Cost-efficient Long-Read Trio-Barcoded Adaptive Sequencing Improves Rare Disease Diagnosis

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. Here the authors present Phenotype-driven Trio-barcoded Adaptive Sequencing (TBAS) that enables cost-efficient long-read sequencing of rare-disease trios on a single flow cell, detecting diverse genetic variants while cutting costs to 32% of standard trio sequencing.