Analysis of transcripts and splice isoforms in red clover (Trifolium pratense L.) by single-molecule long-read sequencing

Chao, Yuehui; Yuan, Jianbo; Li, Sifeng; Jia, Siqiao; Han, Liebao; Xu, Lixin

doi:10.1186/s12870-018-1534-8

Cited by 88 publications

(64 citation statements)

References 33 publications

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“…The human transcriptome was revealed to be much more complex than previously expected owing to the application of TGS technology, which identified an array of novel isoforms that had not yet been annotated [9]. Similar findings were reported in some other species such as pigs [39], rabbits [26], switchgrass [13], red clover [30], and so forth. In the present study, we identified 2405 novel genic loci from unannotated regions and 11,623 new isoforms from various exons in the draft genome based on SMRT data, suggestive of a more complex transcriptome of A. apis .…”

Section: Discussionsupporting

confidence: 52%

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Reconstruction and functional annotation of Ascosphaera apis full-length transcriptome via PacBio single-molecule long-read sequencing

Chen

Fan

et al. 2019

Preprint

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Ascosphaera apis is a widespread fungal pathogen of honeybee larvae that results in chalkbrood disease, leading to heavy losses for the beekeeping industry in China and many other countries. This work was aimed at generating a full-length transcriptome of A. apis using PacBio single-molecule real-time (SMRT) sequencing. Here, more than 23.97 Gb of clean reads was generated from long-read sequencing of A. apis mecylia, including 464,043 circular consensus sequences (CCS) and 394,142 full-length non-chimeric (FLNC) reads. In total, we identified 174,095 high-confidence transcripts covering 5141 known genes with an average length of 2728 bp. We also discovered 2405 genic loci and 11,623 isoforms that have not been annotated yet within the current reference genome. Additionally, 16,049, 10,682, 4520 and 7253 of the discovered transcripts have annotations in the Non-redundant protein (Nr), Clusters of Eukaryotic Orthologous Groups (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, 1205 long non-coding RNAs (lncRNAs) were identified, which have less exons, shorter exon and intron lengths, shorter transcript lengths, lower GC percent, lower expression levels, and fewer alternative splicing (AS) evens, compared with protein-coding transcripts. A total of 253 members from 17 transcription factor (TF) families were identified from our transcript datasets. Finally, the expression of A. apis isoforms was validated using a molecular approach. Overall, this is the first report of a full-length transcriptome of entomogenous fungi including A. apis. Our data offer a comprehensive set of reference transcripts and hence contributes to improving the genome annotation and transcriptomic study of A. apis.

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Section: Discussionsupporting

confidence: 52%

“…TFs are key components involved in the transcriptional regulatory system in various animals, plants, and insects [12,30]. A total of 253 members from 17 TF families were identified from our transcript datasets.…”

Section: Resultsmentioning

confidence: 99%

Reconstruction and functional annotation of Ascosphaera apis full-length transcriptome via PacBio single-molecule long-read sequencing

Chen

Fan

et al. 2019

Preprint

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“…After comparing the identified PASs to the Ensembl reference annotation, we not only found that a large number of PASs are located in the intergenic regions, but also confirmed the existence of partial misannotated genes, which reflected the incompleteness of the current annotation. Single-molecule long-read sequencing technology is sufficient for the complete assembly of most known microbial genomes and has been applied to research studies of many species by now [26,27,30]. With the PacBio SMRT sequencing technology employment to whole-transcriptome profiling in rabbit, Chen et al obtained 36,186 high-confidence transcripts and detected 24,797 alternative splicing (AS) and 11,184 APA events, indicating a significant improvement in the current annotation of the rabbit [26].…”

