Assessment of changes to DNA database interrogation at Forensic Science SA

ABSTRACT

The ultimate goal of database casework is to supply investigative information to authority agencies. Traditionally this has been accomplished by direct matching. Recently, Forensic Science SA (FSSA) made changes to the DNA profile upload criteria for direct matching. This was made possible in-house by the introduction of a new Laboratory Information Management System (LIMS). In conjunction with direct matching for database investigations, FSSA also routinely perform mixture searches on unresolved DNA mixtures using the expert forensic software STRmix. Data from the FSSA no suspect workflow was collected for a 10 month period from September 2017 to June 2018 to determine the impact of these two changes compared with previous direct matching processes over the same period. The data demonstrate an increase in investigative information provided to the South Australian Police (SAPOL) by FSSA.     

A review of DNA profiling success for laser microdissected forensic casework samples

ABSTRACT

Laser microdissection (LMD) is a tool that is used in forensic laboratories for the analysis of DNA from specifically targeted cells. Since 2010, the Institute of Environmental Science and Research Limited’s (ESR) Forensic Biology laboratory has applied LMD DNA testing to a variety of sexual assault casework samples where small numbers of sperm are present in cell mixtures. In this paper we review the DNA profiling results obtained from semen-stained casework samples that have been analysed using the LMD DNA methodology developed in our laboratory. Dissected sperm have been analysed using the AmpFISTR Identifiler amplification kit at 28 cycle PCR, the AmpFISTR MiniFiler amplification kit at 30 cycle PCR, or the AmpFISTR SGM Plus amplification kit at 34 cycle PCR, depending on the number of sperm recovered and on consideration of other circumstantial case information, such as the time since intercourse (TSI). From a review of these data, success rates for different sample numbers of sperm recovered from semen-stained samples are determined. The DNA profiling results obtained from three cases where laser microdissection has been used are also presented to demonstrate the success of the LMD testing strategy in a forensic laboratory.     

Exploring the DNA mixture deconvolution through simulation

ABSTRACT

If an unambiguous single-source DNA profile is obtained from a crime scene, then a potential person of interest can either match or not match the crime scene profile and the likelihood ratio for the single matching genotype can be easily computed. Mixed DNA profiles on the other hand are typically ambiguous and a vast number of different likelihood ratios can be obtained depending on the genotype of a potential person of interest that is compared with the mixture later. In the absence of a person of interest it can be unclear how suitable the profile is for discriminating between donors and non-donors. We introduce a simulation method to explore the range of likelihood ratios that is expected to be obtained when a non-donor or a true donor is compared with the mixed DNA profile. Sampling is conditional on the mixture deconvolution obtained using probabilistic genotyping. These simulations help to decide whether or not a (mixed) profile is suitable for comparison to a person of interest. Moreover, the methods can be used to determine whether a profile is suitable for upload to a database and whether or not potential rework could be advised.     

Separation of SNP profiles from DNA mixtures with two contributors via massively parallel sequencing technology

ABSTRACT

Massively parallel sequencing technology offers the opportunity to analyse forensically challenging samples, such as degraded samples and mixtures. In the current study, we developed a perl-based pipeline to separate the DNA mixture into its components and to predict the most probable single nucleotide polymorphism (SNP) genotypes of each contributor to the mixed profile. We examined the usefulness of this method by detecting both artificially constructed DNA mixtures and mixtures from crime cases using the Precision ID Identity Panel on the Ion PGM platform. The separated genotypes of mixtures were validated by genotypes of each of the donors detected independently. The results indicate that the method performed well in identifications of both the artificially constructed mixtures and case-type mixtures, even when the two contributors are immediate relatives (mother and son), which demonstrated the practical usefulness of this method in forensic casework. Our research presents an efficient and different strategy for identification and paternity testing of DNA mixtures in forensic genetics.     

