scholarly journals High Precision Detection of Rare Splice Isoforms Using Multiplexed Primer Extension Sequencing

2018 ◽  
Author(s):  
Hansen Xu ◽  
Benjamin J. Fair ◽  
Zach Dwyer ◽  
Michael Gildea ◽  
Jeffrey A. Pleiss
Keyword(s):  
Rna Sequencing ◽  
High Precision ◽  
Reverse Transcription ◽  
Mrna Splicing ◽  
Primer Extension ◽  
Splice Isoforms ◽  
Mrna Splice ◽  
Novel Approach ◽  
Areas Of Interest ◽  
Splice Junctions

ABSTRACTTargeted RNA-sequencing aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-sequencing experiments. Here we present a novel approach for targeted RNA-sequencing that uses complex pools of reverse transcription primers to enable sequencing enrichment at user-selected locations across the genome. We demonstrate this approach by targeting hundreds to thousands of pre-mRNA splice junctions, revealing high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.

scholarly journals Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Laura Schulz ◽  
Manuel Torres-Diz ◽  
Mariela Cortés-López ◽  
Katharina E. Hayer ◽  
Mukta Asnani ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Reverse Transcription ◽  
Lymphoblastic Leukemia ◽  
Mrna Splicing ◽  
Exon 2 ◽  
Long Read ◽  
Reporter Assays ◽  
Cryptic Intron

AbstractResistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While “exitrons” are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed “falsitrons,” that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.

scholarly journals Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects

2020 ◽  
Vol 6 (29) ◽  
pp. eaba1773 ◽  
Author(s):  
Sangsin Lee ◽  
Ning Ding ◽  
Yidi Sun ◽  
Tanglong Yuan ◽  
Jing Li ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Precision ◽  
Disease Modeling ◽  
Human Cells ◽  
Sequencing Analysis ◽  
Injection Method ◽  
Cell Embryo ◽  
Human Apobec3g ◽  
Target Effects

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-CC-3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

scholarly journals Detection of splice isoforms and rare intermediates using multiplexed primer extension sequencing

2018 ◽  
Vol 16 (1) ◽  
pp. 55-58 ◽  
Author(s):  
Hansen Xu ◽  
Benjamin J. Fair ◽  
Zachary W. Dwyer ◽  
Michael Gildea ◽  
Jeffrey A. Pleiss
Keyword(s):  
Primer Extension ◽  
Splice Isoforms

scholarly journals Real-time detection of mRNA splicing variants with specifically designed reverse-transcription loop-mediated isothermal amplification

RSC Advances ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 6271-6276
Author(s):  
Fengxia Su ◽  
Guanhao Wang ◽  
Jianing Ji ◽  
Pengbo Zhang ◽  
Fangfang Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Mrna Splicing ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Splicing Variant ◽  
Splicing Variants ◽  
Real Time Detection

A novel splicing variant assay is developed based on specifically designed reverse-transcription (RT) loop-mediated isothermal amplification.

scholarly journals A Computer-Aided Detection System for Digital Chest Radiographs

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Juan Manuel Carrillo-de-Gea ◽  
Ginés García-Mateos ◽  
José Luis Fernández-Alemán ◽  
José Luis Hernández-Hernández
Keyword(s):  
Template Matching ◽  
Detection System ◽  
Local Binary Patterns ◽  
Chest Radiographs ◽  
Computer Aided Detection ◽  
Success Rates ◽  
Novel Approach ◽  
Computer Aided ◽  
Digital Chest ◽  
Areas Of Interest

Computer-aided detection systems aim at the automatic detection of diseases using different medical imaging modalities. In this paper, a novel approach to detecting normality/pathology in digital chest radiographs is proposed. The problem tackled is complicated since it is not focused on particular diseases but anything that differs from what is considered as normality. First, the areas of interest of the chest are found using template matching on the images. Then, a texture descriptor called local binary patterns (LBP) is computed for those areas. After that, LBP histograms are applied in a classifier algorithm, which produces the final normality/pathology decision. Our experimental results show the feasibility of the proposal, with success rates above 87% in the best cases. Moreover, our technique is able to locate the possible areas of pathology in nonnormal radiographs. Strengths and limitations of the proposed approach are described in the Conclusions.

Evidence of the modulation of mRNA splicing fidelity in humans by oxidative stress and p53

Genome ◽  
2007 ◽  
Vol 50 (10) ◽  
pp. 946-953 ◽  
Author(s):  
Kim Disher ◽  
Adonis Skandalis
Keyword(s):  
Oxidative Stress ◽  
Alternative Splicing ◽  
Molecular Mechanisms ◽  
Splice Variants ◽  
Dna Lesions ◽  
Mrna Splicing ◽  
Aberrant Splice ◽  
Biological Phenomenon ◽  
Mrna Splice ◽  
Human Genes

The majority of human genes generate mRNA splice variants and while there is little doubt that alternative splicing is an important biological phenomenon, it has also become apparent that some splice variants are associated with disease. To elucidate the molecular mechanisms responsible for generating aberrant splice variants, we have investigated alternative splicing of the human genes HPRT and POLB following oxidative stress in different genetic backgrounds. Our study revealed that splicing fidelity is sensitive to oxidative stress. Following treatment of cells with H2O2, the overall frequency of aberrant, unproductive splice variants increased in both loci. At least in POLB, splicing fidelity is p53 dependent. In the absence of p53, the frequency of POLB splice variants is elevated but oxidative stress does not further increase the frequency of splice variants. Our data indicate that mis-splicing following oxidative stress represents a novel and significant genotoxic outcome and that it is not simply DNA lesions induced by oxidative stress that lead to mis-splicing but changes in the alternative splicing machinery itself.

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