Short tandem repeats (STRs) are highly polymorphic repetitive DNA sequences extensively used in forensic science for identification of individuals. STR genotyping is usually performed by capillary electrophoresis (CE) or next-generation sequencing (NGS) in centralized laboratories. However, there is an increasing need for a low cost, portable and rapid STR genotyping method. Multiple methods for miniaturization have been explored, all relying on polymerase chain reaction (PCR) for generating amplicons. PCR requires precise thermal cycling, which complicates the design of the STR genotyping microfluidic device. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification method that operates between 37 °C and 42 °C and completes within 40 min. This, along with the robustness of reagents and reduced stutter rate compared to PCR makes RPA a suitable candidate for implementation in an STR genotyping microfluidics device as well as a part of the established STR genotyping work flows. In this proof-of-concept study, we evaluate RPA assay for amplification of forensically relevant STR loci. Thirteen core STR loci of the Combined DNA Index System (CODIS) were amplified using RPA in both singleplex and multiplex formats. The amplicons were then analyzed using three different methods: CE, Illumina and Oxford Nanopore Technologies (ONT). A subset of 5 loci was used for CE analysis. CE, Illumina and ONT sequencing of singleplex RPA each resulted in complete and correct STR profiles across all samples for the standard input of 1 ng. Sensitivity assessment demonstrated that complete and correct genotypes were achieved with DNA inputs of 62 pg and above for all but locus D8S1179. Attempts at multiplex RPA amplification resulted in incomplete or incorrect STR profiles. This outcome highlights a challenge in adapting RPA for simultaneous amplification of multiple STR loci, which is a standard requirement in forensic DNA profiling.

In forensics, bloodstains on dark fabrics might be invisible for the naked eye. Although several visualization, presumptive, and confirmatory blood tests have been developed, all have one or more disadvantages, especially on DNA analysis. We report here the use of a visualization assay that can visually detect blood drops up to 1/20 dilution. In this assay, the fabric is placed between two wet filter papers and covered by glass surfaces on both sides. Pressure is applied on the glass surfaces in which bloodstains transfer onto the filter papers through capillary forces. Detected stains can be tested with other more sensitive presumptive blood tests performed on the filter paper. Even more, DNA analysis can be performed on the transferred bloodstains. The presented visualization assay is easy to perform, extremely cheap, requires little hands on time, and does not affect bloodstain pattern analysis.

Routine use of massively parallel sequencing (MPS) for forensic genomics is on the horizon. The last few years, several algorithms and workflows have been developed to analyze forensic MPS data. However, none have yet been tailored to the needs of the forensic analyst who does not possess an extensive bioinformatics background. We developed our previously published forensic MPS data analysis framework MyFLq (My-Forensic-Loci-queries) into an open-source, user-friendly, web-based application. It can be installed as a standalone web application, or run directly from the Illumina BaseSpace environment. In the former, laboratories can keep their data on-site, while in the latter, data from forensic samples that are sequenced on an Illumina sequencer can be uploaded to Basespace during acquisition, and can subsequently be analyzed using the published MyFLq BaseSpace application. Additional features were implemented such as an interactive graphical report of the results, an interactive threshold selection bar, and an allele length-based analysis in addition to the sequenced-based analysis. Practical use of the application is demonstrated through the analysis of four 16-plex short tandem repeat (STR) samples, showing the complementarity between the sequence- and length-based analysis of the same MPS data.

The success rate of STR profiling of hairs found at a crime scene is quite low and negative results of hair analysis are frequently reported. To increase the success rate of DNA analysis of hairs in forensics, nuclei in hair roots can be counted after staining the hair root with DAPI. Two staining methods were tested: a longer method with two 1h incubations in respectively a DAPI- and a wash-solution, and a fast, direct staining of the hair root on microscope slides. The two staining methods were not significantly different. The results of the STR analysis for both procedures showed that 20 nuclei are necessary to obtain at least partial STR profiles. When more than 50 nuclei were counted, full STR profiles were always obtained. In 96% of the cases where no nuclei were detected, no STR profile could be obtained. However, 4% of the DAPI-negative hair roots resulted in at least partial STR profiles. Therefore, each forensic case has to be evaluated separately in function of the importance of the evidential value of the found hair. The fast staining method was applied in 36 forensic cases on 279 hairs in total. A fast screening method using DAPI can be used to increase the success rate of hair analysis in forensics.

The theoretical advantages of miniSTRs are undeniable. Several studies show that miniSTRs are more sensitive and robust in the analysis of low template and degraded DNA. In this study we want to show the overall benefit of using miniSTRs in real forensic casework samples and show the percentage of samples that benefit from analysis with additional miniSTR loci in terms of resulting in a useful profile. The considered samples were 3064 touch DNA samples, analyzed in our accredited routine forensic DNA profiling laboratory between mid 2009 and mid 2013. Of these 3064 samples, 618 samples were analyzed using 13 loci, 532 samples using 15 loci and 1914 samples using 20 loci of which 5 were the mini- and midi-STR loci that were added to the extended European Standard Set (ESS). The retrospective results show a small increased success rate after implementation of extra loci and an even smaller increase after the implementation of the mini- and midi-STR analysis. The percentage of touch DNA samples that benefit from the analysis of additional mini- and midi-STR loci is limited.

