scholarly journals TRAWLING: a Transcriptome Reference Aware of spLIciNG events

2021 ◽  
Author(s):  
Noemi Di Nanni ◽  
Alejandro Reyes ◽  
Daniel Ho ◽  
Robert Ihry ◽  
Audrey Kauffmann ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Gene Expression Regulation ◽  
Comprehensive Evaluation ◽  
Transcript Level ◽  
Aberrant Splicing ◽  
Rnaseq Data ◽  
Powerful Approach ◽  
Whole Transcriptome Sequencing ◽  
Whole Transcriptome

AbstractAlternative splicing is critical for human gene expression regulation and plays an important role in multiple human diseases. In this context, RNA sequencing has emerged as powerful approach to detect alternative splicing events.In parallel, fast alignment-free methods have emerged as a viable alternative to quantify gene and transcript level abundance from RNAseq data. However, the ability to detect differential splicing events is dependent on the annotation of the transcript reference provided by the user.Here, we introduce a new reference transcriptome aware of splicing events, TRAWLING, which simplifies the detection of aberrant splicing events in a fast and simple way. In addition, we evaluate the performances and the benefits of aligning transcriptome data to TRAWLING using three different RNA sequencing datasets: whole transcriptome sequencing, single cell RNA sequencing and Digital RNA with pertUrbation of Genes.Collectively, our comprehensive evaluation underlines the value of using TRAWLING in transcriptomic data analysis.Availability and implementationOur code is available at https://github.com/Novartis/TRAWLING

scholarly journals Direct nanopore sequencing of mRNA reveals landscape of transcript isoforms in apicomplexan parasites

2020 ◽  
Author(s):  
V Vern Lee ◽  
Louise M. Judd ◽  
Aaron R. Jex ◽  
Kathryn E. Holt ◽  
Christopher J. Tonkin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Intron Retention ◽  
Transcript Level ◽  
Full Length ◽  
Sequencing Platform ◽  
Apicomplexan Parasites ◽  
Oxford Nanopore ◽  
Long Read ◽  
Widespread Phenomenon

AbstractAlternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterised in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AT content of Plasmodium RNA, but also the limitations of short read sequencing in deciphering complex splicing events. In this study, we utilised the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies (ONT) to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum. We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or near full-length transcripts with comparable quantification to Illumina sequencing. By comparing this data with available gene models, we find widespread alternative splicing, particular intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This work highlights a strategy in using long read sequencing to understand splicing events at the whole transcript level, and has implications in future interpretation of RNA-seq studies.

Whole Transcriptome Sequencing in SLE: Deep Sequencing of RNA from SLE Blood Reveals Interferon-regulated Alternative Splicing Events

2010 ◽  
Vol 135 ◽  
pp. S9
Author(s):  
Donna Thibault Flesher ◽  
Christina Chaivorapol ◽  
Kristen Wolslegel ◽  
Alexander Abbas ◽  
Stephen Kingsmore ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Deep Sequencing ◽  
Transcriptome Sequencing ◽  
Whole Transcriptome Sequencing ◽  
Alternative Splicing Events ◽  
Whole Transcriptome

scholarly journals Detection of aberrant events in RNA sequencing data

2020 ◽  
Author(s):  
Vicente A. Yépez ◽  
Christian Mertes ◽  
Michaela F. Mueller ◽  
Daniela S. Andrade ◽  
Leonhard Wachutka ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rare Allele ◽  
Rna Seq ◽  
Sequencing Data ◽  
Aberrant Splicing ◽  
Aberrant Expression ◽  
Powerful Approach ◽  
Disease Entities ◽  
Computational Workflow ◽  
Assess Quality

Abstract RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects for individuals affected with a genetically undiagnosed rare disorder. Pioneer studies have shown that RNA-seq could increase diagnostic rates over DNA sequencing alone by 8% to 36 % depending on disease entities and probed tissues. To accelerate the adoption of RNA-seq among human genetic centers, detailed analysis protocols are now needed. Here, we present a step-by-step protocol that instructs how to robustly detect aberrant expression, aberrant splicing, and mono-allelic expression of a rare allele in RNA-seq data using dedicated statistical methods. We describe how to generate and assess quality control plots and interpret the analysis results. The protocol is based on DROP (Detection of RNA Outliers Pipeline), a modular computational workflow that integrates all analysis steps, can leverage parallel computing infrastructures, and generates browsable web page reports.