Section: (C)mentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Deng

Zou

et al. 2020

BioMed Research International

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Many increasing documents have proved that alternative polyadenylation (APA) events with different polyadenylation sites (PAS) contribute to posttranscriptional regulation. However, little is known about the detailed molecular features of PASs and its role in porcine fast and slow skeletal muscles through microRNAs (miRNAs) and RNA binding proteins (RBPs). In this study, we combined single-molecule real-time sequencing and Illumina RNA-seq datasets to comprehensively analyze polyadenylation in pigs. We identified a total of 10,334 PASs, of which 8734 were characterized by reference genome annotation. 32.86% of PASassociated genes were determined to have more than one PAS. Further analysis demonstrated that tissue-specific PASs between fast and slow muscles were enriched in skeletal muscle development pathways. In addition, we obtained 1407 target genes regulated by APA events through potential binding 69 miRNAs and 28 RBPs in variable 3′ UTR regions and some are involved in myofiber transformation. Furthermore, the de novo motif search confirmed that the most common usage of canonical motif AAUAAA and three types of PASs may be related to the strength of motifs. In summary, our results provide a useful annotation of PASs for pig transcriptome and suggest that APA may serve as a role in fast and slow muscle development under the regulation of miRNAs and RBPs.

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“…One important reason behind the lack of functional perspective in splicingdedicated bioinformatics tools is the inability of RNAseq to correctly capture isoform expression 51 . Recently, third generation sequencing technologies have demonstrated their power in detecting full-length transcript [52][53][54][55][56] and identifying expressed isoforms. Options for quantification are found in the combination with short-reads 55 or the utilization of the newest high throughput instruments.…”

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confidence: 99%

tappAS: a comprehensive computational framework for the analysis of the functional impact of differential splicing

Fuente

Arzalluz-Luque

Tardáguila

et al. 2019

Preprint

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Traditionally, the functional analysis of gene expression data has used pathway and network enrichment algorithms. These methods are usually gene rather than transcript centric and hence fall short to unravel functional roles associated to posttranscriptional regulatory mechanisms such as Alternative Splicing (AS) and Alternative PolyAdenylation (APA), jointly referred here as Alternative Transcript Processing (AltTP). Moreover, short-read RNA-seq has serious limitations to resolve full-length transcripts, further complicating the study of isoform expression. Recent advances in long-read sequencing open exciting opportunities for studying isoform biology and function. However, there are no established bioinformatics methods for the functional analysis of isoform-resolved transcriptomics data to fully leverage these technological advances. Here we present a novel framework for Functional Iso-Transcriptomics analysis (FIT). This framework uses a rich isoform-level annotation database of functional domains, motifs and sites -both coding and noncoding-and introduces novel analysis methods to interrogate different aspects of the functional relevance of isoform complexity. The Functional Diversity Analysis (FDA) evaluates the variability at the inclusion/exclusion of functional domains across annotated transcripts of the same gene. Parameters can be set to evaluate if AltTP partially or fully disrupts functional elements. FDA is a measure of the potential of a multiple isoform transcriptome to have a functional impact. By combining these functional labels with expression data, the Differential Analysis Module evaluates the relative contribution of transcriptional (i.e. gene level) and post-transcriptional (i.e. transcript/protein levels) regulation on the biology of the system. Measures of inclusion of NLS, transmembrane domains or DNA binding motifs, for example.Some of these findings were experimentally validated by others and us.In summary, we propose a novel framework for the functional analysis of transcriptomes at isoform resolution. We anticipate the tappAS tool will be an important resource for the adoption of the Functional Iso-Transcriptomics analysis by functional genomics community.

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Analysis of transcripts and splice isoforms in red clover (Trifolium pratense L.) by single-molecule long-read sequencing

Cited by 88 publications

References 33 publications

Reconstruction and functional annotation of Ascosphaera apis full-length transcriptome via PacBio single-molecule long-read sequencing

Reconstruction and functional annotation of Ascosphaera apis full-length transcriptome via PacBio single-molecule long-read sequencing

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

tappAS: a comprehensive computational framework for the analysis of the functional impact of differential splicing

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