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

Background: 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 RNAsequencing. This overcomes limitations of using expression values alone as a metric to predict RNA-sequencing utility.
Results: 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 are the optimum source of RNA, of these four biosamples, 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.
Conclusions: We demonstrate the utility and benefits of MRSD as a metric to informfunctional assessment of splicing aberrations, in particular in the context of Mendelian genetic disorders to improve diagnostic yield.