PacBio full‐length cDNA sequencing integrated with RNA‐seq reads drastically improves the discovery of splicing transcripts in rice

Abstract

In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and the validation rate of splicing isoforms, and integrated a comprehensive pipeline of splicing transcripts analysis by Iso-Seq and RNA-seq (STAIR) to identify the full-length multi-exon isoforms in rice seedling transcriptome (Oryza sativa L. ssp. japonica). STAIR discovered 11 733 full-length multi-exon isoforms, 6599 more than the SMRT Portal RS_Iso-Seq pipeline did. Of these splicing isoforms identified, 4453 (37.9%) were missed in assembled transcripts from RNA-seq reads, and 5204 (44.4%), including 268 multi-exon long non-coding RNAs (lncRNAs), were not reported in the MSU_osa1r7 annotation. Some randomly selected unreported splicing junctions were verified by polymerase chain reaction (PCR) amplification. In addition, we investigated alternative polyadenylation (APA) events in transcripts and identified 829 major polyadenylation [poly(A)] site clusters (PACs). The analysis of splicing isoforms and APA events will facilitate the annotation of the rice genome and studies on the expression and polyadenylation of AS genes in different developmental stages or growth conditions of rice.

Characterization and Functional Analysis of Polyadenylation Sites in Fast and Slow Muscles

“…Especially, the Iso-seq library built with the oligo(dT) primer ensures the integrity of 3′ terminal. So far, the PacBio protocol Iso-Seq ™ has been successfully applied to characterize the posttranscriptional APA events in various eukaryotes [26][27][28][29][30]. Our study used the PacBio dataset to annotate PASs of pigs and analyzed differential usages and functions of APA genes under the regulation of miRNAs and RBPs between fast and slow muscles.…”

IsoSeq transcriptome assembly of C3 panicoid grasses provides tools to study evolutionary change in the Panicoideae

“…As a result, while published whole genome sequence assemblies exist for at least 14 grasses within the PACMAD clade (Table ), only one of these ( Dichanthelium oligosanthes , a wild species) (Studer et al, ) utilizes C 3 photosynthesis. Long‐read sequencing can effectively generate sequence for large numbers of full‐length cDNAs even in species lacking reference genome assemblies (An, Cao, Li, Humbeck, & Wang, ; Zhang et al, ). One concern with utilizing this technology for comparative genetic studies is that the higher error rate, particularly the frequencies of insertion and deletion errors, makes data from long‐read‐based sequencing of non‐model species unsuitable for use in comparative evolutionary analyses (Gonzalez‐Garay, ).…”

“…Long-read sequencing can effectively generate sequence for large numbers of full-length cDNAs even in species lacking reference genome assemblies (An, Cao, Li, Humbeck, & Wang, 2018;Zhang et al, 2019). One concern with utilizing this technology for comparative genetic studies is that the higher error rate, particularly the frequencies of insertion and deletion errors, makes data from long-read-based sequencing of non-model species unsuitable for use in comparative evolutionary analyses (Gonzalez-Garay, 2016).…”

Extending rnaSPAdes functionality for hybrid transcriptome assembly

“…In the past few years, several research projects involving long-read RNA sequencing were carried out [3][4][5][6][7]. All of them, however, were performed for species with relatively well-sequenced genomes.…”