Identification of organ tissue types and skin from forensic samples by microRNA expression analysis

The identification of organ tissues in traces recovered from scenes and objects with regard to violent crimes involving serious injuries can be of considerable relevance in forensic investigations. Molecular genetic approaches are provably superior to histological and immunological assays in characterizing organ tissues, and micro-RNAs (miRNAs), due to their cell type specific expression patterns and stability against degradation, emerged as a promising molecular species for forensic analyses, with a range of tried and tested indicative markers.Thus, herein we present the first miRNA based approach for the forensic identification of organ tissues. Using quantitative PCR employing an empirically derived strategy for data normalization and unbiased statistical decision making, we assessed the differential expression of 15 preselected miRNAs in tissues of brain, kidney, lung, liver, heart muscle, skeletal muscle and skin. We show that not only can miRNA expression profiling be used to reliably differentiate between organ tissues but also that this method, which is compatible with and complementary to forensic DNA analysis, is applicable to realistic forensic samples e.g. mixtures, aged and degraded material as well as traces generated by mock stabbings and experimental shootings at ballistic models.     

Microbial DNA in human nucleic acid extracts: Recoverability of the microbiome in DNA extracts stored frozen long-term and its potential and ethical implications for forensic investigation

Human DNA samples can remain unaltered for years and preserve important genetic information for forensic investigations. In fact, besides human genetic information, these extracts potentially contain additional valuable information: microbiome signatures. Forensic microbiology is rapidly becoming a significant tool for estimating post-mortem interval (PMI), and establishing cause of death and personal identity. To date, the possibility to recover unaltered microbiome signatures from human DNA extracts has not been proven. This study examines the microbiome signatures within human DNA extracts obtained from six cadavers with different PMIs, which were stored frozen for 5–16 years. Results demonstrated that the microbiome can be co-extracted with human DNA using forensic kits designed to extract the human host’s DNA from different tissues and fluids during decomposition. We compared the microbial communities identified in these samples with microbial DNA recovered from two human cadavers donated to the Forensic Anthropology Center at Texas State University (FACTS) during multiple decomposition stages, to examine whether the microbial signatures recovered from “old” (up to 16 years) extracts are consistent with those identified in recently extracted microbial DNA samples. The V4 region of 16 S rRNA gene was amplified and sequenced using Illumina MiSeq for all DNA extracts. The results obtained from the human DNA extracts were compared with each other and with the microbial DNA from the FACTS samples. Overall, we found that the presence of specific microbial taxa depends on the decomposition stage, the type of tissue, and the depositional environment. We found no indications of contamination in the microbial signatures, or any alterations attributable to the long-term frozen storage of the extracts, demonstrating that older human DNA extracts are a reliable source of such microbial signatures. No shared Core Microbiome (CM) was identified amongst the total 18 samples, but we identified certain species in association with the different decomposition stages, offering potential for the use of microbial signatures co-extracted with human DNA samples for PMI estimation in future. Unveiling the new significance of older human DNA extracts brings with it important ethical-legal considerations. Currently, there are no shared legal frameworks governing the long-term storage and use of human DNA extracts obtained from crime scene evidence for additional research purposes. It is therefore important to create common protocols on the storage of biological material collected at crime scenes. We review existing legislation and guidelines, and identify some important limitations for the further development and application of forensic microbiomics.     

Application of thiopropyl sepharose 6B for removal of PCR inhibitors from DNA extracts of a thigh bone recovered from the sea

PCR amplification of DNA from forensic samples often proves difficult due to the presence of inhibitors of the polymerase chain reaction. One possible way to remove PCR inhibitors from a DNA extract is the use of the affinity resin thiopropyl sepharose 6B (TS), which has been used previously for the removal of PCR inhibitors in DNA extracts originating from stains on clothing. Here we show that TS is efficient also for the removal of inhibitors from PCR extracts from a highly decomposed human thigh bone. TS treatment, however, leads to a substantial loss of DNA making the technique best suited when substantial amounts of DNA are present.     

