Casanova Adán, LucíaMosquera Miguel, AnaGonzález Bao, JavierAmbroa Conde, AdriánRuiz Ramírez, JorgeCabrejas Olalla, AmaiaGonzález Martín, E.Freire Aradas, Ana MaríaRodríguez López, AmeliaPhillips, Christopher PaulLareu Huidobro, María VictoriaPuente Vila, María del Carmen de la2024-03-182024-03-182023Forensic Science International: Genetics, Volume 67, 2023, 1029371872-4973http://hdl.handle.net/10347/33244We have adapted an established Ampliseq microhaplotype panel for nanopore sequencing with the Oxford Nanopore Technologies (ONT) system, as a cost-effective and highly scalable solution for forensic genetics applications. For this purpose, we designed a protocol combining direct PCR amplification from unextracted DNA with ONT library construction and sequencing using the MinION device and workflow. The analysis of reference samples at input amounts of 5–10 ng of DNA demonstrates stable coverage patterns, allele balance, and strand bias, reaching profile completeness and concordance rates of ∼95%. Similar levels were achieved when using direct-PCR from blood, buccal and semen swabs. Dilution series results indicate sensitivity is maintained down to 250 pg of input DNA, and informative profiles are produced down to 62.5 pg. Finally, we demonstrated the forensic utility of the nanopore workflow by analyzing two third degree pedigrees that showed low likelihood ratio values after the analysis of an extended panel of 38 STRs, achieving likelihood ratios 2–3 orders of magnitude higher when testing with the MinION–based haplotype dataeng© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)Atribución 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/Nanopore sequencingMinIONMicrohaplotypesKinship testingDirect PCRField laboratoriesjournal article10.1016/j.fsigen.2023.102937open access