Applications of epigenetic methylation in body fluid identification, age determination and phenotyping

McCord, Bruce; Gauthier, Quentin; Alghanim, Hussain; Antunes, Joana; Tejero, Nicole Fernández; Duncan, George; Balamurugan, Kuppareddi

doi:10.1016/j.fsigss.2019.10.061

Cited by 4 publications

(4 citation statements)

References 22 publications

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“…The study demonstrated the identification of semen samples using ZC3H12D on levels as low as 0.1 ng of input DNA, supporting previous reports from Silva et al [56]. As the current pyrosequencer can provide a quantitative result, the integration of an automated laboratory workflow, while providing confirmatory results, will be useful for casework samples [57]. In another study, markers were developed using high resolution melt analysis and pyrosequencing.…”

Section: Body Fluid Identificationsupporting

confidence: 80%

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Pyrosequencing: Current forensic methodology and future applications—a review

et al. 2022

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The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science. Pyrosequencing is a sequencing by synthesis technique, based on chemiluminescent inorganic pyrophosphate detection. This review explains the pyrosequencing workflow and illustrates the step-by-step chemistry, followed by a description of the assay design and factors to keep in mind for an exemplary assay. Existing and potential forensic applications are highlighted using this technology. Current applications include identifying species, identifying bodily fluids, and determining smoking status. We also review progress in potential applications for the future, including research on distinguishing monozygotic twins, detecting alcohol and drug abuse, and other phenotypic characteristics such as diet and body mass index. Overall, the versatility of the pyrosequencing technologies renders it a useful tool in forensic genomics.

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Section: Body Fluid Identificationsupporting

confidence: 80%

“…[56]. As the current pyrosequencer can provide a quantitative result, the integration of an automated laboratory workflow, while providing confirmatory results, will be useful for casework samples [57]. In another study, markers were developed using high resolution melt analysis and pyrosequencing.…”

Section: Current Forensic Applicationsmentioning

confidence: 99%

Pyrosequencing: Current forensic methodology and future applications—a review

et al. 2022

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“…Furthermore, given the nanogram-to-sub-nanogram sensitivity of DNA typing, it is important to develop newer, more sensitive assays for body fluids [3]. We have been developing epigenetic methods for this purpose based on tissue-specific DNA methylation [4][5][6].…”

Section: Introductionmentioning

confidence: 99%

An examination of differences in epigenetic methylation of saliva type samples based on collection method

Ghemrawi,

Fernandez‐Tejero,

Vaquero

et al. 2024

Electrophoresis

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In the context of forensic casework, it is imperative to both establish a DNA profile from biological specimens and accurately identify the specific bodily fluid source. To achieve this, DNA methylation markers have been developed for the differentiation of blood, semen, vaginal epithelial secretions, and saliva samples. Saliva, alternatively referred to as oral fluid, is recognized for its heterogeneous cellular composition, characterized by a mixture of epithelial, leukocytic, and bacterial cells. Consequently, our research has revealed variations in methylation percentages that correlate with the method employed for collecting saliva samples. To investigate these concepts, we scrutinized four CpG markers situated within or in proximity to the BCAS4, SLC12A8, SOX2OT, and FAM43A genes. Subsequently, we designed primers based on bioinformatically transformed reference sequences for these markers and rigorously assessed their quality by examining dimer and hairpin formation, melting temperature, and specificity. These loci were identified as saliva markers based on either buccal swabs or spit collection. Yet, there has been minimal or no research conducted to explore the variations in methylation between different collection methods. For this study, buccal, lip, tongue, spit, and nasal swabs were collected from 20 individuals (N = 100). Mock forensic samples, which include chewing gum (N = 10) and cigarettes (N = 10), were also tested. DNA was extracted, bisulfite converted, then amplified using in‐house designed assays, and pyrosequenced. The methylation levels were compared to other body fluids (semen, blood, vaginal epithelia, and menstrual blood [N = 32]). A total of 608 pyrosequencing results demonstrated that sampling location and collection method can greatly influence the level of methylation, highlighting the importance of examining multiple collection/deposition methods for body fluids when developing epigenetic markers.

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“…In our group and others, work has been carried out to identify tDMRs capable of determining methylation differences across multiple forensically relevant tissues [10][11][12][13][14]; however, there is still a need to identify additional tDMRs, to increase specificity when mixtures of body fluids are present. Body fluids left at crime scenes can sometimes be mixed or contain minute amounts of DNA.…”

Section: Introductionmentioning

confidence: 99%

A data‐driven, high‐throughput methodology to determine tissue‐specific differentially methylated regions able to discriminate body fluids

et al. 2021

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Tissue-specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high-resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.

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Applications of epigenetic methylation in body fluid identification, age determination and phenotyping

Cited by 4 publications

References 22 publications

Pyrosequencing: Current forensic methodology and future applications—a review

Pyrosequencing: Current forensic methodology and future applications—a review

An examination of differences in epigenetic methylation of saliva type samples based on collection method

A data‐driven, high‐throughput methodology to determine tissue‐specific differentially methylated regions able to discriminate body fluids

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