Dataset Information

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples.

ABSTRACT: Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice.

SUBMITTER: Zubakov D

PROVIDER: S-EPMC2226061 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples.

Zubakov Dmitry D Hanekamp Eline E Kokshoorn Mieke M van Ijcken Wilfred W Kayser Manfred M

International journal of legal medicine 20070620 2

Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that ...[more]

PMID: 17579879

Similar Datasets

Project description:Evaluating microbial community composition through next-generation sequencing has become increasingly accessible. However, metagenomic sequencing data sets provide researchers with only a snapshot of a dynamic ecosystem and do not provide information about the total microbial number, or load, of a sample. Additionally, DNA can be detected long after a microorganism is dead, making it unsafe to assume that all microbial sequences detected in a community came from living organisms. By combining relic DNA removal by propidium monoazide (PMA) with microbial quantification with flow cytometry, we present a novel workflow to quantify live microbial load in parallel with metagenomic sequencing. We applied this method to unstimulated saliva samples, which can easily be collected longitudinally and standardized by passive collection time. We found that the number of live microorganisms detected in saliva was inversely correlated with salivary flow rate and fluctuated by an order of magnitude throughout the day in healthy individuals. In an acute perturbation experiment, alcohol-free mouthwash resulted in a massive decrease in live bacteria, which would have been missed if we did not consider dead cell signal. While removing relic DNA from saliva samples did not greatly impact the microbial composition, it did increase our resolution among samples collected over time. These results provide novel insight into the dynamic nature of host-associated microbiomes and underline the importance of applying scale-invariant tools in the analysis of next-generation sequencing data sets.IMPORTANCE Human microbiomes are dynamic ecosystems often composed of hundreds of unique microbial taxa. To detect fluctuations over time in the human oral microbiome, we developed a novel workflow to quantify live microbial cells with flow cytometry in parallel with next-generation sequencing, and applied this method to over 150 unstimulated, timed saliva samples. Microbial load was inversely correlated with salivary flow rate and fluctuated by an order of magnitude within a single participant throughout the day. Removing relic DNA improved our ability to distinguish samples over time and revealed that the percentage of sequenced bacteria in a given saliva sample that are alive can range from nearly 0% up to 100% throughout a typical day. These findings highlight the dynamic ecosystem of the human oral microbiome and the benefit of removing relic DNA signals in longitudinal microbiome study designs.

Project description: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.

Project description:Despite the increasing numbers of studies investigating hepatitis A diagnostic through saliva, the frequency and the pattern of hepatitis A virus (HAV) markers in this fluid still remains unknown. To address this issue, we carried on a longitudinal study to examine the kinetics of HAV markers in saliva, in comparison with serum samples. The present study followed-up ten patients with acute hepatitis A infection during 180 days post diagnosis (dpd). Total anti-HAV was detected in paired serum and saliva samples until the end of the follow-up, showing a peak titer at 90th. However, total anti-HAV level was higher in serum than in saliva samples. This HAV marker showed a probability of 100% to be detected in both serum and saliva during 180 dpd. The IgM anti-HAV could be detected in saliva up to 150 dpd, showing the highest frequency at 30th, when it was detected in all individuals. During the first month of HAV infection, this acute HAV marker showed a detection probability of 100% in paired samples. The detection of IgM anti-HAV in saliva was not dependent on its level in serum, HAV-RNA detection and/or viral load, since no association was found between IgM anti-HAV positivity in saliva and any of these parameter (p>0.05). Most of the patients (80%) were found to contain HAV-RNA in saliva, mainly at early acute phase (30th day). However, it was possible to demonstrate the HAV RNA presence in paired samples for more than 90 days, even after seroconversion. No significant relationship was observed between salivary HAV-RNA positivity and serum viral load, demonstrating that serum viral load is not predictive of HAV-RNA detection in saliva. Similar viral load was seen in paired samples (on average 104 copies/mL). These data demonstrate that the best diagnostic coverage can be achieved by salivary anti-HAV antibodies and HAV-RNA tests during 30-90 dpd. The long detection and high probability of specific-HAV antibodies positivity in saliva samples make the assessment of salivary antibodies a useful tool for diagnosis and epidemiological studies. The high frequency of HAV-RNA in saliva and the probability of detection of about 50%, during the first 30 dpd, demonstrate that saliva is also useful for molecular investigation of hepatitis A cases, mainly during the early course of infection. Therefore, the collection of saliva may provide a simple, cheap and non-invasive means of diagnosis, epidemiological surveys and monitoring of hepatitis A infection purposes.

Project description:BackgroundMitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome.MethodsWe used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method 'immortalises' the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS).ResultsWe sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old).ConclusionsThis approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging samples.

Dataset Information

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples.

Publications

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets