expression of Myosin Heavy Chains

“…In addition, KEGG analysis demonstrated that myhc isoform and hsp74 were significantly enriched in the tight junction in this study, which was once considered to be tightly related to meat hardness ( 32 ). Regretfully, due to the homology differences in myosin heavy chain nucleotide sequences in fish and higher vertebrates and the reduced isoform discovery effect resulting from the short read-sequencing in the transcriptome ( 33 ), the subtypes ( myh1, myh2 , and myh4 ) of myosin heavy chain, fast skeletal muscle, could not be annotated in grass carp transcripts in this study and previous studies ( 34 ). Instead, the expression status of myhc isoforms presented in Figure 2E and the relation between meat hardness and myhc isoforms in Appendix Table 3 clearly supported that the myhc isoform profile affected muscle hardness.…”

Comparative Analysis of PacBio and Oxford Nanopore Sequencing Technologies for Transcriptomic Landscape Identification of Penaeus monodon

“…The rapid development of these technologies has produced a large number of new tools and software that should be comprehensively evaluated to allow the scientific community to identify and choose adequately between them. Some open-source resources and repositories for long-read software and tools are available on GitHub ( , accessed on 1 August 2020) and on the long-read-tools.org database [ 4 ].…”

“…Thus, many other “-omics” are quickly adapting to these technologies, further improving the accumulated scientific knowledge obtained during the past decades using short-read sequencing methods. Although Illumina (short read) is still a widely used sequencing platform for transcriptomic studies, it has some technological limitations that can be overcome using long-read sequencing [ 2 , 3 , 4 ]. The main limitations of short-read sequencing are that assembled transcripts from short reads do not cover full-length transcripts in eukaryotic genomes, and PCR amplification bias can be introduced during library construction [ 5 , 6 ].…”

A de novo Full-Length mRNA Transcriptome Generated From Hybrid-Corrected PacBio Long-Reads Improves the Transcript Annotation and Identifies Thousands of Novel Splice Variants in Atlantic Salmon

“…Our pipeline aimed to generate a full-length mRNA transcriptome with reference level sequence accuracy from a variety of organ samples by processing PacBio long-reads that were hybrid error-corrected with Illumina paired-end reads from the same samples. Similar approaches have been used to generate high quality transcriptomes in other species ( Feng et al, 2019 ; Puglia et al, 2020 ), but this is the first of its kind in Atlantic salmon. The strategy and the main functions of the pipeline is illustrated in Figures 1 – 3 .…”