Self-reported throwing volumes are not a valid tool for monitoring throwing loads in elite Australian cricket players: An observational cohort study

ObjectivesTo determine the concurrent validity of player self-reported and independently observed throwing volume. Examine whether sex, playing position, or time to upload self-reported data post training influences the accuracy of self-reported throwing loads.DesignCross-sectional cohort study.MethodsA total of 8 female and 18 male elite cricket players participated in the study. Overarm throws from 12 training sessions during the 2020–21 cricket year were observed. Player self-reported throwing volume data were retrieved post training, with the time difference between session completion and self-reported data upload recorded.ResultsA moderate positive correlation was found between self-reported and observed throwing loads (rho = 0.65), however only 22 % of players reported values within a 10 % level of error. Players reported a mean (SD) absolute inaccuracy of 11.17 (9.77) throws, and a mean (SD) relative inaccuracy of 24.76 (16.04) percent. Sex did not influence reporting accuracy (p = 0.41). Females tended to upload self-reported data the day of training, whereas men report the day following. Players who uploaded their data greater than one day after training were the most inaccurate with a mean relative inaccuracy of 36 %.ConclusionsWhile there is a clear relationship between observed and self-reported throwing volumes, the findings of this study question the validity of using player self-reported throwing load as a marker of true throwing loads with most players recording in excess of 10 % error. High performance staff and players should consider whether the current accuracy of self-reported throwing load justifies the additional reporting burden on the players during training.     

SNP-microarrays can accurately identify the presence of an individual in complex forensic DNA mixtures

Common forensic and mass disaster scenarios present DNA evidence that comprises a mixture of several contributors. Identifying the presence of an individual in such mixtures has proven difficult. In the current study, we evaluate the practical usefulness of currently available “off-the-shelf” SNP microarrays for such purposes. We found that a set of 3000 SNPs specifically selected for this purpose can accurately identify the presence of an individual in complex DNA mixtures of various compositions. For example, individuals contributing as little as 5% to a complex DNA mixture can be robustly identified even if the starting DNA amount was as little as 5.0 ng and had undergone whole-genome amplification (WGA) prior to SNP analysis. The work presented in this study represents proof-of-principle that our previously proposed approach, can work with real “forensic-type” samples. Furthermore, in the absence of a low-density focused forensic SNP microarray, the use of standard, currently available high-density SNP microarrays can be similarly used and even increase statistical power due to the larger amount of available information.     

Evaluation of salivary DNA obtained from dental prosthesis and its applicability in forensic investigations

AimThis study evaluated the salivary DNA obtained from dental prosthesis after a period of storage and its applicability in human identification.Material and methodsIn first phase, DNA was extracted after a span of 1 week and 1 month from the dental prosthesis dipped in whole saliva for 15 min. It was followed by PCR and electrophoresis.In second phase, from extracted DNA samples 15 STRs (short tandem repeats) of human genomic DNA were amplified via polymerase chain reaction.Results & conclusionDNA isolated from saliva stained dental prosthesis after a period of storage and the techniques employed are adequate for further forensic analysis.     

Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage

Purpose: Epidemiological evidence regarding the radiosensitivity of the lens of the eye and radiation cataract development has led to changes in the EU Basic Safety Standards for protection of the lens against ionizing radiation. However, mechanistic details of lens radiation response pathways and their significance for cataractogenesis remain unclear. Radiation-induced DNA damage and the potential impairment of repair pathways within the lens epithelium, a cell monolayer that covers the anterior hemisphere of the lens, are likely to be involved.

Materials and Methods: In this work, the lens epithelium has been analyzed for its DNA double-strand break (DSB) repair response to ionizing radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to at the very periphery of the monolayer (germinative and transitional zones). Described here are the different responses in the two regions and across four strains (C57BL/6, 129S2, BALB/c and CBA/Ca) over a low dose (0–25 mGy) in-vivo whole body X-irradiation range up to 24?hours post exposure.

Results: DNA damage and repair as visualized through 53BP1 staining was present across the lens epithelium, although repair kinetics appeared non-uniform. Epithelial cells in the central region have significantly more 53BP1 foci. The sensitivities of different mouse strains have also been compared.

Conclusions: 129S2 and BALB/c showed higher levels of DNA damage, with BALB/c showing significantly less inter-individual variability and appearing to be a more robust model for future DNA damage and repair studies. As a result of this study, BALB/c was identified as a suitable radiosensitive lens strain to detect and quantify early low dose ionizing radiation DNA damage effects in the mouse eye lens specifically, as an indicator of cataract formation.     

