David Van Hoofstat

Forensic STR analysis using massive parallel sequencing

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.

DNA typing of fingerprints using capillary electrophoresis: effect of dactyloscopic powders.

DNA typing is a useful tool in crime solving, not only for blood samples, sperm, or sa liva but also for traces of DNA left on tools or pieces of clothing used in burglaries or thefts. On these kinds of samples, the sources of DNA are extremely small amounts of skin debris left after gripping tools. When a sensitive technique such as polymerase chain reaction (PCR) coupled with capillary electrophoresis is used, it is possible to get a profile from these low amounts of DNA. The classic technique in such cases, used in forensic sciences, is to reveal fingerprints by different dactyloscopic powders. Therefore, DNA profiling was performed on physical fingerprints left on glass and wooden plates, in order to establish eventual problems or interferences involved by using both techniques simultaneously. Eleven dactyloscopic powders were investigated on their influence on DNA typing. The results show that some can be used together with DNA profiling but that serious precautions have to be taken to avoid contamination.

Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science

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.

Automatic detection of spermatozoa for laser capture microdissection

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.

Presence and potential of cell free DNA in different types of forensic samples

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.

Suspension fluorescence in situ hybridization (S-FISH) combined with automatic detection and laser microdissection for STR profiling of male cells in male/female mixtures

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.

Comparison of slab gel electrophoresis and capillary electrophoresis for the detection of the fluorescently labeled polymerase chain reaction products of short tandem repeat fragments

The sizing capability of slab gel electrophoresis for short tandem repeat (STR) fragments was compared to the sizing capability of capillary electrophoresis (CE). Both systems used automated laser fluorescence detection to detect four fluorescent dyes, enabling the use of an internal lane standard within each sample. The STR fragments were amplified using a multiplex polymerase chain reaction (PCR) in which the STR fragments Hum CD-4, Hum TH01, Hum D21S11 and Hum SE33 were amplified simultaneously. The reproducibility of the size calling was determined for both systems. The average standard deviation obtained for the slab gel system was 0.2, which was comparable to the standard deviation of 0.12 obtained for the CE system. The CE system produced results comparable to those obtained on the slab gel system, with a level of precision of +/- 1.0 bp (between instruments).

Evaluation of three DNA extraction protocols for forensic STR typing after laser capture microdissection

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.

Forensic massively parallel sequencing data analysis tool: Implementation of MyFLq as a standalone web- and Illumina BaseSpace(®)-application.

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.

Sources of DNA Contamination and Decontamination Procedures in the Forensic Laboratory

The sensitivity of forensic DNA typing techniques can cause problems when evidence samples are inadvertently contaminated with DNA from another source. Therefore, precautions need to be taken to minimize the risk of contamination . In this study, laboratory air and surfaces, tools and equipment we re evaluated as potential sources of contaminating DNA. Subsequently, two decontamination procedures, i.e. the conventionally used sodium hypochlorite and the commercially available DNA decontamination solution DNA ZAP TM (Applied Biosystems), wer e compared for their use in removing potentially contaminating DNA from the laboratory working environment. From our results, it can be concluded that air is unlikely to be the source of observed DNA contamination in the laboratory whereas DNA accumulating on surfaces, tools and equipment within the laboratory environment may potentially be transferred to evidence samples. DNA ZAP TM outperformed the conventionally used sodium hypochlorite decontamination procedure. Stringent preventive measures and decontamination of equipment and laboratory surfaces is important to avoid secondary transfer of this contaminating DNA to evidence samples.