Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis
(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.
(Less)
- author
- organization
- publishing date
- 2022-01
- 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
- 2025-02-09 21:47:36
@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 (> 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}}, }