1. MRSD: A quantitative approach for assessing suitability of RNA-seq in the investigation of mis-splicing in Mendelian disease.
- Author
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Rowlands CF, Taylor A, Rice G, Whiffin N, Hall HN, Newman WG, Black GCM, O'Keefe RT, Hubbard S, Douglas AGL, Baralle D, Briggs TA, and Ellingford JM
- Subjects
- B-Lymphocytes metabolism, B-Lymphocytes pathology, Blood Cells metabolism, Blood Cells pathology, Cell Line, Fibroblasts metabolism, Fibroblasts pathology, Genetic Diseases, Inborn classification, Genetic Diseases, Inborn metabolism, Genetic Diseases, Inborn pathology, Genetic Variation, Humans, Muscle, Skeletal metabolism, Muscle, Skeletal pathology, RNA, Messenger metabolism, Research Design, Exome Sequencing statistics & numerical data, Genetic Diseases, Inborn genetics, RNA Splicing, RNA, Messenger genetics, Sequence Analysis, RNA statistics & numerical data, Software
- Abstract
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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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