Characterisation of forward stutter in the AmpFlSTR® SGM Plus® PCR

PCR amplification of tetrameric short tandem repeats (STRs) can lead to Taq enzyme slippage and artefact products typically one repeat unit less in size than the parent STR. These back stutter or n ? 4 amplification products are low-level relative to the amplification of the parent STR but are widely seen in the forensic community where tetrameric STRs are employed in the generation of DNA profiles. To aid the interpretation of DNA mixtures where minor contributor(s) might be present in comparable amounts to the back stutter products, the typical amounts of back stutter generated have been well characterised and guidelines for interpretation are in place. However, further artefacts thought to be Taq enzyme slippage leading to products with one repeat unit greater than the parent sequence (n + 4 or forward stutter) or two repeats less (n ? 8 or double back stutter) also occur, but these have not been well characterised despite their potential influence in mixture interpretations. Here we present findings with respect to these additional artefacts from a study of 10,000 alleles and include guidelines for interpretation.     

The use of Hemastix® severely reduces DNA recovery using the BioRobot® EZ1

Abstract: The choice of reagents for presumptive tests for blood, and subsequent extraction methodologies, can significantly affect both the quantity and quality of purified DNA. Blood samples directly tested with Hemastix^® yielded <1% of the DNA recovered from untested samples when purified using the Qiagen BioRobot^® EZ1 and EZ1^® DNA Investigator kit. Full short tandem repeat profiles were obtained from both tested and untested samples, suggesting that the Hemastix^® reagent(s) affect DNA binding, rather than produce DNA damage. The Hemastix^® inhibition of DNA yield could be overcome by the addition of MTL buffer to the sample prior to extraction. Laboratories may wish to modify current procedures for extracting blood samples, utilize other extraction/purification methodologies, or inform their submitting agencies to avoid direct exposure of questioned bloodstains to Hemastix^® reagents.     

Performance of the Emit® II Plus 6-Acetylmorphine Assay on the Viva-E® analyzer

We evaluated the performance of Emit(?) II Plus 6-Acetylmorphine Assay for human urine screening on the Viva-E(?) analyzer. Precision was evaluated using the cutoff and ±25% controls. Recovery and linearity were studied by spiking 6-acetylmorphine (6-AM) into human urine pools. Accuracy was evaluated using urine specimens and the results were compared to those from GC/MS. Cross-reactivity with structurally related drugs was assessed at different cross-reactant concentrations. Interferences were assessed in the presence of 7.5 and 12.5 ng/mL of 6-AM. The qualitative repeatability coefficients of variation (CV's) ranged from 0.3% to 0.4% and the within-lab CV's ranged from 2.0% to 2.2%. In analyte units (ng/mL), the repeatability CV's ranged from 1.3% to 2.2% and the within-lab CV's ranged from 2.6% to 4.3%. The limit of detection of the assay was found to be 2.1 ng/mL. Recovery was within 15% of expected value. Linearity was 2.1-20 ng/mL. Method comparison showed 99% agreement with GC/MS. The assay had minimal cross-reactivity with morphine, morphine-3-glucuronide, morphine-6-glucuronide and other opioids. No interference was observed with endogenous interferences and structurally unrelated drugs. The assay correctly classified CAP survey samples. The Emit(?) II Plus 6-Acetylmorphine Assay will be a suitable screening method for urine specimens in both qualitative and semi-quantitative analyses.     

Validating TrueAllele® DNA mixture interpretation

Abstract: DNA mixtures with two or more contributors are a prevalent form of biological evidence. Mixture interpretation is complicated by the possibility of different genotype combinations that can explain the short tandem repeat (STR) data. Current human review simplifies this interpretation by applying thresholds to qualitatively treat STR data peaks as all‐or‐none events and assigning allele pairs equal likelihood. Computer review, however, can work instead with all the quantitative data to preserve more identification information. The present study examined the extent to which quantitative computer interpretation could elicit more identification information than human review from the same adjudicated two‐person mixture data. The base 10 logarithm of a DNA match statistic is a standard information measure that permits such a comparison. On eight mixtures having two unknown contributors, we found that quantitative computer interpretation gave an average information increase of 6.24 log units (min = 2.32, max = 10.49) over qualitative human review. On eight other mixtures with a known victim reference and one unknown contributor, quantitative interpretation averaged a 4.67 log factor increase (min = 1.00, max = 11.31) over qualitative review. This study provides a general treatment of DNA interpretation methods (including mixtures) that encompasses both quantitative and qualitative review. Validation methods are introduced that can assess the efficacy and reproducibility of any DNA interpretation method. An in‐depth case example highlights 10 reasons (at 10 different loci) why quantitative probability modeling preserves more identification information than qualitative threshold methods. The results validate TrueAllele^® DNA mixture interpretation and establish a significant information improvement over human review.     

