PseudoRA: A Novel Pseudogene ReAligner for Detecting SBDS Variants in Shwachman-Diamond Syndrome.

Accurate detection of pathogenic variants in genes with highly homologous pseudogenes remains challenging in next-generation sequencing (NGS). SBDS, the causative gene for Shwachman-Diamond syndrome (SDS), is challenging to analyze because of interference from its pseudogene, SBDSP1, causing frequent read misalignment and false-negative variant calls, especially for low-variant-allele-frequency (VAF) variants such as c.183_184delinsCT. We developed Pseudogene ReAligner (PseudoRA), a novel bioinformatics algorithm that resolves gene-pseudogene misalignment through read realignment and haplotype-based reassignment. PseudoRA was retrospectively applied to targeted NGS binary alignment map files from 846 patients with suspected hematological disorders, and its performance was compared with that of conventional analysis, HapICE, and Sanger sequencing. Conventional NGS identified SBDS variants in six patients (0.7%), whereas PseudoRA detected variants in 10 patients (1.2%), representing a 1.7-fold increase in diagnostic yield. PseudoRA identified seven additional cases, including low-VAF c.183_184delinsCT variants that had been missed or misclassified when assessed using conventional analysis. All variants detected via PseudoRA showed 100% concordance with Sanger sequencing. PseudoRA demonstrated comparable sensitivity to HapICE but enabled broader detection across multiple exons. PseudoRA improves the molecular diagnosis of SDS by recovering mismapped low-VAF variants and offers a scalable solution that can be integrated into conventional clinical pipelines.