Automated extraction of DNA from biological stains on fabric from crime cases. A comparison of a manual and three automated methods

The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit either with the sample pre-treatment recommended by Qiagen or an in-house optimized sample pre-treatment on a QIAsymphony SP) and one manual method (Chelex) with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable STR-profiles. A total of 480 samples were processed. The highest DNA recovery was obtained with the PrepFiler Express kit on an AutoMate Express while the lowest DNA recovery was obtained using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen. Extraction using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen resulted in the lowest percentage of PCR inhibition (0%) while extraction using manual Chelex resulted in the highest percentage of PCR inhibition (51%). The largest number of reportable STR-profiles was obtained with DNA from samples extracted with the PrepFiler Express kit (75%) while the lowest number was obtained with DNA from samples extracted using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen (41%).     

Ancestry informative markers: Inference of ancestry in aged bone samples using an autosomal AIM-Indel multiplex

Ancestry informative markers (AIMs) can be useful to infer ancestry proportions of the donors of forensic evidence. The probability of success typing degraded samples, such as human skeletal remains, is strongly influenced by the DNA fragment lengths that can be amplified and the presence of PCR inhibitors. Several AIM panels are available amongst the many forensic marker sets developed for genotyping degraded DNA. Using a 46 AIM Insertion Deletion (Indel) multiplex, we analyzed human skeletal remains of post mortem time ranging from 35 to 60 years from four different continents (Sub-Saharan Africa, South and Central America, East Asia and Europe) to ascertain the genetic ancestry components. Samples belonging to non-admixed individuals could be assigned to their corresponding continental group. For the remaining samples with admixed ancestry, it was possible to estimate the proportion of co-ancestry components from the four reference population groups. The 46 AIM Indel set was informative enough to efficiently estimate the proportion of ancestry even in samples yielding partial profiles, a frequent occurrence when analyzing inhibited and/or degraded DNA extracts.     

A comparison of four methods for PCR inhibitor removal

Biological samples collected from the crime scenes often contain some compounds that can inhibit the polymerase chain reaction (PCR). The removal of PCR inhibitors from the extracts prior to the PCR amplification is vital for successful forensic DNA typing. This paper aimed to evaluate the ability of four different methods (PowerClean^® DNA Clean-Up kit, DNA IQ™ System, Phenol–Chloroform extraction and Chelex^®-100 methods) to remove eight commonly encountered PCR inhibitors including: melanin, humic acid, collagen, bile salt, hematin, calcium ions, indigo and urea. Each of these PCR inhibitors was effectively removed by the PowerClean^® DNA Clean-Up kit and DNA IQ™ System as demonstrated by generating more complete short tandem repeat (STR) profiles from the cleaned up inhibitor samples than from the raw inhibitor samples. The Phenol–Chloroform extraction and Chelex^®-100 methods, however, could only remove some of eight PCR inhibitors. Our results demonstrated that the PowerClean^® DNA Clean-Up kit and DNA IQ™ System were very effective for the removal of known PCR inhibitors that are routinely found in DNA extracts from forensic samples.     

Insertion–deletion polymorphisms—utilization on forensic analysis

Insertion–deletion (INDEL) markers are very frequent in the human genome and present several advantages for population and forensic studies, such as low mutation rates, easy interpretation, small amplicons, easy genotyping, and the possibility of using multiplex PCR. The great adaptability of INDELs for amplification of low copy number or degraded DNA allows its using as an interesting platform of genetic identity by DNA in forensic cases. In the present study, we tested the ability of 48 diallelic INDEL markers on genotyping forensic samples collected from different biological samples related to criminal cases. Moreover, we evaluated the lowest DNA concentration with which there was amplification of all markers from each one of three indel-plex panels. When comparing the performances obtained by the indel-plex panels described in this study with results obtained using Identifiler? kit (Applied Biosystems) related to forensic samples, as well as to control samples with different concentrations of DNA, we observed superior efficiency on samples with low copy number or in the presence of inhibitors.     

Comparison of DNA polymerases for improved forensic analysis of challenging samples

Inhibitors of polymerase chain reaction (PCR) amplification often present a challenge in forensic investigations of e.g., terrorism, missing persons, sexual assaults and other criminal cases. Such inhibitors may be counteracted by dilution of the DNA extract, using different additives, and selecting an inhibitory resistant DNA polymerase. Additionally, DNA in forensic samples is often present in limited amounts and degraded, requiring special analyses of short nuclear targets or mitochondrial DNA. The present study evaluated the enzymes AmpliTaq Gold, HotStarTaq Plus, KAPA3G Plant, and KAPA2G Robust, with regard to their ability to overcome inhibitory effects. Our data showed that diluting the extracts and adding bovine serum albumin may increase the yield of the PCR product. However, the largest impact was observed when alternative enzymes were utilized, instead of the commonly used AmpliTaq Gold. KAPA2G Robust presented the highest amplification efficiency in the presence of the inhibitor ammonium nitrate. Moreover, the KAPA3G Plant enzyme had the highest efficiency in amplifying degraded DNA from old buried bone material. KAPA3G Plant and KAPA2G Robust may thus be useful for counteracting inhibitors and improving the analysis of challenging samples.     