Internal validation study of the PowerPlex® Y23 system

The PowerPlex Y23 System is a 23-loci, 5-color Y-STR multiplex designed for genotyping forensic casework samples, database samples and paternity samples. The kit contains: all 12 loci in the current PowerPlex Y System, the additional 5 loci found in AmpFlSTR Y-filer, plus 6 new loci. An internal validation study of the PowerPlex Y23 kit was therefore conducted including the following aspects: sensitivity, mixture studies of male–male and female–male DNA, performance with simulated inhibition and stutter calculations. 100 Caucasians living in Switzerland were also typed using the PowerPlex Y23 kit.     

Detection and analysis of DNA mixtures with the MiSeq FGx®

Recognizing and interpreting mixtures are challenges that occur frequently in forensic casework. Therefore, any new analysis methods that are implemented must handle the challenges of mixed forensic samples. Next generation sequencing offers advantages over capillary electrophoresis in amplicon multiplexing and degraded sample analysis; however, advantages with mixed samples rely heavily on the advancement of user-friendly analysis software. This research analyzed samples with the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx® and compared them with the GlobalFiler™ STR Kit for capillary electrophoresis. Metrics tested for both chemistries included concordance, limits of detection, and mixture analysis. Data analysis for mixture samples was completed with the MixtureAce™ plug-in and ArmedXpert™ software. Next generation sequencing offered distinct advantages in limits of detection and isoallele heterozygosity but suffered from increased variability in stutter and allele count ratios compared to capillary electrophoresis.     

Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples

Abstract: The AmpF?STR^® Identifiler^® Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single‐source blood and buccal samples on FTA^® card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay’s sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA^® cards, and the assay’s specificity was verified by establishing minimal cross‐reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA^® substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler^® Direct Kit for forensic standards and database samples genotyping.     

The neuroendocrine effects of the TASER X26®: A brief report

IntroductionLaw enforcement officers use conducted electrical weapons (CEW) such as the TASER X26^® to control violently resistive subjects. There are no studies in the medical literature examining the effects of these weapons on the human stress response. This is the first study to compare the human stress response to conducted electrical weapons, oleoresin capsicum (O.C.), a cold-water tank immersion, and a defensive tactics drill.MethodsSubjects were randomized to one of the four interventions studied. Subjects received either a 5-s exposure from the TASER X26 CEW with the probes fired into the back from 7 ft, a 5-s spray of O.C., a skin and mucous membrane irritant, to the eyes, a 45-s exposure of the hand and forearm in a 0 °C cold water tank, or a 1-min defensive tactics drill.ResultsAlpha-amylase had the greatest increase from baseline at 10–15 min with the defensive tactics drill. Cortisol had the greatest increase at 15–20 min with O.C. Cortisol remained most elevated at 40–60 min in the defensive tactics drill group.ConclusionsOur preliminary data suggests that physical exertion during custodial arrest may be most activating of the human stress response, particularly the sympathetic–adrenal–medulla axis. This may suggest that techniques to limit the duration of this exertion may be the safest means to apprehend subjects, particularly those at high-risk for in-custody death. Conducted electrical weapons were not more activating of the human stress response than other uses of force.     

Comparisons of protocols for enhanced DNA profile quality from FTA®-deposited urine samples

