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Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis

Heidegger, A. ; Pisarek, A. ; de la Puente, M. ; Niederstätter, H. ; Pośpiech, E. ; Woźniak, A. ; Schury, N. ; Unterländer, M. ; Sidstedt, M. LU and Junker, K. , et al. (2022) In Forensic Science International: Genetics 56.
Abstract

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE)... (More)

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.

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type
Contribution to journal
publication status
published
subject
keywords
Age prediction, Bisulfite sequencing, DNA methylation, Forensic DNA phenotyping, Massively parallel sequencing, Semen
in
Forensic Science International: Genetics
volume
56
article number
102596
publisher
Elsevier
external identifiers
  • scopus:85118801860
  • pmid:34763164
ISSN
1872-4973
DOI
10.1016/j.fsigen.2021.102596
language
English
LU publication?
yes
additional info
Publisher Copyright: © 2021 The Authors
id
7f095b4f-c90f-4e64-85f1-f230dd97d65a
date added to LUP
2021-12-02 16:45:58
date last changed
2024-04-20 17:03:44
@article{7f095b4f-c90f-4e64-85f1-f230dd97d65a,
  abstract     = {{<p>The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (&gt; 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.</p>}},
  author       = {{Heidegger, A. and Pisarek, A. and de la Puente, M. and Niederstätter, H. and Pośpiech, E. and Woźniak, A. and Schury, N. and Unterländer, M. and Sidstedt, M. and Junker, K. and Ventayol Garcia, M. and Laurent, F. X. and Ulus, A. and Vannier, J. and Bastisch, I. and Hedman, J. and Sijen, T. and Branicki, W. and Xavier, C. and Parson, W.}},
  issn         = {{1872-4973}},
  keywords     = {{Age prediction; Bisulfite sequencing; DNA methylation; Forensic DNA phenotyping; Massively parallel sequencing; Semen}},
  language     = {{eng}},
  publisher    = {{Elsevier}},
  series       = {{Forensic Science International: Genetics}},
  title        = {{Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis}},
  url          = {{http://dx.doi.org/10.1016/j.fsigen.2021.102596}},
  doi          = {{10.1016/j.fsigen.2021.102596}},
  volume       = {{56}},
  year         = {{2022}},
}