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

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.

RNA-sequencing improves diagnosis for neurodevelopmental disorders by identifying pathogenic non-coding variants and reinterpretation of coding variants

“…We found that, when using fibroblasts as a source of RNA instead of whole blood RNA, RNA and data quality was higher, based on our control parameters, and more genes known to be involved in NDD could be detected. This matches a recent study showing that fibroblasts have broader gene expression than whole blood, potentially allowing identification of mis-splicing events in more individuals (64).…”

Clinical implementation of RNA sequencing for Mendelian disease diagnostics

“…Three samples had a high number of splicing outlier genes, out of which two corresponded to the ones with the highest sequencing depth. This indicates that sensitivity to splicing outliers could increase with deeper sequencing, which is in line with analyses in simulated and real datasets [ 29 , 90 ]. Power analysis for FRASER has been described in the original publication on suprapubic skin tissue from the GTEx dataset, which has a similar sequencing depth to this study’s dataset.…”

Targeted RNAseq Improves Clinical Diagnosis of Very Early-Onset Pediatric Immune Dysregulation

“…In order to determine if the coverage extended across the entire gene, however, it is important to look at splice junction coverage. A recent paper developed a calculation for the minimum read sequencing depth (MRSD) needed to express a given gene or genes at a level sufficient for splicing [ 63 ]. Because we did not have a PBMC whole mRNA control dataset for direct comparison of MRSD with our targeted panel, we used the MRSD web tool and combined the results for whole blood and lymphoblastoid cell lines (LCLs), acknowledging that a handful of genes were likely to be specific to a given biotype.…”

“…This generally leads to arguments that the disease-relevant tissue is a necessity for RNAseq and/or that sequencing depth should be at least 50–100 M reads per sample [ 5 , 38 , 39 ]. The recent minimum read sequencing depth (MRSD) study identified whole blood (over LCL, cultured fibroblasts, and skeletal muscle) as the worst option for most gene panels [ 63 ]. However, we show that our targeted gene panel outperformed expectations and allowed us to analyze at least 20% more genes of interest than we would have been able to with whole mRNA.…”