Disputes over the identity of a urine sample donor have been reported, and urine authentication by genetic profiling has helped resolved the cases. However, since genotyping of urine is not always required, many drug-testing laboratories may face sample storage issues. Several studies have investigated the use of FTA® cards as a convenient tool for keeping specimen at room temperature for extended periods of time. However, generating complete STR profile from some FTA®-deposited urine samples remains challenging due to low levels of genetic material content, necessitating amendments to the laboratory’s standard protocols. This work therefore aims to evaluate the effects of two DNA template preparation methods, both employing FTA® cards as the storage medium, on the success rates of STR profiling from urine. Specimen from a female volunteer, representing a particularly low-yield sample, was employed. Aliquots of 1 and 2 mL were used as the starting material to evaluate DNA template preparation using the FTA® manufacturer’s protocol for disc purification against elution of DNA from the FTA® using Prepfiler™ Forensic DNA Extraction Kit. AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit was used to amplify the STR markers, and the PCR products were analysed using Applied Biosystems™ 3500xL Genetic Analyzer. The DNA profile qualities were examined in terms of number of loci detected and peak height balance. Comparisons with the profiles obtained from DNA isolated using QIAamp® DNA Micro Kit from 1 and 2 mL of the same batch of urine were also made. The optimised protocol was then tested on urine samples from three male volunteers. The results showed that the purification of FTA® punches according to the manufacturer’s protocol enabled full DNA profiles to be obtained from both 1 and 2 mL of urine from all samples tested, including male samples. In contrast, no DNA profile could be generated from the DNA eluted with the Prepfiler™ kit. When compared with the more conventional solid-phase DNA extraction method, the profiles generated from the FTA® punches exhibited similar reproducibility and quality to those from the template isolated by the QIAamp® Kit. This work further demonstrated the feasibility of FTA® cards as a tool for specimen storage and DNA template preparation from small volumes of urine for authentication by STR profiling. Full STR profiles could be generated from sample from both sexes without modification of the PCR conditions or injection time.     

The effect of NucleoSpin® Forensic Filters on DNA recovery from trace DNA swabs

Forensic DNA profiling is a globally accepted method for human identification, however, obtaining full DNA profiles from trace DNA can be challenging. The optimal recovery of DNA from trace DNA swabs is therefore crucial. Methods for extracting DNA from swabs often make use of a spin basket combined with a centrifugation step, to enhance the release of cells from the swab prior to DNA extraction. The NucleoSpin® Forensic Filter (Macherey-Nagel, Düren) is a type of spin basket, but it has not been thoroughly assessed on trace DNA samples. This study aimed to assess if the inclusion of the NucleoSpin® Forensic Filter significantly improved DNA recovery and DNA profiling success from cotton and flocked swabs used to collect trace DNA and buccal cells (control). Buccal cells and trace DNA samples were collected from 25 volunteers using each swab type (cotton and flocked) in duplicate. DNA was extracted from the samples using the NucleoSpin® DNA Forensic kit, one set with, and the other set without, NucleoSpin® Forensic Filters. DNA concentration was assessed using real time PCR, and DNA profiling was done using the PowerPlex® ESX 16 system. The inclusion of the NucleoSpin® Forensic Filters significantly improved DNA concentration for buccal cells that were collected using flocked swabs (p = 0.035). However, no significant differences were noted for trace DNA samples for either swab type. There was also no significant difference in DNA profiling success when NucleoSpin® Forensic Filters were used, regardless of swab and sample type. These results may be helpful for laboratories that are considering the NucleoSpin® Forensic Filters in the DNA extraction workflow, particularly for trace DNA samples.     

The use of the M-Vac® wet-vacuum system as a method for DNA recovery

Collecting sufficient template DNA from a crime scene sample is often challenging, especially with low quantity samples such as touch DNA (tDNA). Traditional DNA collection methods such as double swabbing have limitations, in particular when used on certain substrates which can be found at crime scenes, thus a better collection method is advantageous. Here, the effectiveness of the M-Vac® Wet-Vacuum System is evaluated as a method for DNA recovery on tiles and bricks. It was found that the M-Vac® recovered 75% more DNA than double swabbing on bricks. However, double swabbing collected significantly more DNA than the M-Vac® on tiles. Additionally, it was found that cell-free DNA is lost in the filtration step of M-Vac® collection. In terms of peak height and number of true alleles detected, no significant difference was found between the DNA profiles obtained through M-Vac® collection versus double swabbing of tDNA depositions from 12 volunteers on bricks. The results demonstrate that the M-Vac® has potential for DNA collection from porous surfaces such as bricks, but that alterations to the filter apparatus would be beneficial to increase the amount of genetic material collected for subsequent DNA profiling. These results are anticipated to be a starting point to validate the M-Vac® as a DNA collection device, providing an alternative method when DNA is present on a difficult substrate, or if traditional DNA collection methods have failed.     