scholarly journals Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
V. Vern Lee ◽  
Louise M. Judd ◽  
Aaron R. Jex ◽  
Kathryn E. Holt ◽  
Christopher J. Tonkin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Intron Retention ◽  
Transcript Level ◽  
Full Length ◽  
Nanopore Sequencing ◽  
Content Type ◽  
Sequencing Platform ◽  
Sequencing Studies ◽  
Long Read

ABSTRACT Alternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterized in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AU content of Plasmodium RNA, but also the limitations of short-read sequencing in deciphering complex splicing events. In this study, we utilized the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum. We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or nearly full-length transcripts with comparable quantification to Illumina sequencing. By comparing these data with available gene models, we find widespread alternative splicing, particularly intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This study highlights a strategy in using long-read sequencing to understand splicing events at the whole-transcript level and has implications in the future interpretation of transcriptome sequencing studies. IMPORTANCE We have used a novel nanopore sequencing technology to directly analyze parasite transcriptomes. The very long reads of this technology reveal the full-length genes of the parasites that cause malaria and toxoplasmosis. Gene transcripts must be processed in a process called splicing before they can be translated to protein. Our analysis reveals that these parasites very frequently only partially process their gene products, in a manner that departs dramatically from their human hosts.

Mammary-type Myofibroblastoma with Leiomyomatous Differentiation: A Rare Variant with Potential Pitfalls

2021 ◽  
pp. 106689692110313
Author(s):  
Alexander M. Strait ◽  
Julia A. Bridge ◽  
Anthony J. Iafrate ◽  
Marilyn M. Li ◽  
Feng Xu ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Transcriptome Sequencing ◽  
Smooth Muscle Actin ◽  
The Body ◽  
Muscle Actin ◽  
Mature Adipose Tissue ◽  
Spindle Cells ◽  
Whole Transcriptome Sequencing ◽  
Whole Transcriptome

Myofibroblastoma is a rare, benign stromal tumor with a diverse morphologic spectrum. Mammary-type myofibroblastoma (MTMF) is the extra-mammary counterpart of this neoplasm and its occurrence throughout the body has become increasingly recognized. Similar morphologic variations of MTMF have now been described which mirror those seen in the breast. We describe a case of intra-abdominal MTMF composed of short fascicles of eosinophilic spindle cells admixed with mature adipose tissue. The spindle cells stained diffusely positive for CD34, desmin, smooth muscle actin, and h-caldesmon by immunohistochemistry. Concurrent loss of RB1 (13q14) and 13q34 loci were confirmed by fluorescence in situ hybridization whereas anchored multiplex PCR and whole transcriptome sequencing did not reveal any pathognomonic fusions suggesting an alternative diagnosis. To the best of our knowledge this is the first documented case of leiomyomatous variant of MTMF.

scholarly journals Splicing Genomics Events in Cervical Cancer: Insights for Phenotypic Stratification and Biomarker Potency

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 130
Author(s):  
Flavia Zita Francies ◽  
Sheynaz Bassa ◽  
Aristotelis Chatziioannou ◽  
Andreas Martin Kaufmann ◽  
Zodwa Dlamini
Keyword(s):  
Cervical Cancer ◽  
Alternative Splicing ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Therapy Resistance ◽  
Splicing Factors ◽  
Aberrant Splicing ◽  
Molecular Alteration ◽  
Common Cancer ◽  
Potential Biomarkers

Gynaecological cancers are attributed to the second most diagnosed cancers in women after breast cancer. On a global scale, cervical cancer is the fourth most common cancer and the most common cancer in developing countries with rapidly increasing mortality rates. Human papillomavirus (HPV) infection is a major contributor to the disease. HPV infections cause prominent cellular changes including alternative splicing to drive malignant transformation. A fundamental characteristic attributed to cancer is the dysregulation of cellular transcription. Alternative splicing is regulated by several splicing factors and molecular changes in these factors lead to cancer mechanisms such as tumour development and progression and drug resistance. The serine/arginine-rich (SR) proteins and heterogeneous ribonucleoproteins (hnRNPs) have prominent roles in modulating alternative splicing. Evidence shows molecular alteration and expression levels in these splicing factors in cervical cancer. Furthermore, aberrant splicing events in cancer-related genes lead to chemo- and radioresistance. Identifying clinically relevant modifications in alternative splicing events and splicing variants, in cervical cancer, as potential biomarkers for their role in cancer progression and therapy resistance is scrutinised. This review will focus on the molecular mechanisms underlying the aberrant splicing events in cervical cancer that may serve as potential biomarkers for diagnosis, prognosis, and novel drug targets.

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