Multisite Evaluation of an Amplification-based Nanopore Sequencing Solution to Analyze Challenging Clinically Relevant Variants in Genes Associated with Hereditary Diseases
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Purpose
Carrier screening for hereditary conditions is challenged by genes with complex genomic architecture, where short-read sequencing can fail to detect clinically relevant variants. This study evaluated a unified, amplification-based nanopore sequencing workflow across multiple laboratories for comprehensive analysis of such loci.
Methods
A modular long-read sequencing assay was evaluated across five laboratories using targeted PCR enrichment, Oxford Nanopore sequencing, and automated variant analysis. The workflow interrogated genes associated with spinal muscular atrophy, thalassemia, cystic fibrosis, fragile X syndrome, congenital adrenal hyperplasia, Gaucher disease, and hemophilia A. Performance was assessed against orthogonal methods for single nucleotide variants (SNVs), indels, copy-number variants, repeat expansions, and structural rearrangements.
Results
Across 882 unique samples (1,266 tests), overall agreement with comparator methods exceeded 96% for variant-level detection and 97% for genotype status classification. Long-read sequencing enabled phasing of paralogous loci, integrated sizing and interruption analysis for FMR1 repeats, and simultaneous detection of SNVs and structural variants in globin loci and CYP21A2 - TNXB region, reducing reliance on multiple workflows.
Conclusion
This multisite evaluation suggests that targeted long-read sequencing can consolidate complex variant detection into a single workflow, improving analytical completeness and operational efficiency for carrier screening.
