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

Am J Hum Genet. 2022 Feb 3;109(2):210-222. doi: 10.1016/j.ajhg.2021.12.014. Epub 2022 Jan 21.


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.

Keywords: Mendelian disease; RNA; RNA-seq; diagnostics; gene expression; splicing; transcriptomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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 / genetics*
  • Genetic Diseases, Inborn / metabolism
  • Genetic Diseases, Inborn / pathology
  • Genetic Variation
  • Humans
  • Muscle, Skeletal / metabolism
  • Muscle, Skeletal / pathology
  • RNA Splicing*
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism
  • Research Design
  • Sequence Analysis, RNA / statistics & numerical data*
  • Software*
  • Whole Exome Sequencing / statistics & numerical data


  • RNA, Messenger