Exploring a multiplex DNA methylation-based SNP typing method for body fluids identification: As a preliminary report

The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. Lactobacillus crispatus and Streptococcus salivarius were selected because of high abundance within vaginal fluid and saliva respectively. While Fusobacterium nucleatum and Gardnerella vaginalis, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected L. crispatus and G. vaginalis in vaginal fluid while F. nucleatum and S. salivarius was detected in saliva. Multiplex PCR detected F. nucleatum, S. salivarius and L. crispatus in mixed body fluid samples while, G. vaginalis was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, L. crispatus and G. vaginalis were detected in vaginal swabs while only S. salivarius was detected in saliva swabs. The limit of detection was 0.06 copies/µl for F. nucleatum (2.5 ×10−9 ng/µl) and S. salivarius (2.5 ×10−6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 ×10−4 ng/µl) and 0.48 copies/µl (2.5 ×10−7 ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.

Recent advances in whole-genome epigenetic analysis indicate that chromosome segments called tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to cell or tissue type. Therefore, body fluid-specific differential DNA methylation is a promising indicator for body fluid identification. However, DNA methylation patterns are susceptible to change in response to environmental factors and aging. Therefore, we investigated age-related methylation changes in semen-specific tDMRs using body fluids from young and elderly men. After confirming the stability of the body fluid-specific DNA methylation profile over time, two different multiplex PCR systems were constructed using methylation-sensitive restriction enzyme PCR and methylation SNaPshot, in order to analyze the methylation status of specific CpG sites from the USP49, DACT1, PRMT2, and PFN3 tDMRs. Both multiplex systems could successfully identify semen with spermatozoa and could differentiate menstrual blood and vaginal fluids from blood and saliva. Although including more markers for body fluid identification might be necessary, this study adds to the support that body fluid identification by DNA methylation profiles could be a valuable tool for forensic analysis of body fluids.

RNA analysis has become a reliable method of body fluid identification for forensic use. Previously, we developed a combination of four multiplex quantitative PCR (qRT-PCR) probes to discriminate four different body fluids (blood, semen, saliva, and vaginal secretion). While those makers successfully identified most body fluid samples, there were some cases of false positive and negative identification. To improve the accuracy of the identification further, we tried to use multiple markers per body fluid and adopted the NanoString nCounter system instead of a multiplex qRT-PCR system. After measuring tens of RNA markers, we evaluated the accuracy of each marker for body fluid identification. For body fluids, such as blood and semen, each body fluid-specific marker was accurate enough for perfect identification. However, for saliva and vaginal secretion, no single marker was perfect. Thus, we designed a logistic regression model with multiple markers for saliva and vaginal secretion and achieved almost perfect identification. In conclusion, the NanoString nCounter is an efficient platform for measuring multiple RNA markers per body fluid and will be useful for forensic RNA analysis.

Identification of body fluids found at crime scenes provides important information that can support a link between sample donors and actual criminal acts. Previous studies have reported that DNA methylation analysis at several tissue-specific differentially methylated regions (tDMRs) enables successful identification of semen, and the detection of certain bacterial DNA can allow for identification of saliva and vaginal fluid. In the present study, a method for detecting bacterial DNA was integrated into a previously reported multiplex methylation-sensitive restriction enzyme-polymerase chain reaction. The developed multiplex PCR was modified by the addition of a new semen-specific marker and by including amplicons for the 16S ribosomal RNA gene of saliva- and vaginal fluid-specific bacteria to improve the efficacy to detect a specific type of body fluid. Using the developed multiplex system, semen was distinguishable by unmethylation at the USP49, DACT1, and PFN3 tDMRs and by hypermethylation at L81528, and saliva could be identified by detection of saliva-specific bacteria, Veillonella atypica and/or Streptococcus salivarius. Additionally, vaginal fluid and menstrual blood were differentiated from other body fluids by hypomethylation at the PFN3 tDMR and the presence of vaginal fluid-specific bacteria, Lactobacillus crispatus and/or Lactobacillus gasseri. Because the developed multiplex system uses the same biological source of DNA for individual identification profiling and simultaneously analyses various types of body fluid in one PCR reaction, this method will facilitate more efficient body fluid identification in forensic casework.

DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood

In forensic sciences, the serological methods used to determine which body fluid was collected from the crime scene are merely presumptive or labor intensive since they rely on protein detection or on microscopic identification of cells. Given that certain forensic cases may need the precise identification of a body fluid to determine criminal contact, such is the example of a suspected sexual assault of a minor; certainty in the body fluid of origin may depict a precise picture of the events. The identification of loci that show differences in methylation according to the tissue of origin can aid forensic analysts in determining the origin of a DNA sample. The process of DNA methylation occurs naturally in the genome of living organisms and consists in the presence of a methyl group on the carbon 5 of a cytosine, which is typically followed by a guanine (CpG). Analyzing patterns of DNA methylation in body fluids collected from a crime scene is preferential to the analysis of proteins or mRNA since the same extracted DNA used for STR typing can be used for DNA methylation analysis. We have validated and identified loci able to discriminate blood, saliva, semen and vaginal epithelia. In the current study, we have also established the minimum amount of DNA able to provide reliable results using methodologies such as pyrosequencing and high-resolution melt (HRM) analysis for the different markers identified. Lastly, we performed an alternative bioinformatic analysis of data collected using an array that studied methylation in over 450,000 individual cytosines on the human genome. We were able to sort the locations that showed potentially higher methylation differences between body fluids and investigated over 100 of them using HRM analysis. The results of that study, allowed the identification of three new loci able to distinguish blood and two new loci able to distinguish saliva and vaginal epithelia, respectively. The use of DNA methylation patterns to aid forensic investigations started with a publication in 2010, therefore each small contribution such as this work may, similarly to what occured in the biochemistry field, result in the discovery of a method able to put the technology in the hands of forensic analysts.

Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs

Microbial communities present in body fluids can assist in distinguishing between types of body fluids. Metagenomic studies have reported bacterial genera which are core to specific body fluids and are greatly influenced by geographical location and ethnicity. Bacteria in body fluids could also be due to bacterial infection; hence, it would be worthwhile taking into consideration bacterial species associated with diseases. The present review reports bacterial species characteristic of diseased and healthy body fluids across geographical locations, and bacteria described in forensic studies, with the aim of collating a set of bacteria to serve as the core species-specific markers for forensic body fluid identification. The most widely reported saliva-specific bacterial species are Streptococcus salivarius, Prevotella melaninogenica, Neisseria flavescens, with Fusobacterium nucleatum associated with increased diseased state. Lactobacillus crispatus and Lactobacillus iners are frequently dominant in the vaginal microbiome of healthy women. Atopobium vaginae, Prevotella bivia, and Gardnerella vaginalis are more prevalent in women with bacterial vaginosis. Semen and urine-specific bacteria at species level have not been reported, and menstrual blood bacteria are indistinguishable from vaginal fluid. Targeting more than one bacterial species is recommended for accurate body fluid identification. Although metagenomic sequencing provides information of a broad microbial profile, the specific bacterial species could be used to design biosensors for rapid body fluid identification. Validation of microbial typing methods and its application in identifying body fluids in a mixed sample would allow regular use of microbial profiling in a forensic workflow.

Identification of coding region SNPs from specific and sensitive mRNA biomarkers for the deconvolution of the semen donor in a body fluid mixture

Traces of body fluids can be found at a crime scene. Being able to identify and differentiate the body fluids while preserving DNA is fundamentally important for forensic applications. Saliva is an important body fluid that can be found on bite marks, cigarette butts, and more, being an excellent source for DNA extraction. Current tests for saliva and other body fluids are destructive and body fluid specific, and are prone to false positives. Our laboratory has developed a universal method for identification of all main body fluids; saliva, semen, sweat, peripheral blood, and vaginal fluid, using Raman spectroscopy combined with chemometrics (Muro et al.)3. The objective of this study is to evaluate the developed method for potential false positives of saliva. The potential false positives chosen for this study include compounds, which result in false positive reaction for current biochemical tests or have a similar appearance to dry saliva. It was found that Raman spectroscopy was able to differentiate saliva from all tested samples effectively by both visual inspection of spectral data and using statistical analysis.