Robust detection of individual forensic profiles in DNA mixtures

For a forensic identification method to be admissible in international courts, the probability of false match must be quantified. For comparison of individuals against complex mixtures using a panel of single nucleotide polymorphisms (SNPs), the probability of a random man not excluded, P(RMNE) is one admissible standard. While the P(RMNE) of SNP alleles has been previously studied, it remains to be rigorously defined and calculated for experimentally genotyped mixtures. In this report, exact P(RMNE) values were calculated for a range of complex mixtures, verified with Monte Carlo simulations, and compared alongside experimentally determined detection probabilities.     

Radiation-induced DNA Damage and Its Repair

Summary

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes.

A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment ³²P-labelled at one end, it has been shown that the alkali—labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.     

单细胞凝胶电泳用于放射工作者DNA损伤评价的研究

目的 评价不同级别医院放射工作人员的DNA损伤情况,探讨不同工种、工龄与DNA损伤程度之间的关系。方法 用碱性单细胞凝胶电泳技术检测放射组和对照组外周血淋巴细胞的DNA单链断裂水平。CASP软件分析彗星图像,主要观察彗星尾部DNA百分含量(TDNA%)、彗星尾长(TL)、尾矩(TM)和Olive尾矩(OTM)等指标。 结果 放射组的TDNA%,TL、TM、OTM明显高于对照组(F=3.93, P<0.01),不同工种、工龄间均有统计学差异(F=1.83, P<0.05),不同级别医院之间放射工作者上述指标也差异存在统计学意义(F=1.91,P<0.05)。结论 所观察医院的放射工作者外周血淋巴细胞DNA均存在不同程度的损伤,高级别医院略好于低级别医院,而且DNA损伤程度与放射工龄和工种有一定的相关性。     

Diagnostic radiographers’ experience of COVID-19, Gauteng South Africa

IntroductionAs of July 2020, South Africa (SA) had the fifth highest number of COVID-19 infections in the world, with the greatest contributor of these infections, being the province of Gauteng. Diagnostic radiographers in Gauteng providing chest CT, chest radiograph and MRI services are frontline workers experiencing these unprecedented times. Therefore, this study undertook to explore diagnostic radiographers’ experiences of COVID-19.MethodsA qualitative approach using an asynchronous opened-ended online questionnaire was used to explore diagnostic radiographers’ experiences of COVID-19. Responses from purposively sampled diagnostic radiographers in Gauteng SA, underwent thematic analysis.ResultsSixty diagnostic radiographers representing both the private and public health sector responded to the questionnaire. Thematic analysis revealed three themes: new work flow and operations, effect on radiographer well-being and radiographer resilience.ConclusionBesides experiencing a shift in their professional work routine and home/family dynamics, diagnostic radiographers’ well-being has also been impacted by COVID-19. Adapting to the “new way of work” has been challenging yet their resilience and dedication to their profession, providing quality patient care and skill expertise is their arsenal to combat these challenges.Implications for practiceUnderstanding the impact of COVID-19 on diagnostic radiographers will allow radiology departments’ management, hospital management, professional bodies and educational institutions to re-evaluate provision of resources, training, employee wellness programs as well as policies and procedures.     

DNA recovery from fired hollow point ammunition

ABSTRACT

Firearm-related exhibits are often found at crime scenes. These exhibits may include the firearm, cartridges, cartridges cases or bullets. As ammunition needs to be handled to load the weapon, regardless of the action or loading type, DNA may be deposited onto the ammunition via touch. As reproducible DNA profiles have been obtained from fired cartridge casings and Improvised Explosive Device (IED) fragments, it is possible that quantifiable amounts of DNA could be recovered from fired bullets. A series of 40 Winchester PowerPoint 22LR 42 grain HP Copper Plated bullets were loaded with serially diluted cell suspensions obtained from a female donor. These were shot into 500 sheet reams of A3 paper for capture and returned to a sterile DNA laboratory for removal, extraction and quantification of DNA. Repeatable partial profiles with five reportable loci pairs consistent with the cell donor were obtained in one replicate of the neat sample. Weak partial profiles were also present in 1:2, 1:5 and 1:10 dilutions. To our knowledge, this is the first reported evidence of DNA surviving the cycle of fire and being recovered from a fired bullet under controlled conditions.     

