Heidegger, A.Xavier, C.Niederstätter, H.Puente Vila, María del Carmen de laPospiech, E.Pisarek, A.Kayser, M.Branicki, W.Parson, W.2026-01-282026-01-282020-06-06A. Heidegger, C. Xavier, H. Niederstätter, M. de la Puente, E. Pośpiech, A. Pisarek, M. Kayser, W. Branicki, W. Parson, Development and optimization of the VISAGE basic prototype tool for forensic age estimation, Forensic Science International: Genetics, Volume 48, 2020, 102322, ISSN 1872-4973, https://doi.org/10.1016/j.fsigen.2020.102322https://hdl.handle.net/10347/45546The VISAGE (VISible Attributes through GEnomics) consortium aims to develop, optimize and validate prototype tools to broaden the use of DNA intelligence methods in forensic routine laboratories. This includes age estimation based on the quantification of DNA methylation at specific CpG sites. Here, we present the VISAGE basic prototype tool for age estimation targeting 32 CpGs from five genes ELOVL2, MIR29B2CHG (herein, MIR29B2C), FHL2, TRIM59 and KLF14. The assay interrogates these well described age markers by multiplex PCR for bisulfite converted DNA and massively parallel sequencing on a MiSeq FGx instrument. We describe protocol optimizations including tests on five bisulfite conversion kits and an evaluation of the assay’s reproducibility and sensitivity with artificially methylated DNA standards. We observed robust quantification of methylation levels with a mean standard deviation of 1.4 % across ratios. Sensitivity tests showed no increase of variability down to 20 ng DNA input into bisulfite conversion with a median difference below 1.6 % between technical replicateseng© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND licensehttp://creativecommons.org/licenses/by-nc-nd/4.0/Bisulfite PCR multiplex developmentTargeted bisulfite sequencingAge estimationMiSeq FGx sequencing3203 Medicina forensejournal article10.1016/j.fsigen.2020.1023221878-0326open access