Using oral microbial DNA analysis to identify expirated bloodspatter

Distinguishing expirated bloodstains (blood forced by airflow out of the nose, mouth or a chest wound) from impact spatter (blood from gunshots, explosives, blunt force trauma and/or machinery accidents) is an important challenge in forensic science. Streptococcal bacteria are only found in the human mouth and saliva. This study developed a polymerase chain reaction (PCR) method that detects DNA from these bacteria as a sensitive tool to detect the presence of saliva. The PCR method was very specific to human oral streptococci, with no PCR product being made from human DNA or DNA from other microbes that were tested. It was also very sensitive, detecting as little as 60 fg of target DNA. The PCR amplification gave product with 99 out of 100 saliva samples tested. PCR was not inhibited by the presence of blood and could detect target DNA in expirated bloodstains in a range of materials and for up to 92 days after deposit on cardboard or cotton fabric. In a blind trial, the PCR method was able to distinguish three mock forensic samples that contained expirated blood from four that did not. Our data show that bacteria present in the oral cavity can be detected in bloodstains that contain saliva and therefore can potentially be used as a marker in forensic work to distinguish mouth-expirated bloodstains from other types of bloodstains.     

Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples

Though shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, DNA testing has historically focused on the recovery of just about 600 base pairs of the mitochondrial DNA control region. Here, we describe our success in recovering complete mitochondrial genome (mtGenome) data (∼16,569 bp) from single shed hairs. By employing massively parallel sequencing (MPS), we demonstrate that particular hair samples yield DNA sufficient in quantity and quality to produce 2–3 kb mtGenome amplicons and that entire mtGenome data can be recovered from hair extracts even without PCR enrichment. Most importantly, we describe a small amplicon multiplex assay comprised of sixty-two primer sets that can be routinely applied to the compromised hair samples typically encountered in forensic casework. In all samples tested here, the MPS data recovered using any one of the three methods were consistent with the control Sanger sequence data developed from high quality known specimens. Given the recently demonstrated value of complete mtGenome data in terms of discrimination power among randomly sampled individuals, the possibility of recovering mtGenome data from the most compromised and limited evidentiary material is likely to vastly increase the utility of mtDNA testing for hair evidence.     

Responsibility for Misleading Expert Testimony

The development of molecular biological tools and their application to the study of human DNA has had a widespread impact on forensic science in terms of greater accuracy of results and increased use of biological materials. Novel techniques in DNA fingerprinting coupled with traditional methods in forensic haemogenetics will in time greatly increase the contribution of forensic science to the legal process. As technologies for the recovery and analysis of the genetic component of biological material are progressively refined, so will the ability of law enforcement agencies to analyse evidence obtained from scenes of violent crime. This review briefly describes some of the traditional methods used in forensic science as well as the novel DNA techniques rapidly superseding them.     

Whole mitochondrial genomes assembled from thermally altered forensic bones and teeth

The recovery and analysis of genetic material obtained from thermally altered human bones and teeth are increasingly important to forensic investigations, especially in cases where soft-tissue identification is no longer possible. Although little is known about how these fire-related processes affect DNA degradation over time, next-generation sequencing technology in combination with traditional osteobiographical applications may provide us clues to these questions. In this study, we compare whole mitochondrial genome data generated using two different DNA extraction methods from 27 thermally altered samples obtained from fire victims (Maricopa County, Arizona) . DNA extracts were converted to double-stranded DNA libraries and enriched for whole mitochondrial DNA (mtDNA) using synthetic biotinylated RNA baits, then sequenced on an Illumina MiSeq. We processed the mitochondrial data using an in-house computational pipeline (MitoPipe1.0) composed of ancient DNA and modern genomics applications, then compared the resulting information across the two extraction types and five burn categories. Our analysis shows that DNA fragmentation increases with temperature, but that the acute insult from fire combined with the lack of water is insufficient to produce 5′ and 3′ terminal deamination characteristic of ancient DNA. Our data also suggest an acute and significant point of DNA degradation between 350 °C and 550 °C, and that the likelihood of generating high quality mtDNA haplogroup calls decreases significantly at temperatures > 550 °C. This research is part of a concerted effort to understand how fire affects our ability to generate genetic profiles suitable for forensic identification purposes.     

