Spitting in the wind?-The challenges of RNA sequencing for biomarker discovery from saliva.

Forensic trace contextualization, i.e., assessing information beyond who deposited a biological stain, has become an issue of great and steadily growing importance in forensic genetic casework and research. The human transcriptome encodes a wide variety of information and thus has received increasing interest for the identification of biomarkers for different aspects of forensic trace contextualization over the past years. Massively parallel sequencing of reverse-transcribed RNA ("RNA sequencing") has emerged as the gold standard technology to characterize the transcriptome in its entirety and identify RNA markers showing significant expression differences not only between different forensically relevant body fluids but also within a single body fluid between forensically relevant conditions of interest. Here, we analyze the quality and composition of four RNA sequencing datasets (whole transcriptome as well as miRNA sequencing) from two different research projects (the RNAgE project and the TrACES project), aiming at identifying contextualizing forensic biomarker from the forensically relevant body fluid saliva. We describe and characterize challenges of RNA sequencing of saliva samples arising from the presence of oral bacteria, the heterogeneity of sample composition, and the confounding factor of degradation. Based on these observations, we formulate recommendations that might help to improve RNA biomarker discovery from the challenging but forensically relevant body fluid saliva.

Selecting mRNA markers in blood for age estimation of the donor of a biological stain.

RNA has gained a substantial amount of attention within the forensic field over the last decade. There is evidence that RNAs are differentially expressed with biological age. Since RNA can be co-extracted with DNA from the same piece of evidence, RNA-based analysis appears as a promising molecular alternative for predicting the biological age and hence inferring the chronological age of a person. Using RNA-Seq data we searched for markers in blood potentially associated with age. We used our own RNA-Seq data from dried blood stains as well as publicly available RNA-Seq data from whole blood, and compared two different approaches to select candidate markers. The first approach focused on individual gene analysis with DESeq2 to select the genes most correlated with age, while the second approach employed lasso regression to select a set of genes for optimal prediction of age. We present two lists with 270 candidate markers, one for each approach.

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.

"The acid test"-validation of the ParaDNA® Body Fluid ID Test for routine forensic casework.

The identification of the cellular origin and composition of crime scene-related traces can provide crucial insight into a crime scene reconstruction. In the last decade, especially mRNA-based body fluid and tissue identification (BFI) has been vigorously examined. Besides capillary electrophoretic (CE) and real-time quantitative PCR (RT-qPCR)-based approaches for mRNA detection, melt curve analysis bears potential as a simple-to-use method for BFI. The ParaDNA® Body Fluid ID Test relies on HyBeacon® probes and was developed as a rapid test for mRNA-based BFI of six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual, and peripheral blood. The herein presented work was performed as an "acid test" of the system and should clarify whether the approach matches the requirements of forensic routine casework in German police departments. Tested samples consisted of single source as well as of mixed samples.

Forensic SNP Genotyping with SNaPshot: Development of a Novel In-house SBE Multiplex SNP Assay.

This study introduces a newly developed in-house SNaPshot single-base extension (SBE) multiplex assay for forensic single nucleotide polymorphism (SNP) genotyping of fresh and degraded samples. The assay was validated with fresh blood samples from four different populations. In addition, altogether 24 samples from skeletal remains were analyzed with the multiplex. Full SNP profiles could be obtained from 14 specimens, while ten remains showed partial SNP profiles. Minor allele frequencies (MAF) of bone samples and different populations were compared and used for association of skeletal remains with a certain population. The results reveal that the SNPs of the bone samples are genetically close to the Pathan population. The findings show that the new multiplex system can be utilized for SNP genotyping of degraded and forensic relevant skeletal material, enabling to provide additional investigative leads in criminal cases.

As solid as a rock-comparison of CE- and MPS-based analyses of the petrosal bone as a source of DNA for forensic identification of challenging cranial bones.

Short tandem repeat (STR) typing from skeletal remains can be a difficult task. Dependent on the environmental conditions of the provenance of the bones, DNA can be degraded and STR typing inhibited. Generally, dense and compact bones are known to preserve DNA better. Several studies already proved that femora and teeth have high DNA typing success rates. Unfortunately, these elements are not present in all cases involving skeletal remains. Processing partial or singular skeletal elements, it is favorable to select bone areas where DNA preservation is comparably higher. Especially, cranial bones are often accidentally discovered during criminal investigations. The cranial bone is composed of multiple parts. In this examination, we evaluated the potential of the petrous bone for human identification of skeletal remains in forensic case work. Material from different sections of eight unknown cranial bones and-where available-additionally other skeletal elements, collected at the DNA department of the Institute of Legal Medicine in Ulm, Germany, from 2010 to 2017, were processed with an optimized DNA extraction and STR typing strategy. The results highlight that STR typing from the petrous bones leads to reportable profiles in all individuals, even in cases where the analysis of the parietal bone failed. Moreover, the comparison of capillary electrophorese (CE) typing to massively parallel sequencing (MPS) analysis shows that MPS has the potential to analyze degraded human remains and is even capable to provide additional information about phenotype and ancestry of unknown individuals.

Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues.

In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination.