Comparison of massively parallel sequencing to capillary electrophoresis for short tandem repeat genotyping of trace DNA.

In forensic genetics, massively parallel sequencing (MPS) offers several advantages over the current golden standard, capillary electrophoresis (CE): additional sequence information, shorter amplicon lengths, and the simultaneous analysis of many markers. These benefits result in a reduced number of reactions necessary while improving the amount of data obtained, thereby conserving valuable sample extracts. This proves particularly advantageous for the analysis of trace DNA. This study assessed the suitability of MPS for short tandem repeat (STR) typing of low template samples compared with results obtained through CE. The MPS genotypes showed higher concordance to reference genotypes, with donor alleles being more frequently assigned to be the major contributor, meeting the requirements for database entry. However, the MPS workflow is more time-consuming and associated with higher costs.

The diversity of shedder tests and a novel factor that affects DNA transfer.

Since the first shedder test was formulated almost 20 years ago, a plethora of different test strategies has emerged. The amount of data generated so far is considerable. However, because of the limited reproducibility of its results, the reliability of the shedder concept is frequently questioned. This study provides a literature overview of applied shedder tests that capture the diversity of the concept. It is pointed out to what extent different classification criteria, workflows, and trace evaluation can impair the classification outcome. The robustness of shedder status was assessed by applying a promising approach established by Fonneløp et al. (Forensic Sci Int Genet 29:48-60, 21). Data provide similar results to those in recent studies but also ambiguous shedder classifications. The applied shedder test was adapted based on our own as well as the reviewed data. With novel classification parameters, promising results were achieved. This study reveals uncertainties and inconsistencies of the shedder concept. Recommendations for harmonization and transparency are proposed. Implementation of the recommendations may result in an increased impact on casework and transfer studies, including activity-level assessments. Furthermore, this study shows that moisturizers affect participants' shedder status as well as DNA transfer. The impact appears to remain relevant even 60 min post ointment application but depends greatly on the type of moisturizer applied.

Validation of an immunochromatographic D-dimer test to presumptively identify menstrual fluid in forensic exhibits.

Identifying the biological source of a crime scene stain can be crucial for police investigations in many scenarios. Blood is one of the most common fluids found, and accurate differentiation between peripheral blood and menstrual fluid could provide valuable information regarding the issue of consent in sexual assault cases. For the detection of menstrual fluid, no easy-to-use presumptive test is available to date. Therefore, this study aimed to validate a simple immunochromatographic test for the indication of menstrual fluid, focusing on a D-dimer assay. The Clearview® rapid D-dimer test provides a diagnostic assay for the detection of fibrin degradation products. We validated the sensitivity and robustness of the assay using fresh and dried menstrual fluid samples, body fluid mixtures, diluted samples, and casework swabs. Cross reactivity was tested for saliva, semen, vaginal fluid, and blood. No false positive results were obtained; it was possible to successfully analyze mixtures, highly diluted samples, and casework swabs. The results of this study indicate that the D-dimer assay reliably detects menstrual fluid in forensic exhibits and is easy to implement into the current workflow of body fluid identification.

Casework-related DNA transfer on footwear in consideration of the shedder status.

DNA evidence on shoes can play an important role in solving a variety of crimes. We investigated the transfer, persistence, prevalence and recovery of DNA (DNAtppr) on shoes (sneakers) and their soles in realistic handling scenarios taking into account the shedder status. This study aims to increase the understanding of the expected composition of DNA profiles and their probative value, providing a basis for activity level assessments. Samples were analyzed using a direct lysis method, suggesting its versatility and increasing the DNA typing success compared to previous studies on footwear. The data showed surface-dependent background DNA (bDNA) levels on shoe soles and prevalence of bDNA on the upper parts of the shoe. The owner of the shoe was allocatable to the mixture for almost every shoe and sampling location. Alternating scenarios of shoe handling were simulated through different pairs of shedders to distinguish shoe owner and subsequent user. Secondary users were attributable to DNA mixtures regardless of shedder status after wearing shoes a single time. The influence of the shedder status follows specific trends in this context. However, particularly intermediate shedders show inconsistent results. The prevalence of bDNA appears to have a greater effect on the impact of the shedder status on DNA profile composition than previously reported. The data help researchers to better resolve suspect statements and determine if a person of interest wore the shoes relevant to the investigation.

Best of both: A simultaneous analysis of mRNA and miRNA markers for body fluid identification.

The body fluid identification of traces found at crime scenes is crucial in relation of the circumstance of crime. For this reason, the body fluid identification (BFI) by molecular biological methods has been increasingly investigated in recent decades. Especially the use of messenger RNA (mRNA) has been established and validated by various studies. mRNAs can resist degradation for several decades under dry and dark environmental conditions, but degradation increases greatly e.g., in humid environments and UV radiation. In contrast, the shorter and protein-protected micro RNAs (miRNAs) are less susceptible to degradation, but not all potential markers are tissue-specific. The aim of this study was to develop a simultaneous mRNA/miRNA multiplex assay to take advantage of both types of RNA. The final assay was tested for various body fluids, dilutions, and mixtures. To demonstrate the advantage of a combined mRNA/miRNA assay, older and mostly degraded samples were examined and compared to an established mRNA assay. Initial results from degraded samples show that tissue-specific miRNAs expected could be detected for 93% of the degraded samples compared to mRNA markers with 25% of the mRNA assay. The result is a simultaneous mRNA/miRNA multiplex assay on capillary electrophoresis (CE) for the first time.

Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs, assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies.

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.

Development and validation of an mRNA-based multiplex body fluid identification workflow and a rectal mucosa marker pilot study.

Molecular identification of body fluids and tissues is crucial in order to understand the circumstances of crimes. For that reason, molecular investigations used to identify body fluids/tissues have increasingly been examined recently. Various studies have proved that messenger RNA (mRNA) profiling is a sensitive and robust method for body fluid/tissue identification. The forensically relevant body fluids/tissues blood, semen, saliva, vaginal secretion, menstrual blood and skin have all been detected successfully by applying suitable mRNA assay. However, rectal mucosa, which can be found as evidence in sexual assault cases, has been neglected in forensic investigations. So far there is no mRNA marker to detect rectal mucosa, although anal penetration occurs in a large number of sexual assaults (23.2% of female victims and 50% of male victims). In this study, specific and sensitive mRNA markers for forensically relevant body fluids were adapted and validated in an mRNA multiplex assay for routine casework. This included the implementation of a DNA/RNA re-extraction method for automated extraction that can be integrated into casework without loss of DNA. This re-extraction method and the mRNA multiplex assay were tested using casework samples. PCR-primers were designed for the identification of rectal mucosa and the more effective marker MUC12 was integrated into an extended multiplex assay. The result of our study is a highly specific and sensitive mRNA multiplex assay plus an automated DNA/RNA re-extraction method, that can be integrated into casework and identify rectal mucosa for the first time.

Looking for the pinpoint: Optimizing identification, recovery and DNA extraction of micro traces in forensic casework.

Many challenges are encountered in the analysis of micro traces such as touch DNA or telogen hair samples. Although DNA typing methods have become immensely more sensitive over the last years, recovery of the minute amounts of biological material in micro traces requires further enhancement. For example felony cases, where an offender oftentimes only contributes minor amounts of touch DNA, separation of victim and offender DNA poses difficulties. Whereas complete sampling of evidence generates admixed profiles, single particle collection is labor- and time-intensive. Besides optimization of identification and collection of bio particles during this study, a novel sampling strategy for enhanced DNA yield as well as success rate but simultaneous avoidance of mixture creation is proposed for tapings in forensic casework. Improvement of another crucial step in micro trace analysis, DNA extraction, involved evaluation of efficiency and DNA recovery of different extraction methods, namely magnetic bead based Maxwell extraction, Chelex, Casework Direct Kit and Investigator Casework GO! kit. Direct lysis approaches seemed to be most suitable for low template traces. Recently developed commercial kits even allow the presence of inhibitors. Improvement of embraced aspects successfully facilitates processing of micro traces in terms of time and labor.