Deep RNA sequencing reveals a high frequency of alternative splicing events in the fungus Trichoderma longibrachiatum

AbstractBackground and ObjectivesCastor (Ricinus communis L.) is an important non-edible oilseed crop. Lm type female strains and normal amphiprotic strains are important castor cultivars, and are mainly different in inflorescence structures and leaf shapes. To better understand the mechanisums underling these differences at the molecular level, we performed comparative transcriptional analysis.Materials and MethodsFull-length transcriptome sequencing and short-read RNA sequencing were employed.ResultsA total of 76,068 and 44,223 non-redundant transcripts were obtained from high-quality transcripts of Lm type female strains and normal amphiprotic strains, respectively. In Lm female strain and normal amphiprotic strains 51,613 and 20,152 alternative splicing events were found, respectively. There were 13,239 transcription factors identified from the full-length transcriptomes. Comparative analysis showed great different gene expression of common and unique transcription factors between the two cultivars. Meanwhile, functional analysis of isoform was conducted. Full-length sequences were used as a reference genome, and short-read RNA sequencing analysis was performed to conduct differential gene analysis. Furthermore, the function of DEGs were performed to annotation analysis.ConclusionsThe results revealed considerable difference and expression diversity between two cultivars, well beyond what was reported in previous studies, likely reflecting the differences in architecture between these two cultivars.HighlightUsing the full-length transcriptome sequencing technology, we performed comparative analysis of transcription factors of two castor cultivars, analyzed alternative splicing events, and identified their lncRNAs.

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Identifying Differential Alternative Splicing Events from RNA Sequencing Data Using RNASeq-MATS

Methods in Molecular Biology - Deep Sequencing Data Analysis ◽

10.1007/978-1-62703-514-9_10 ◽

2013 ◽

pp. 171-179 ◽

Cited By ~ 31

Author(s):

Juw Won Park ◽

Collin Tokheim ◽

Shihao Shen ◽

Yi Xing

Keyword(s):

Alternative Splicing ◽

Rna Sequencing ◽

Sequencing Data ◽

Alternative Splicing Events

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Strand-Specific Total RNA Sequencing Establishes the Complete Transcriptome and Alternative Splicing Repertoire in Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood.v124.21.864.864 ◽

2014 ◽

Vol 124 (21) ◽

pp. 864-864

Author(s):

Adrienne Greenough ◽

Andrea Moffitt ◽

Dereje Jima ◽

Jane Healy ◽

Amee Patel ◽

...

Keyword(s):

Alternative Splicing ◽

B Cell ◽

Rna Sequencing ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Total Rna ◽

Large B Cell Lymphoma ◽

Non Coding Rnas ◽

Alternative Splicing Events ◽

Large B Cell

Abstract Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of lymphoma in adults. Gene expression profiling has demonstrated that DLBCL can be classified into two distinct subgroups – activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL. These subgroups arise through distinct normal cells of origin, activate different oncogenic pathways and display markedly different clinical outcomes. Deregulation of the transcriptome is believed to play a key role in the malignant transformation of B cells that culminates in the development of either ABC or GCB DLBCL. Here we describe global differences in RNA expression, mutation and splicing in relation to the pathogenesis of these subgroups of DLBCL. Methods: RNA sequencing (RNAseq) has emerged as a powerful tool for defining the cancer transcriptome. While mRNA sequencing is the most widely applied method for RNAseq, it overlooks non-coding RNAs, requires high-quality RNA and lacks strand-specificity. To overcome these limitations, we developed a method for strand-specific total RNA sequencing (ssRNAseq) to characterize the transcriptomes of 112 DLBCL tumors. Results: Through this work, we defined the entire spectrum of coding and non-coding RNAs expressed in DLBCLs including hundreds of lincRNAs, snoRNAs and microRNAs in addition to mRNAs. We found that the strand-specificity of our method was greater than 95% in all cases. This strand-specific sequencing strategy allowed us to maintain the orientation of the transcript to enable more accurate transcript annotation and better prediction of novel transcripts. Furthermore, we showed that our method had equal efficacy on frozen and FFPE tumor specimens from the sample patient in 24 cases. In addition, through simultaneous measurement of expression of diverse RNA types combined with mutations in MYD88, GNA13, EZH2, and BCL2, we demonstrated that we could distinguish the clinically important subgroups of DLBCL. Finally, we applied ssRNAseq to distinct training and validation sets of DLBCL cases (N=86 and N=112) to define alternative splicing events in DLBCL and found 1,021 genes that were preferentially spliced in a subgroup-specific manner. These alternatively spliced genes were selectively enriched in a number of different pathways important in lymphomas including those related to immune function, cell cycle progression and focal adhesion pathways, suggesting that alternative splicing regulates a number of important oncogenic processes in DLCBL. Conclusions: Strand-specific total RNA sequencing is a powerful method for defining the transcriptome and alternative splicing events in DLBCL. Here we define a complete coding and non-coding transcriptome of DLBCL and report the first characterization of subgroup-specific alternative splicing in DLBCL using high throughput sequencing. Our data demonstrate the power of our ssRNAseq method in defining the molecular patterns underlying DLBCLs and provide a starting point for defining the role of alternative splicing in this complex and heterogeneous disease. Disclosures Mann: Quiagen: Research Funding.

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Identification of alternative splicing events by RNA sequencing in early growth tomato fruits

BMC Genomics ◽

10.1186/s12864-015-2128-6 ◽

2015 ◽

Vol 16 (1) ◽

Cited By ~ 23

Author(s):

Yuan Sun ◽

Han Xiao

Keyword(s):

Alternative Splicing ◽

Rna Sequencing ◽

Early Growth ◽

Tomato Fruits ◽

Alternative Splicing Events

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Analysis of alternative splicing events by RNA sequencing in the ovaries of Xiang pig at estrous and diestrous

Theriogenology ◽

10.1016/j.theriogenology.2018.06.022 ◽

2018 ◽

Vol 119 ◽

pp. 60-68 ◽

Cited By ~ 4

Author(s):

Liang-Ting Tang ◽

Xue-Qin Ran ◽

Ning Mao ◽

Fu-Ping Zhang ◽

Xi Niu ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Sequencing ◽

Alternative Splicing Events

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Quick and easy: 1-day-protocol for detection of normal alternative splicing events and aberrant spliceogenic transcripts with deep RNA sequencing

10.22541/au.161574220.03730539/v1 ◽

2021 ◽

Author(s):

Vita Šetrajčič Dragoš ◽

Vida Stegel ◽

Ana Blatnik ◽

Gašper Klančar ◽

Mateja Krajc ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Sequencing ◽

Single Gene ◽

Stk11 Gene ◽

Hands On ◽

Nbn Gene ◽

Splicing Isoforms ◽

Alternative Splicing Events ◽

Aberrant Rna

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting all normal splicing isoforms of STK11 gene that were previously published. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetics variants. Due to the simplicity of our approach it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

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New Approach for Detection of Normal Alternative Splicing Events and Aberrant Spliceogenic Transcripts with Long-Range PCR and Deep RNA Sequencing

Biology ◽

10.3390/biology10080706 ◽

2021 ◽

Vol 10 (8) ◽

pp. 706

Author(s):

Vita Šetrajčič Dragoš ◽

Vida Stegel ◽

Ana Blatnik ◽

Gašper Klančar ◽

Mateja Krajc ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Sequencing ◽

Single Gene ◽

Stk11 Gene ◽

Uncertain Significance ◽

Nbn Gene ◽

Splicing Isoforms ◽

Alternative Splicing Events ◽

Long Range Pcr ◽

Aberrant Rna

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

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