You can’t hide encoded evidence: DNA recovery from different fabrics after washing

Bloodstains are extremely important forensic evidence for DNA profiling. The current study has been planned to quantify DNA recovery from a list of fabrics tested after washing. On each fabric, spots of blood was dotted, and the dried-bloodstained fabrics were hand washed with tap water, soaked in tap water with detergent and then hand washed, for 5 and 10 min. DNA was extracted through Chelex100 from these fabrics and quantified through Real Time PCR using the Quantifiler Human DNA Quantification kit. Results showed that DNA can be recovered from fabrics after they have been washed. The amount of recovered DNA is comparable among these fabrics and did not show significant variations with washing methods. Blends of fabrics showed the highest recovery while non-absorbent fabrics showed least DNA recovery. Knowledge of the different DNA quantities recovered from washed fabrics will be useful in solving criminal cases. The ability to recover DNA from washed fabrics could prove important in forensic casework.     

STRs, mini STRs and SNPs--a comparative study for typing degraded DNA

Short tandem repeat (STR) systems are the most powerful and widely used genetic marker systems in forensic DNA typing. Optimized amplification conditions and PCR reagents in combination with laser fluorescence based detection methods have increased the sensitivity and decreased the detection threshold down to approximately 100 pg. The quality of human DNA from forensic samples can be influenced by environmental factors. These may cause different degrees of degradation which have a negative impact on the amplification process especially of STR systems with large amplicons. Therefore, methods which need only small amplicon sizes to detect DNA markers are a better choice for typing degraded DNA. Here we report investigations on different types of DNA markers and typing methods which should all be applicable for analysing degraded DNA. These are two commercially available mini STR kits and five SNP markers which were analysed with two self established assays, a 5' nuclease assay and a minisequencing (SNaPshot) assay. The investigations comprised sensitivity studies, different types of stain material, as well as intact and degraded DNA. Results indicate that mini STRs are superior to standard STR typing methods, especially for typing old stain material with small amounts of degraded DNA. SNP typing based on the minisequencing (SNaPshot) assay achieved a better success rate in typing aged blood and saliva stains compared to standard STRs and SNP typing using the 5' nuclease assay.     

A custom hybridisation enrichment forensic intelligence panel to infer biogeographic ancestry,hair and eye colour,and Y chromosome lineage

Massively parallel sequencing can provide genetic data for hundreds to thousands of loci in a single assay for various types of forensic testing. However, available commercial kits require an initial PCR amplification of short-to-medium sized targets which limits their application for highly degraded DNA. Development and optimisation of large PCR multiplexes also prevents creation of custom panels that target different suites of markers for identity, biogeographic ancestry, phenotype, and lineage markers (Y-chromosome and mtDNA). Hybridisation enrichment, an alternative approach for target enrichment prior to sequencing, uses biotinylated probes to bind to target DNA and has proven successful on degraded and ancient DNA. We developed a customisable hybridisation capture method, that uses individually mixed baits to allow tailored and targeted enrichment to specific forensic questions of interest. To allow collection of forensic intelligence data, we assembled and tested a custom panel of hybridisation baits to infer biogeographic ancestry, hair and eye colour, and paternal lineage (and sex) on modern male and female samples with a range of self-declared ancestries and hair/eye colour combinations. The panel correctly estimated biogeographic ancestry in 9/12 samples (75%) but detected European admixture in three individuals from regions with admixed demographic history. Hair and eye colour were predicted correctly in 83% and 92% of samples respectively, where intermediate eye colour and blond hair were problematic to predict. Analysis of Y-chromosome SNPs correctly assigned sex and paternal haplogroups, the latter complementing and supporting biogeographic ancestry predictions. Overall, we demonstrate the utility of this hybridisation enrichment approach to forensic intelligence testing using a combined suite of biogeographic ancestry, phenotype, and Y-chromosome SNPs for comprehensive biological profiling.     

