# Isoform analysis of heterozygous putative splicing variants at the allele level using nanopore long-read sequencing

## TL;DR

This paper introduces a new method using nanopore sequencing to study how heterozygous splicing variants affect gene isoforms at the allele level, which is important for understanding rare diseases and personalized medicine.

## Contribution

The novel contribution is an allele-separative bioinformatics pipeline for analyzing splicing variants using nanopore long-read sequencing data.

## Key findings

- The pipeline successfully identified heterozygous splicing variants with significant isoform differences between alleles.
- A novel pathogenic splicing variant in PYGM was found to affect isoforms in a McArdle disease case.
- Nanopore sequencing proved effective for allele-level isoform analysis.

## Abstract

One of the challenges in clinical genetics for rare diseases and personalized medicine is evaluating isoform alterations arising from heterozygous putative splicing variants at the allele level. Our aim was to analyze these variants by dividing cDNA or direct RNA nanopore long reads into two alleles, referencing whole-genome sequencing data containing allele-informative single nucleotide variants and then comparing the allele-separated reads using Full-Length Alternative Isoform analysis of RNA (FLAIR), a previously published bioinformatics tool for isoform analysis. In this study, we developed an allele-separative bioinformatics pipeline and described its performance. We applied our pipeline to previously published nanopore direct RNA sequencing data, as well as 5’ cap-trapping full-length cDNA nanopore sequencing (CTR-seq) data from blood samples of three individuals. We successfully identified heterozygous splicing variants associated with significant isoform differences between alleles. Furthermore, we uncovered the effects of a novel pathogenic splicing variant in PYGM on isoforms in a compound-heterozygous case of McArdle disease using nanopore cDNA amplicon and targeted genomic sequencing. This study demonstrates the utility of nanopore long-read sequencing for isoform analysis at the allele level, providing a valuable approach to evaluating the direct consequences of heterozygous splicing variants in individuals.

The online version contains supplementary material available at 10.1038/s41598-025-14566-z.