Comparison of new Ampac bags and FireDebrisPAK® bags as packaging for fire debris analysis

Abstract: The FireDebrisPAK^® bags that were produced by Kapak were considered to be one of the best containers for fire debris. Kapak bags were discontinued; however, from July 2010, Ampac is offering a new packaging material. The aim of the presented research was to compare the properties (durability, background interferences, and permeability) of Kapak bags and packaging material offered by Ampac. The analysis was conducted by passive adsorption from the headspace with subsequent thermal desorption and analysis by GC‐MS. The results proved that the properties of the compared materials are similar. Their greatest advantage is that they are impermeable for components of flammable liquids, so there is no danger of losing analytes or cross‐contamination. Their one significant drawback is that they should not be exposed to temperatures above 80°C. At this temperature, they become soft, their inner layer is compromised (becomes sticky), and they emit some volatile organic compounds. Among them, there are compounds that constitute the components of some of flammable liquids.     

GeneMarker® HID: A reliable software tool for the analysis of forensic STR data

Abstract: GeneMarker^® HID was assessed as a software tool for the analysis of forensic short tandem repeat (STR) data and as a resource for analysis of custom STR multiplexes. The software is easy to learn and use, and includes design features that have the potential to reduce user fatigue. To illustrate reliability and accuracy, STR data from both single‐source and mixture profiles were analyzed and compared to profiles interpreted with another software package. A total of 1898 STR profiles representing 28,470 loci and more than 42,000 alleles were analyzed with 100% concordance. GeneMarker HID was also used to successfully analyze data generated from a custom STR multiplex, with simplified and rapid implementation. Finally, the impact of the user‐friendly design features of the software was assessed through a time scale study. The results suggest that laboratories can reduce the time required for data analysis by at least 25% when using GeneMarker HID.     

Powerplex® Y 23 System: Molecular characterization of a null allele at locus DYS549

We observed a null allele pattern at locus DYS549 in a male subject from North-East Italy typed with the PowerPlex^® Y 23 System (Promega). To investigate whether this pattern was due to the presence of a microdeletion/mutation in primer binding sites or in the locus target region, the sample was amplified with our designed DYS549 primers obtained from GenBank sequence (GDB: 515022). After amplification, a normal hemizygous genotype at this locus was generated, thus indicating the presence of a point mutation in the binding site of the original primer set of PowerPlex^® Y 23 System (Promega). This was further confirmed by sequence analysis, carried out with the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), according to the manufacturer's instructions. Sequences were run on the ABI Prism 3130 Genetic Analyzer (Applied Biosystems) and analyzed using the Sequencing Analysis v.5.3.1 and the SeqScape v2.6 softwares (Applied Biosystems). Ascertainment of the frequency of null alleles generated from variations at primer binding sites of short tandem repeats loci is of great importance in forensic genetics.     

Forensic genetic value of 27 Y-STR loci (Y-Filer® Plus) in the South African population

South Africa has one of the highest rape statistics in the world, with an average of 117 rapes reported daily. Y-STR genotyping is becoming a popular tool in the analysis of DNA evidence collected after a crime of a sexual nature has been committed, but has yet to be implemented in South Africa’s forensic laboratories. This study aimed to investigate the forensic value of the 27 Yfiler™ Plus loci in the South African population. A total of 271 samples from the African, Asian/Indian, Mixed Ancestry¹, and Caucasian populations at the University of the Free State in Bloemfontein, South Africa were amplified and analysed using ThermoFisher Scientific’s Yfiler™ Plus PCR Amplification kit. Of the 271 samples, 261 were identified to be unique, with an overall discrimination capacity of 98.15%. Discrimination capacities ranged from 91.67% for the Asian/Indian population to 100% for the Mixed Ancestry population. The haplotype diversity across the four populations is 0.9999, with an average gene diversity across all loci of 0.717. The forensic parameters estimated in this study provide evidence for the potential use of the commercial Yfiler™ Plus PCR amplification kit in a forensic application in South Africa.     

DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones.     

Validation of the Seratec® SeraQuant™ for the quantitation of prostate-specific antigen levels on immunochromatographic membranes

Abstract: Use of immunochromatographic membranes for the detection of prostate‐specific antigen (PSA) has become commonplace in forensic laboratories. Experiments were designed to test the newly developed Seratec^® SeraQuant? for accuracy, precision, and consistency in the quantitation of PSA. PSA standards were diluted with buffers and run on the instruments. Values obtained were examined for accuracy (was the correct value obtained?) and precision (were multiple sample values consistent?). To test for variation between instruments, large volumes of diluted PSA standard were run repeatedly on six units and the values obtained were plotted against the known PSA values to obtain a standard curve for each instrument. Fifty membranes having negative or weak positive results were then run on the six units, and the adjusted values were recorded and compared. Results of these experiments indicate that the instruments are accurate and precise in the quantitation of low levels of PSA.