Cost-efficient long-read trio-barcoded adaptive sequencing improves rare disease diagnosis

Abstract Rare diseases often remain unsolved because causal genetic changes can be complex and thus missed by standard sequencing or difficult to prioritize. Long-read sequencing can reveal structural variants, repeat expansions, DNA methylation and inherited haplotypes, but trio sequencing of an affected child and both parents remains costly. Here we show that phenotype-driven Trio-barcoded Oxford Nanopore Adaptive Sequencing (TBAS) enables cost-efficient long-read analysis of rare-disease trios on one flow cell. TBAS workflow uses clinical features to select broad disease-gene panels, barcodes all three family members and enriches these regions during sequencing rather than targeting a known causal locus. In benchmark regions, TBAS increased coverage and accurately detected small variants, structural variants, tandem repeat expansions, methylation and read-backed phasing, while reducing estimated sequencing consumable costs to 32.2% of conventional three-flow-cell trio long-read sequencing. Across 13 trios, TBAS recovered all five known diagnoses and prioritized candidates in five of eight unresolved cases.