Development and optimization of the VISAGE basic prototype tool for forensic age estimation

•Assay development for chronological age estimation based on quantitative DNA methylation analysis.•A bisulfite multiplex PCR was optimized to analyze 32 age informative CpGs at ELOVL2, MIR29B2C, FHL2, TRIM59 and KLF14.•Targeted bisulfite sequencing was performed using the MiSeq FGx platform.•Reprod...

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Bibliographic Details
Published in:Forensic science international : genetics 2020-09, Vol.48, p.102322-102322, Article 102322
Main Authors: Heidegger, A., Xavier, C., Niederstätter, H., de la Puente, M., Pośpiech, E., Pisarek, A., Kayser, M., Branicki, W., Parson, W.
Format: Article
Language:English
Subjects:
Age estimation
Bisulfite PCR multiplex development
MiSeq FGx sequencing
Targeted bisulfite sequencing
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Summary:•Assay development for chronological age estimation based on quantitative DNA methylation analysis.•A bisulfite multiplex PCR was optimized to analyze 32 age informative CpGs at ELOVL2, MIR29B2C, FHL2, TRIM59 and KLF14.•Targeted bisulfite sequencing was performed using the MiSeq FGx platform.•Reproducibility and sensitivity were tested with artificially methylated DNA standards. The 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 replicates.
ISSN:1872-4973
1878-0326
DOI:10.1016/j.fsigen.2020.102322