October 2025
Authors:
Yilei Fu, Adam C. English, Luis F. Paulin, Shalini N Jhangiani, George Weissenberger, Vanessa Vee, Yi Han, Heer H. Mehta, Donna M. Muzny, Richard A. Gibbs, Jennifer E. Posey, Daniel G. Calame, Fritz J. Sedlazeck
Abstract:
We present Trio-barcoded ONT Adaptive Sampling (TBAS), a cost-efficient long-read sequencing strategy combining sample barcoding and adaptive enrichment to sequence rare disease trios on a single PromethION flow cell. TBAS achieved near-complete variant phasing and detection of small variants, structural variants, and tandem repeats with high accuracy and 77% potential solve rate. This scalable approach retains methylation data and enables clinically relevant, phenotype-guided long-read diagnostics at a fraction of current costs.
Sage Science Products:
PippinHT High-Pass size selection (>6kb) for ONT library prep.
Methods Excerpt:
“Genomic DNA was diluted to 30 ng/µl. Starting with 1500 ng for each sample, DNA was shared using g-tubes (Covaris 520079) to achieve an average size of ~10 kb. The sheared DNA was size-selected on the PippinHT instrument (Sage Science) using the 6-10 kb High-Pass definition with a minimum size selection threshold of 6 kb. Libraries for ONT were prepared using the Native Barcoding Kit 96 V14 (SQK-NBD114.96) wherein each trio was barcoded and pooled into 1 library. Libraries were sequenced on the ONT PromethION 24 device using R10.4.1 flow cells with the adaptive sampling option enabled.”
Author Affiliations:
Human Genome Sequencing Center, Baylor College of Medicine
Department of Molecular and Human Genetics, Baylor College of Medicine
Departments of Pediatrics and Medicine, Columbia University Vagelos College of Physicians and Surgeons
Section of Pediatric Neurology and Developmental Neurosciences, Department of Pediatrics, Baylor College of Medicine
Texas Children’s Hospital
Department of Computer Science, Rice University
medRxiv preprint
DOI: 10.1101/2025.10.21.25338483