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Estimating the Time Since Deposition of Saliva Stains With a Targeted Bacterial DNA Approach: A Proof-of-Principle Study.

ABSTRACT: Information on the time when a stain was deposited at a crime scene can be valuable in forensic investigations. It can link a DNA-identified stain donor with a crime or provide a post-mortem interval estimation in cases with cadavers. The available methods for estimating stain deposition time have limitations of different types and magnitudes. In this proof-of-principle study we investigated for the first time the use of microbial DNA for this purpose in human saliva stains. First, we identified the most abundant and frequent bacterial species in saliva using publicly available 16S rRNA gene next generation sequencing (NGS) data from 1,848 samples. Next, we assessed time-dependent changes in 15 identified species using de-novo 16S rRNA gene NGS in the saliva stains of two individuals exposed to indoor conditions for up to 1 year. We selected four bacterial species, i.e., Fusobacterium periodonticum, Haemophilus parainfluenzae, Veillonella dispar, and Veillonella parvula showing significant time-dependent changes and developed a 4-plex qPCR assay for their targeted analysis. Then, we analyzed the saliva stains of 15 individuals exposed to indoor conditions for up to 1 month. Bacterial counts generally increased with time and explained 54.9% of the variation (p = <2.2E-16). Time since deposition explained ≥86.5% and ≥88.9% of the variation in each individual and species, respectively (p = <2.2E-16). Finally, based on sample duplicates we built and tested multiple linear regression models for predicting the stain deposition time at an individual level, resulting in an average mean absolute error (MAE) of 5 days (ranging 3.3-7.8 days). Overall, the deposition time of 181 (81.5%) stains was correctly predicted within 1 week. Prediction models were also assessed in stains exposed to similar conditions up to 1 month 7 months later, resulting in an average MAE of 8.8 days (ranging 3.9-16.9 days). Our proof-of-principle study suggests the potential of the DNA profiling of human commensal bacteria as a method of estimating saliva stains time since deposition in the forensic scenario, which may be expanded to other forensically relevant tissues. The study considers practical applications of this novel approach, but various forensic developmental validation and implementation criteria will need to be met in more dedicated studies in the future.

SUBMITTER: Diez Lopez C

PROVIDER: S-EPMC8206545 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methylcytosine to detect the presence of 5 methylcytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used. A method that detects DNA methylation that is not restricted to specific sequences would provide a useful platform to analyse methylation distribution among the genes in any given phenotypic condition.Availability of additional approaches to examine DNA methylation would lead to increase in our understanding of the methylome and would help define the intrinsic and extrinsic factors which govern it. Microarray based high throughput methods have been extensively used for gene expression analysis. The method described uses a microarray to analyse DNA methylation levels in a gene/cDNA/oligo specific manner. Here, genomic DNA is fragmented and used for hybridization to microarray slides. The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC( 5-methyl cytidine) antibody (monoclonal). The resolution provided by this method is dependent upon the microarray platform used. This method can be adapted to any platform thus enabling seamless integration with existing technology of gene expression analysis

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Estimating the Time Since Deposition of Saliva Stains With a Targeted Bacterial DNA Approach: A Proof-of-Principle Study.

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