The screening and validation process of miR-223-3p for saliva identification

Identification and the time since deposition (TsD) estimation of body fluid stains from a crime scene could provide valuable information for solving the cases and are always difficult for forensics. Microbial characteristics were considered as a promising biomarker to address the issues. However, changes in the microbiota may damage the specific characteristics of body fluids. Correspondingly, incorrect body fluid identification may result in inaccurate TsD estimation. The mutual influence is not well understood and limited the codetection. In the current study, saliva, semen, vaginal secretion, and menstrual blood samples were exposed to indoor conditions and collected at eight time points (from fresh to 30 days). High-throughput sequencing based on the 16S rRNA gene was performed to characterize the microbial communities. The results showed that a longer TsD could decrease the discrimination of different body fluid stains. However, the accuracies of identification still reached a quite high value even without knowing the TsD. Correspondingly, the mean absolute error (MAE) of TsD estimation significantly increased without distinguishing the types of body fluids. The predictive TsD of menstrual blood reached a quite low MAE (1.54 ± 0.39 d). In comparison, those of saliva (6.57 ± 1.17 d), semen (6.48 ± 1.33 d), and vaginal secretion (5.35 ± 1.11 d) needed to be further improved. The great effect of individual differences on these stains limited the TsD estimation accuracy. Overall, microbial characteristics allow for codetection of body fluid identification and TsD estimation, and body fluids should be identified before estimating TsD in microbiome-based stain analyses.IMPORTANCEEmerged evidences suggest microbial characteristics could be considered a promising tool for identification and time since deposition (TsD) estimation of body fluid stains. However, the two issues should be studied together due to a potential mutual influence. The current study provides the first evidence to understand the mutual influence and determines an optimal process for codetection of identification and TsD estimation for unknown stains for forensics. In addition, we involved aged stains into our study for identification of body fluid stains, rather than only using fresh stains like previous studies. This increased the predictive accuracy. We have preliminary verified that individual differences in microbiotas limited the predictive accuracy of TsD estimation for saliva, semen, and vaginal secretion. Microbial characteristics could provide an accurate TsD estimation for menstrual blood. Our study benefits the comprehensive understanding of microbiome-based stain analyses as an essential addition to previous studies.

MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene expression. miRNAs have tissue-specific expression and are also present in various extracellular body fluids, including blood, tears, semen, vaginal fluid, and urine. Additionally, the expression of miRNAs in body fluids is linked to various pathological diseases, including cancer and neurodegenerative diseases. Examination of body fluids is important in forensic medicine as they serve as a valuable form of evidence. Due to its stability, miRNA offers an advantage for body fluid identification, which can be detected even after several months or from compromised samples. Identification of unique miRNA profiles for different body fluids enables the identification of the body fluids. Furthermore, miRNAs profiling can be used to estimate post-mortem interval. Various biochemical and molecular methods used for the identification of miRNAs have shown promising results. We discuss different miRNAs as specific biomarkers and their clinical importance in different pathological conditions, as well as their medicolegal importance.