Selective quantification of human DNA by real-time PCR of FOXP2

We established a simple quantitative PCR procedure with high specificity and sensitivity using TaqMan probes targeting the FOXP2 sequence. This assay distinguished human and nonhuman, including primates, samples with the exception of mouse, turtle, lizard, and fishes. However, the specific amplification of mouse, lizard, and turtle fragments of FOXP2 could be confirmed by electrophoresis after real-time PCR. Because the C(T) values obtained for human DNA were not affected by contaminating animal DNA at concentrations up to 30 times that of human DNA, we were able to estimate the concentration of human DNA in mixed specimens. This assay provides a reliable and useful method for routine quantification of human-specific DNA in forensic practice.     

Semi-quantitative PCR Analysis of DNA Degradation

Natural variables, including environmental conditions and post-mortem interval (PMI), affect the course of tissue decay in forensic and archaeological contexts. Compared with soft tissue, skeletal materials provide longer protection against DNA degradation. However environmental factors, including temperature and humidity, may affect the longevity of DNA survival in bone structure. In this study we describe the development of a semi-quantitative multiplex PCR based method to assess the level of DNA degradation with applications in analysis of forensic specimens. The assay was then applied to assess DNA survival in rib bone samples taken from 12 corpses in the mortuary. Samples were de-fleshed and stored in two different environmental conditions, including ambient conditions and underground burial from 103 to 445 days. Bone DNA was extracted and then assessed by the optimized semi-quantitative PCR and capillary electrophoresis of three products from the human mitochondrial DNA. The results show DNA survived for longer periods in samples stored in ambient conditions compared with underground burial conditions. There was no correlation found between the level of DNA degradation and PMI in our samples. Results also confirm that post-mortem alteration to DNA in skeletal tissues can occur very soon after death, with direct implications to the forensic industry.     

Improvements,factors, and influences on DNA recovery from firearms

Touch DNA recovery from firearms can be central to many criminal investigations, yet the generation of DNA profiles from these items remains poor. Currently in Australia, published casework data highlights extremely poor DNA success from samples recovered from firearms. Only between 5% and 25% of samples result in useful DNA data and therefore increasing the success of DNA recovered from firearms is highly important but has not yet been explored in-depth. This study focused on increasing the recovery of DNA from ten firearm components that were held for 15 s. Multiple recovery methods were used, and the resulting genetic data compared. DNA evidence may be deliberately removed from firearms after discharge to hamper forensic investigations, therefore this study examined the effect of wiping down the components or handling them with gloves. A standard double swab and rinse swab recovery method resulted in an average of 73% cellular recovery. A cumulative swab process had the highest average recovery at 86%, although it was found that increasing the DNA yield led to an increase in mixture complexity. Wiping over the components was observed to remove on average 69% of cellular material, compared with 33% when handed with gloves. However, the size and texture of the components affected the efficiency of cellular material removal. The results from this study allow for prioritisation of areas to sample on firearms, as well as suggesting techniques that can be applied for the optimum process of cellular recovery and subsequent generation of STR DNA data.     

Efficiency of a novel forensic room-temperature DNA storage medium

The success of forensic genetics has led to considerable numbers of DNA samples that must be stored. Thus, the ability to preserve the integrity of forensic samples is essential. The possibility of retesting these samples after many years should be guaranteed. DNA storage typically requires the use of freezers. Recently, a new method that enables DNA to be stored at room temperature was developed. This technology is based on the principles of anhydrobiosis and thus permits room-temperature storage of DNA. This study evaluates the ability of this technology to preserve DNA samples mimicking true mixture casework samples for long periods of time. Mixed human DNA from 2 or 3 persons and at low concentrations was dried and stored for a period ranging from 6 months to 2 years in the presence of a desiccant. The quality of the stored DNA was evaluated based on quantitative peak height results from Short Tandem Repeat (STR) genotyping and the number of observed alleles. Furthermore, we determined whether this matrix has a potential inhibitory or enhancing effect on the PCR genotyping reactions. In our previous work, we demonstrated the considerable potential of this new technology. The present study complements our previous work. Our results show that after 2 years of aging at room temperature, there is a decrease in the number of observed alleles and in the peak height of these alleles.