Feasibility of Oxford Nanopore sequencing for newborn screening using dried blood spot cards | LC26

Abstract
In Connecticut, newborn screening for approximately 70 conditions is performed using biochemical approaches from dried blood spot (DBS) cards. Advances in long-read (LR) sequencing offer advantages over short-read (SR) technologies for newborn whole-genome sequencing and early identification of actionable genetic conditions. However, integration of efficient sample collection and LR sequencing into clinical workflows presents a barrier to widespread adoption. We evaluated DNA extraction and sequencing strategies for 10 Neonatal Intensive Care Unit (NICU) newborns, comparing fresh and frozen DBS cards with venous blood (VB). Samples were sequenced using Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) platforms, with Illumina SR sequencing for comparison. Data was aligned to the human reference genome CHM13 to assess the quality and yield of data, chromosome coverage, unmapped regions, and platform-specific bias. Across extraction methods, ONT sequencing without amplification from frozen DBS card spots yielded exceptional results, achieving N50s up to 32 kb and outputs up to 53 Gb from a single flow cell load. ONT LR sequencing demonstrated equal or better chromosome coverage and significantly better N50s relative to PacBio. Targeting ~460 medically actionable genes associated with symptoms of disease by age five, we find ample coverage from ONT LR to accurately score variants and validate ONT-derived methylation calls across select imprinted loci and regions linked to non-imprinting disorders. These results demonstrate that frozen DBS cards yield sufficient high molecular-weight DNA for a single ONT flow cell (24 h run) with sequencing results comparable to fresh VB, offering promise for rapid implementation of newborn genomic screening using LR sequencing technologies.

Biography
Gabrielle Hartley (Gabby) earned a B.S. in Forensic Science (2017) and a B.S. in Biology (2017) from the University of New Haven and a certificate in Nonprofit Management (2020) and PhD in Molecular and Cell Biology (2023) from the University of Connecticut (UConn). She contributes to the Telomere-to-Telomere (T2T) Consortium (CHM13, Primates, and Ruminants), UConn’s partnership with Colossal Biosciences, and ancient/museum sample processing in varying collaborations with the state archaeologist. Her PhD work explored the mechanisms of rapid karyotype evolution in gibbons.