Extracellular or cell free DNA has been found to exist in many biological media such as blood and saliva. To check whether cell free DNA is present in the supernatant which is normally discarded during several DNA extraction processes, such as Chelex® extraction, DNA profiles of cell pellet and concentrated supernatant from 30 artificial case like samples and from 100 real forensic samples were compared. Presence of cell free DNA was shown in all investigated sample types. Moreover, in some samples additional alleles, not detected during analysis of the cell pellet, were detected, offering valuable information which would normally have been discarded together with the supernatant. The results presented here indicate that cell free DNA deserves further consideration since it has the potential to increase the DNA yield in forensic casework samples in general and in contact traces in particular.

In forensic sciences, short tandem repeat (STR) analysis is a valuable tool in identifying the donor(s) of biological stains. Laser capture microdissection (LCM) can be used as a cell separating technique to isolate specific cell types in mixed samples. An important challenge lies in the development of a DNA isolation method appropriate for laser microdissected cells, as these samples usually contain minute amounts of cells. In this study three different DNA isolation methods for LCM collected cells were compared. The PicoPure DNA extraction method outperformed both other methods (IQ™ system and short alkaline method). Consequently, the minimal number of LCM collected cells necessary for STR typing was determined. Using the PicoPure DNA extraction method, full DNA profiles could be obtained from as little as 10 cells. Nevertheless, despite the occurrence of allelic drop out in some of the samples, lower amounts of cells gave rise to useful DNA profiles.

In forensic casework analysis, identification of the biological matrix and the species of a forensic trace, preferably without loss of DNA, is of major importance. The biological matrices that can be encountered in a forensic context are blood (human or non-human), saliva, semen, vaginal fluid, and to a lesser extent nasal secretions, feces, and urine. All these matrices were applied on swabs and digested with trypsin in order to obtain peptides. These peptides were injected on a mass spectrometer (ESI Q-TOF) resulting in the detection of several biomarkers that were used to build a decision tree for matrix identification. Saliva and blood were characterized by the presence of alpha-amylase 1 and hemoglobin, respectively. In vaginal fluid, cornulin, cornifin, and/or involucrin were found as biomarkers while semenogelin, prostate-specific antigen, and/or acid phosphatase were characteristic proteins for semen. Uromodulin or AMBP protein imply the presence of urine, while plunc protein is present in nasal secretions. Feces could be determined by the presence of immunoglobulins without hemoglobin. The biomarkers for the most frequently encountered biological matrices (saliva, blood, vaginal fluid, and semen) were validated in blind experiments and on real forensic samples. Additionally, by means of this proteomic approach, species identification was possible. This approach has the advantage that the analysis is performed on the first "washing" step of the chelex DNA extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing. This technique can be considered as a useful additional tool for biological matrix identification in forensic science and holds the promise of further automation.

We explore the applicability of second generation sequencing (SGS) to sequence multiplexed forensic STR amplicons, both in a single contributor sample as in multiple-person mixtures with different ratios. We compare the results of a commercial STR profiling kit (Applied Biosystems AmpFlSTR(®) Profiler Plus(®)), analyzed both with capillary electrophoresis and with Roche GS FLX sequencing. An easy to use open-source software pipeline is provided, chaining together the different steps needed to start the analysis from a GS FLX FASTA file, resulting in a FASTA file containing the called and quantified alleles present in the data. Sequencing of multiplexed STR amplicons using Roche GS FLX titanium technology is technically feasible but the technology is not ideal for this purpose. The fraction of full length reads is small and the homopolymer sequencing error rate is high. The pipeline compresses the homopolymers to a single base to avoid false results caused by these homopolymers. The qualitative and quantitative results from the SGS STR analysis pipeline are comparable to the electrophoresis method. Additionally, the SGS method provides extra information and is able to call allele subtypes based on STR sequences in a database. In mixed samples, all alleles were reported from individuals that contributed at least 10% to the mixture.

In sexual assault crimes, differential extraction of spermatozoa from vaginal swab smears is often ineffective, especially when only a few spermatozoa are present in an overwhelming amount of epithelial cells. Laser capture microdissection (LCM) enables the precise separation of spermatozoa and epithelial cells. However, standard sperm-staining techniques are non-specific and rely on sperm morphology for identification. Moreover, manual screening of the microscope slides is time-consuming and labor-intensive. Here, we describe an automated screening method to detect spermatozoa stained with Sperm HY-LITER™. Different ratios of spermatozoa and epithelial cells were used to assess the automatic detection method. In addition, real postcoital samples were also screened. Detected spermatozoa were isolated using LCM and DNA analysis was performed. Robust DNA profiles without allelic dropout could be obtained from as little as 30 spermatozoa recovered from postcoital samples, showing that the staining had no significant influence on DNA recovery.

Laser microdissection is a valuable tool for isolating specific cells from mixtures, such as male cells in a mixture with female cells, e.g., in cases of sexual assault. These cells can be stained with Y-chromosome-specific probes. We developed an automatic screening method to detect male cells after fluorescence in situ hybridization in suspension (S-FISH). To simulate forensic casework, the method was tested on female saliva after cataglottis (a kiss involving tongue-to-tongue contact) and on licking traces (swabs of dried male saliva on female skin) even after drying. After isolation of the detected cells, short tandem repeat profiling was performed. Full DNA profiles could consistently be obtained from as little as ten buccal cells. Isolation of five cells resulted in a mean of 98% (SD of 3.4%) of the alleles detected, showing that the developed S-FISH staining had no significant negative influence on DNA recovery and can be used in forensic casework.