Single-cell genomics to understand disease pathogenesis

Nomura, Seitaro

doi:10.1038/s10038-020-00844-3

Cited by 37 publications

(19 citation statements)

References 113 publications

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“…However, our data suggest that the MRSD model may be readily applicable to RNA-seq generated using alternative methodologies, such as increased read length, with only minor variations in model performance (Figure S6). As other technologies, such as long-read, [40][41][42] single-cell, 43,44 and spatially resolved RNA-seq, [45][46][47][48] become more prevalent in a clinical setting,…”

Section: Figure 4 Expanding Control Datasets and Enforcing Read Count Thresholds Improves Filtering Power When Analyzing Mis-splicing Evementioning

confidence: 99%

MRSD: A quantitative approach for assessing suitability of RNA-seq in the investigation of mis-splicing in Mendelian disease

Rowlands

Taylor

Rice

et al. 2022

The American Journal of Human Genetics

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Variable levels of gene expression between tissues complicates the use of RNA sequencing of patient biosamples to delineate the impact of genomic variants. Here, we describe a gene-and tissue-specific metric to inform the feasibility of RNA sequencing. This overcomes limitations of using expression values alone as a metric to predict RNA-sequencing utility. We have derived a metric, minimum required sequencing depth (MRSD), that estimates the depth of sequencing required from RNA sequencing to achieve user-specified sequencing coverage of a gene, transcript, or group of genes. We applied MRSD across four human biosamples: whole blood, lymphoblastoid cell lines (LCLs), skeletal muscle, and cultured fibroblasts. MRSD has high precision (90.1%-98.2%) and overcomes transcript region-specific sequencing biases. Applying MRSD scoring to established disease gene panels shows that fibroblasts, of these four biosamples, are the optimum source of RNA for 63.1% of gene panels. Using this approach, up to 67.8% of the variants of uncertain significance in ClinVar that are predicted to impact splicing could be assayed by RNA sequencing in at least one of the biosamples. We demonstrate the utility and benefits of MRSD as a metric to inform functional assessment of splicing aberrations, in particular in the context of Mendelian genetic disorders to improve diagnostic yield.

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Section: Figure 4 Expanding Control Datasets and Enforcing Read Count Thresholds Improves Filtering Power When Analyzing Mis-splicing Evementioning

confidence: 99%

MRSD: A quantitative approach for assessing suitability of RNA-seq in the investigation of mis-splicing in Mendelian disease

Rowlands

Taylor

Rice

et al. 2022

The American Journal of Human Genetics

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“…This experimental RNAseq approach currently remains widespread (3,10,(13)(14)(15); however, our model may be readily applicable to RNA-seq generated using alternative methodologies, such as increased read length, with only minor variations in model performance ( Supplementary Figure 3). As other technologies, such as long-read (36)(37)(38), singlecell (39,40) and spatially resolved RNA-seq (41)(42)(43)(44), become more prevalent in a clinical setting, appropriate control datasets must be generated to develop corresponding MRSD models. Similarly, recent research has shown noticeable improvements to diagnostic yield for neuromuscular disorders by conducting RNAseq on in vitro myofibrils generated by a fibroblast-to-myofibril transdifferentiation protocol (45).…”

Section: Discussionmentioning

confidence: 99%

MRSD: a novel quantitative approach for assessing suitability of RNA-seq in the clinical investigation of mis-splicing in Mendelian disease

Rowlands

Taylor

Rice

et al. 2021

Preprint

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BackgroundRNA-sequencing of patient biosamples is a promising approach to delineate the impact of genomic variants on splicing, but variable gene expression between tissues complicates selection of appropriate tissues. Relative expression level is often used as a metric to predict RNA-sequencing utility. Here, we describe a gene- and tissue-specific metric to inform the feasibility of RNA-sequencing, overcoming some issues with using expression values alone.ResultsWe derive a novel metric, Minimum Required Sequencing Depth (MRSD), for all genes across three human biosamples (whole blood, lymphoblastoid cell lines (LCLs) and skeletal muscle). MRSD estimates the depth of sequencing required from RNA-sequencing to achieve user-specified sequencing coverage of a gene, transcript or group of genes of interest. MRSD predicts levels of splice junction coverage with high precision (90.1-98.2%) and overcomes transcript region-specific sequencing biases. Applying MRSD scoring to established disease gene panels shows that LCLs are the optimum source of RNA, of the three investigated biosamples, for 69.3% of gene panels. Our approach demonstrates that up to 59.4% of variants of uncertain significance in ClinVar predicted to impact splicing could be functionally assayed by RNA-sequencing in at least one of the investigated biosamples.ConclusionsWe demonstrate the power of MRSD as a metric to inform choice of appropriate biosamples for the functional assessment of splicing aberrations. We apply MRSD in the context of Mendelian genetic disorders and illustrate its benefits over expression-based approaches. We anticipate that the integration of MRSD into clinical pipelines will improve variant interpretation and, ultimately, diagnostic yield.

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“…In terms of budget, current advances of protocols as well as standardization of bioinformatics pipelines warrants the SCS approach as a realistic option for clinical applications in the near future. Importantly, conventional single-cell RNA-seq analysis may not be sufficient for obtaining the information necessary for a deeper understanding of molecular behavior and therefore combined bulk sequencing analysis will be mandatory for a more precise analysis (66).…”

Section: Real-time Single Cell Transcriptome Profile Of the Heart The...mentioning

confidence: 99%

Tailoring Cardiac Synthetic Transcriptional Modulation Towards Precision Medicine

Schoger

Panàkovà

Zelarayán

2022

Front. Cardiovasc. Med.

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Molecular and genetic differences between individual cells within tissues underlie cellular heterogeneities defining organ physiology and function in homeostasis as well as in disease states. Transcriptional control of endogenous gene expression has been intensively studied for decades. Thanks to a fast-developing field of single cell genomics, we are facing an unprecedented leap in information available pertaining organ biology offering a comprehensive overview. The single-cell technologies that arose aided in resolving the precise cellular composition of many organ systems in the past years. Importantly, when applied to diseased tissues, the novel approaches have been immensely improving our understanding of the underlying pathophysiology of common human diseases. With this information, precise prediction of regulatory elements controlling gene expression upon perturbations in a given cell type or a specific context will be realistic. Simultaneously, the technological advances in CRISPR-mediated regulation of gene transcription as well as their application in the context of epigenome modulation, have opened up novel avenues for targeted therapy and personalized medicine. Here, we discuss the fast-paced advancements during the recent years and the applications thereof in the context of cardiac biology and common cardiac disease. The combination of single cell technologies and the deep knowledge of fundamental biology of the diseased heart together with the CRISPR-mediated modulation of gene regulatory networks will be instrumental in tailoring the right strategies for personalized and precision medicine in the near future. In this review, we provide a brief overview of how single cell transcriptomics has advanced our knowledge and paved the way for emerging CRISPR/Cas9-technologies in clinical applications in cardiac biomedicine.

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Single-cell genomics to understand disease pathogenesis

Cited by 37 publications

References 113 publications

MRSD: A quantitative approach for assessing suitability of RNA-seq in the investigation of mis-splicing in Mendelian disease

MRSD: A quantitative approach for assessing suitability of RNA-seq in the investigation of mis-splicing in Mendelian disease

MRSD: a novel quantitative approach for assessing suitability of RNA-seq in the clinical investigation of mis-splicing in Mendelian disease

Tailoring Cardiac Synthetic Transcriptional Modulation Towards Precision Medicine

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