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

The advent of high-throughput technologies such as microarrays, global gene knockout, and next generation sequencing (NGS) has revolutionized the field of molecular oncology. Large-scale endeavors of the kind represented by the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) are generating comprehensive portraits of human cancers at different molecular levels spanning over genome, epigenome and transcriptome landscapes. These data have made substantial contributions to our knowledge of the molecular biology of cancer, and have highlighted novel factors which had not been acknowledged previously. For example, emerging data indicate that the mutational makeup of tumors can significantly vary based on geographical variations among patients,1 pointing to the contribution of potentially different mechanisms (e.g. environmental) to tumorigenesis in different populations. On the other hand, cancer profiling data have also provided catalogues of cancer-associated molecular variations, which can serve as reliable biomarkers for cancer prevention or personalized management of patients. Successful examples include the refined diagnosis of disease subtype in breast cancer using gene expression signatures and protein markers, and stratifying patients affected by colorectal cancer for anti-EGFR therapy based on KRAS mutations. In recent years, much attention has been focused on epigenetic marks, in particular on the development of DNA methylation-based markers. Abnormal DNA methylation patterns are uniquely attractive for marker development: (i) they are abundant in tumors; (ii) they happen at early stages of tumorigenesis; and (iii) as covalently bound modifications to DNA molecules, they are stable in different body fluids. These characteristics point to the notion that monitoring the presence and abundance of methylated DNA in “liquid biopsy” samples from cancer patients or people who are at high risk for cancer can facilitate non-invasive cancer prognosis and early diagnosis. Indeed, recent studies have reported successful examples of assaying cancer-associated DNA methylation marks in body fluids in order to monitor response to treatment2 or tumor recurrence3 in different cancers. Likewise, new data have shown that DNA methylation markers perform as well as routine clinical procedure (e.g. cytology examination) in the prioritization of patients for clinical management.4 These reports together with several other recent studies have demonstrated that tools and technologies for reliable screening of abnormal DNA methylation patterns in non-invasive manners have become available. An essential step in this respect, however, is the identification of truly informative markers that are to be interrogated in screening procedures. Accordingly, it is of paramount importance to factor for changes in epigenetic patterns that happen naturally during life-time when looking for sensible cancer markers.5 In an earlier study, we had found DNA hypermethylation in the promoter of the growth hormone secretagouge receptor (GHSR) gene as an epigenetic marker of highest accuracy for detection of breast cancer.6 This finding was supported by analyzing tumors as well as an extensive list of control samples including normal-appearing tissue samples from cancer patients and healthy individuals, as well as benign lesions. The gene promoter was significantly hypermethylated in both invasive and non-invasive in situ ductal breast cancer as compared to other samples. Moreover, we noted that GHSR hypermethylation may be involved in an epigenetic field defect in breast cancer, as the level of methylation was higher in normal-appearing tissue samples of cancer patients compared to those from healthy individuals after adjustment for age. These findings showed that while GHSR hypermethylation is a cancer specific pattern, it is detectable in early stage tumors. Motivated by the high accuracy of GHSR hypermethylation for detection of breast cancer (with a sensitivity and specificity of 89.3 and 100%, respectively), we expanded our study to other cancers elucidating if this pattern could represent a pan-cancer marker. We observed that GHSR hypermethylation is able to discriminate cancers of lung, breast, prostate, pancreas, colorectum as well as B-cell chronic lymphocytic leukemia from tissue-matched control samples.7 Similar to the abovementioned cancers, the locus was also hypermethylated in glioblastoma samples. By including tumors of different stages, where available, we showed that GHSR hypermethylation is detected already in early-stage tumors. That is, while all tumors were significantly hypermethylated compared to non-tumoral samples, no significant differences existed for this marker between tumors of lower and higher stages. Collectively, these data indicate that GHSR hypermethylation is a pan-cancer marker regardless of the tissue from which the tumor originates. Therefore, interrogating GHSR methylation may provide a supplemental approach to routine clinical examination for cancer diagnosis. Analyzing this marker in plasma samples and other body fluid from cancer patients will be the next step in verifying its usefulness in non-invasive screening procedures. Mechanistically, it is plausible to assume that GHSR hypermethylation may have a functional role in tumorigenesis in different cancers or there is an inherent susceptibility of the locus to DNA methylation, which is acquired by accelerated cell proliferation. Future studies should address these possibilities.