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1.
Nat Protoc ; 16(2): 1276-1296, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462443

RESUMO

RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects in individuals affected by genetically undiagnosed rare disorders. Pioneering studies have shown that RNA-seq could increase the diagnosis rates over DNA sequencing alone by 8-36%, depending on the disease entity and tissue probed. To accelerate adoption of RNA-seq by human genetics centers, detailed analysis protocols are now needed. We present a step-by-step protocol that details how to robustly detect aberrant expression levels, aberrant splicing and mono-allelic expression 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 the detection of RNA outliers pipeline (DROP), a modular computational workflow that integrates all the analysis steps, can leverage parallel computing infrastructures and generates browsable web page reports.


Assuntos
Sequência de Bases/genética , Expressão Gênica/genética , Análise de Sequência de RNA/métodos , Diagnóstico , Técnicas e Procedimentos Diagnósticos , Doença/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA/genética , Software , Fluxo de Trabalho
2.
Elife ; 82019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31025937

RESUMO

RNA splicing is an essential part of eukaryotic gene expression. Although the mechanism of splicing has been extensively studied in vitro, in vivo kinetics for the two-step splicing reaction remain poorly understood. Here, we combine transient transcriptome sequencing (TT-seq) and mathematical modeling to quantify RNA metabolic rates at donor and acceptor splice sites across the human genome. Splicing occurs in the range of minutes and is limited by the speed of RNA polymerase elongation. Splicing kinetics strongly depends on the position and nature of nucleotides flanking splice sites, and on structural interactions between unspliced RNA and small nuclear RNAs in spliceosomal intermediates. Finally, we introduce the 'yield' of splicing as the efficiency of converting unspliced to spliced RNA and show that it is highest for mRNAs and independent of splicing kinetics. These results lead to quantitative models describing how splicing rates and yield are encoded in the human genome.


Assuntos
Genoma Humano , Processamento de RNA , Perfilação da Expressão Gênica , Humanos , Células K562 , Cinética , Modelos Teóricos , Análise de Sequência de RNA , Spliceossomos/metabolismo
3.
PLoS One ; 13(7): e0199938, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29995917

RESUMO

The accurate quantification of cellular and mitochondrial bioenergetic activity is of great interest in medicine and biology. Mitochondrial stress tests performed with Seahorse Bioscience XF Analyzers allow the estimation of different bioenergetic measures by monitoring the oxygen consumption rates (OCR) of living cells in multi-well plates. However, studies of the statistical best practices for determining aggregated OCR measurements and comparisons have been lacking. Therefore, to understand how OCR behaves across different biological samples, wells, and plates, we performed mitochondrial stress tests in 126 96-well plates involving 203 fibroblast cell lines. We show that the noise of OCR is multiplicative, that outlier data points can concern individual measurements or all measurements of a well, and that the inter-plate variation is greater than the intra-plate variation. Based on these insights, we developed a novel statistical method, OCR-Stats, that: i) robustly estimates OCR levels modeling multiplicative noise and automatically identifying outlier data points and outlier wells; and ii) performs statistical testing between samples, taking into account the different magnitudes of the between- and within-plate variations. This led to a significant reduction of the coefficient of variation across plates of basal respiration by 45% and of maximal respiration by 29%. Moreover, using positive and negative controls, we show that our statistical test outperforms the existing methods, which suffer from an excess of either false positives (within-plate methods), or false negatives (between-plate methods). Altogether, this study provides statistical good practices to support experimentalists in designing, analyzing, testing, and reporting the results of mitochondrial stress tests using this high throughput platform.


Assuntos
Mitocôndrias/metabolismo , Análise Serial de Tecidos/métodos , Linhagem Celular , Respiração Celular , Metabolismo Energético , Fibroblastos/citologia , Modelos Estatísticos , Consumo de Oxigênio
4.
Transcription ; 8(2): 75-80, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-27841720

RESUMO

We give an overview of experimental and computational methods to estimate RNA metabolism rates genome-wide. We then advocate a local definition of RNA metabolism rate at the level of individual phosphodiester bonds. Rates of formation and disappearance of individual bonds are unambiguously defined, in contrast to rates of complete transcripts. We show that over previous approaches, the recently developed transient transcriptome sequencing (TT-seq) protocol allows for estimation of metabolism rates of individual bonds with least positional bias.


Assuntos
RNA/metabolismo , Éxons , Genoma , Humanos , Cinética , RNA/química , Processamento de RNA , Análise de Sequência de RNA , Transcriptoma
5.
Mol Syst Biol ; 12(2): 857, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26883383

RESUMO

To decrypt the regulatory code of the genome, sequence elements must be defined that determine the kinetics of RNA metabolism and thus gene expression. Here, we attempt such decryption in an eukaryotic model organism, the fission yeast S. pombe. We first derive an improved genome annotation that redefines borders of 36% of expressed mRNAs and adds 487 non-coding RNAs (ncRNAs). We then combine RNA labeling in vivo with mathematical modeling to obtain rates of RNA synthesis and degradation for 5,484 expressed RNAs and splicing rates for 4,958 introns. We identify functional sequence elements in DNA and RNA that control RNA metabolic rates and quantify the contributions of individual nucleotides to RNA synthesis, splicing, and degradation. Our approach reveals distinct kinetics of mRNA and ncRNA metabolism, separates antisense regulation by transcription interference from RNA interference, and provides a general tool for studying the regulatory code of genomes.


Assuntos
Regulação Fúngica da Expressão Gênica , Genoma Fúngico , RNA Fúngico/genética , RNA Mensageiro/genética , Schizosaccharomyces/genética , Íntrons , Interferência de RNA , Processamento de RNA , RNA Antissenso/genética , Análise de Sequência de RNA , Transcrição Genética
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