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

Abstract

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.

Recommendations for clinical interpretation of variants found in non-coding regions of the genome

“…Strong weighting of PS3 for identified aberrant splicing should only be used when (1) expression profiles in the biosample used match those of the primary disease tissue of the candidate genes [ 62 ], and (2) the sequencing data generated is appropriate for the detection of aberrant splicing events (e.g. exceeds the 95% confidence interval recommendations from MRSD (minimum required sequencing depth) using appropriate parameters for the laboratory and bioinformatics approaches applied [ 63 ]). In addition, we recommend that BS3 is only used in the absence of aberrant splicing events when there is evidence that both alleles are being expressed (e.g.…”

Strategies to Uplift Novel Mendelian Gene Discovery for Improved Clinical Outcomes

“…Minimum Required Sequencing Depth (MRSD) informs biosample selection (whole blood, lymphoblastic cell lines, and skeletal muscle) by estimating the minimum sequencing depth required from RNA-sequencing to achieve desired coverage across a given gene or gene panel. The authors reported high precision, and their results suggest that lymphoblastic cell lines may be suitable for ∼70% of established disease gene panels (Rowlands et al, 2021).…”

Splicing in the Diagnosis of Rare Disease: Advances and Challenges

“…A study targeting genes well expressed in the chosen tissue will need fewer sequencing reads to survey splicing than a study with genes that are poorly expressed. A recent preprint by Rowlands et al presents a web tool 2 that can be used to calculate the minimum required sequencing depth to profile splicing based on the level of expression of genes of interest in three different CATs, which will assist in choosing the appropriate sequencing depth (and target tissue type) in study design ( Rowlands et al, 2021b ). An additional consideration here is that disruption to splicing often leads disruption of the transcript’s open reading frame and the introduction of a premature termination codon.…”