Duintjer, Annelotte JImholz, SandraPico-Knijnenburg, IngridHeuperman, AdindaHodemaekers, HennieDeutekom, Eva SVoorhoeve, ElsDollé, Martijn ETvan der Burg, Mirjam2025-10-012025-10-012025-09-034098130510.3390/ijns11030075https://rivm.openrepository.com/handle/10029/655698Due to rapid technical advancements and increasing cost-effectiveness, the potential application of next-generation sequencing (NGS) in newborn screening (NBS) has raised great interest worldwide. Genomic NBS offers the possibility to improve current NBS programs when applied as follow-up tier, and, as first-tier, allows for inclusion of conditions lacking a detectable biomarker for conventional NBS. Obtaining enough high-quality DNA from typically limited dried blood spot (DBS) material to meet NGS requirements can be challenging. Selecting a DNA isolation method for genomic NBS requires balancing technical performance and laboratory feasibility with optimal cost-effectiveness. Ten DNA isolation protocols, including two column-based, five lysis-based, and three semi-automated magnetic bead-based protocols, were evaluated on technical outcomes and performance in targeted amplicon sequencing. Additionally, estimated costs, hands-on time, turnaround time, scalability, and plastic footprint were assessed. Although technical outcomes, including yield, purity, and molecular weight, differed between methods, qualitative results in amplicon sequencing, as defined by read output, mapping, and coverage depth, were found sufficient and comparable for various protocols. In conclusion, both technical requirements and operational parameters are crucial when selecting a DNA isolation protocol and will depend on the NGS application as well as the NBS approach, as either first-tier or follow-up tier.enDBSDNA isolationDNA preparationNBSNGSdried blood spotgenomic newborn screeningnewborn screeningnext-generation sequencingJournal ArticleInt J Neonatal Screen 2025; 11(3):75ijns11030075