New Approach for Detection of Normal Alternative Splicing Events and Aberrant Spliceogenic Transcripts with Long-Range PCR and Deep RNA Sequencing

Abstract

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

Alternative Splicing, RNA Editing, and the Current Limits of Next Generation Sequencing

“…In addition, validation and cloning of any alternate spliced forms identified can be undertaken with RNA isolation, gene-specific amplification of transcripts, and RNA-seq together with shotgun cloning and DNA-seq, giving a representation of all transcripts present that are related to a particular gene. Of course, this works for when the expression of only a handful of genes is of interest [38]. When it comes to identifying novel splice variants from RNAseq on a larger scale, the past several years have seen a vast increase in the number of algorithms to detect novel alternately spliced transcripts.…”

Identification of Spliceogenic Variants beyond Canonical GT-AG Splice Sites in Hereditary Cancer Genes

“…RNAseq was performed using an in-house developed approach as previously described [8]. In short, primer pairs aligning to the 5 and 3 UTR regions of gene of interest were designed.…”

“…Patients harboring VUS with predicted impact on splicing were eligible for additional RNA analysis. Using our approach previously published [8] using long-range PCR and deep sequencing, we have functionally characterized 12 variants positioned outside of GT-AG splicing motif that were predicted to cause splicing abnormalities in APC, ATM, FH, LZTR1, MSH6, PALB2, RAD51C, and TP53 genes. Furthermore, the study aimed to explore the impact of RNA analysis on variant classification.…”