Isoform Sequencing Provides a More Comprehensive View of the Panax ginseng Transcriptome

Abstract

Korean ginseng (Panax ginseng C.A. Meyer) has been widely used for medicinal purposes and contains potent plant secondary metabolites, including ginsenosides. To obtain transcriptomic data that offers a more comprehensive view of functional genomics in P. ginseng, we generated genome-wide transcriptome data from four different P. ginseng tissues using PacBio isoform sequencing (Iso-Seq) technology. A total of 135,317 assembled transcripts were generated with an average length of 3.2 kb and high assembly completeness. Of those unigenes, 67.5% were predicted to be complete full-length (FL) open reading frames (ORFs) and exhibited a high gene annotation rate. Furthermore, we successfully identified unique full-length genes involved in triterpenoid saponin synthesis and plant hormonal signaling pathways, including auxin and cytokinin. Studies on the functional genomics of P. ginseng seedlings have confirmed the rapid upregulation of negative feed-back loops by auxin and cytokinin signaling cues. The conserved evolutionary mechanisms in the auxin and cytokinin canonical signaling pathways of P. ginseng are more complex than those in Arabidopsis thaliana. Our analysis also revealed a more detailed view of transcriptome-wide alternative isoforms for 88 genes. Finally, transposable elements (TEs) were also identified, suggesting transcriptional activity of TEs in P. ginseng. In conclusion, our results suggest that long-read, full-length or partial-unigene data with high-quality assemblies are invaluable resources as transcriptomic references in P. ginseng and can be used for comparative analyses in closely related medicinal plants.

Transcriptome Atlas by Long-Read RNA Sequencing: Contribution to a Reference Transcriptome

“…The high quality of RNA with enough purity and integrity is critical to reduce the amplification cycles required in large-scale PCR and improve the sequencing diversity. RNA extraction is usually done through an easy-spin RNA extraction kit, or RNAiso Pure RNA Isolation kit [20][21][22]. In general, 2-5 μg of total RNA with an RNA integrity number (RIN) greater than 7 is required.…”

“…The Iso-Seq raw reads are usually called polymerase reads or continuous long reads (CLRs) and have an average length of 10 kb (Figure 1). Considering the average length of a transcript is 1-2 kb, the same copies of the inserts are contained in a single polymerase that could be split into several subreads by removing the adaptor sequences by PacBio SMRT link analysis [20]. The circular consensus sequences or ROIs are generated from several subreads.…”

“…The full-length non-chimeric read (FLNC) is defined not only when the polyA tail signal preceding the 30-primer is present, but also when both 50-and 30-cDNA primers are present. To enhance consensus accuracy and remove the redundancy of FLNC without any additional sequence data, ICE and Quiver can be applied [20]. The Iso-Seq classify tool is used for classifying the ROIs into full-length nonchimeric and non-full-length reads by identifying the 50 and 30 adapters used in library preparation.…”

“…Additionally, the CD-HIT program [26] is likely to be helpful to cluster the high and low quiver consensus isoforms from ROIs with high sequence identity threshold (i.e. 0.98-0.99) [20,21].…”

“…Long-read transcriptome sequencing generates longer and improved transcripts with a high level of assembly completeness and gene annotation. Moreover, it prevents obtaining artifacts such as chimeras, structural errors, incomplete assembly, and base errors [20].…”