Are radiographers suffering from symptoms of compassion fatigue due to occupational stress: A systematic review

IntroductionRepeated exposure to challenging or traumatic situations can lead to a phenomenon called compassion fatigue (CF). This can present as increased stress and anxiety in staff and a reduced patient relationship. If untreated it can lead to sickness and attrition from the profession. This systematic review aims to investigate the evidence of stressors leading to CF in diagnostic radiographers.MethodA review protocol was developed and registered on PROSPERO. Database and grey literature searches were carried out. Studies were selected against pre-defined inclusion and exclusion criteria for review. No meta-analysis was possible therefore the data were presented as a narrative.ResultsFifteen studies were selected for review published between 1982 and 2020. Evidence demonstrates that diagnostic radiographers suffer from high levels of occupational stress, however, stress is perceived rather than defined. Common causes of occupational stress were identified as poor patient interactions and a lack of time to spend with patients. There is a lack of evidence to show how this stress affects radiographer health or their ability to provide compassionate care.ConclusionDiagnostic radiographers are prone to suffering from symptoms that can be attributed to CF. This has been present for an extended period, and the main changes have been a decrease in job satisfaction and accomplishment. Patient interaction was identified as a cause, but it is unclear if this affects staff ability to be compassionate. Further work is required to find ways to mitigate these effects and prevent continued deterioration.Implications for practiceThis review has highlighted that the issue of CF may be getting worse for some radiographers and that work is required to design and implement workable interventions to try and mitigate these issues.     

Molecular efficacy of radio- and chemotherapy sequences from direct DNA damage measurements

Purpose: To investigate the molecular aspects of the synergy between ionizing radiation and platinum (Pt) chemotherapeutic agents in cancer treatment with chemoradiation therapy (CRT) by measuring damages induced by low-energy electrons (LEE) to DNA bound to cisplatin. LEE are produced abundantly by any type of ionizing radiation and cisplatin represents a typical Pt-chemotherapeutic agents.

Materials and methods: Our strategy involves two parallel administrations of cisplatin and irradiation with a 4.6 and 9.6?eV electron fluence of 1.1?×?10¹²: (1) LEE bombardment of supercoiled DNA and its subsequent reaction with cisplatin; (2) the reaction of DNA with cisplatin followed by LEE irradiation. The damage yields for the loss of supercoiled (LS), single-strand breaks (SSB) and double-strand breaks (DSB) were obtained from gel electrophoresis analysis. Base modifications were revealed by treating the samples with Escherichia coli base excision repair endonuclease (Nth and Fpg).

Results: The yields were deduced from the respective time–response for the reaction of DNA with cisplatin. The results show that binding cisplatin to DNA followed by LEE irradiation, consistently yields more DNA damages than the reverse order. In comparison to non-treated DNA, administration (2) results in an increase of LS and SSB of 1.4–3.3 folds and of DSB by more than an order of magnitude. Furthermore, after enzyme treatment, the yields of DSB rise by factors of 5.3–15.4, indicating a large increase of clustered damages, which should at least partially translate into an increase of lethal damages in cancer cells during the CRT.

Conclusions: Our results demonstrate that a strong synergy between radiation and cisplatin can only be achieved at the molecular level, if the drug is present at the time of irradiation. Furthermore, this work confirms the LEE mechanism previously proposed to explain the synergy between radiation and Pt drugs in CRT. It involves chemical sensitization of DNA prior to irradiation, to facilitate strand breaks and clustered damages induced by the highly reactive LEE.     

DNA investigations on fetal material from paternity cases

Summary Three paternity cases have been investigated where DNA was extracted from fetuses (age: 8–10 weeks old) after interruption of pregnancy. In each case it was possible to clearly identify the putative father using 5 or 6 single locus probes (SLP's). Fetal bands (SLP) could be clearly identified from mixtures of placental and fetal DNA by comparison with the maternal and paternal bands. However, it was very difficult to resolve the fragment patterns of tissue mixtures with one multi locus probe (MLP), because of band overlap. Another advantage of using SLP's was that biostatistical calculations could be carried out and very informative Essen-Möller values for the probability of paternity were obtained.     

Validation of JCountPro software for efficient assessment of ionizing radiation-induced foci in human lymphocytes

Purpose: Ionizing radiation-induced foci (IRIF) known also as DNA repair foci represent the most sensitive and specific assay for assessing DNA double-strand break (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to phosphorylated γH2AX and 53BP1. Although several approaches and software packages were developed for quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, JCountPro software was validated for IRIF enumeration in two independent laboratories.

Materials and methods: Human lymphocytes were γ-irradiated at doses of 0, 2, 5, 10 and 50 cGy. The cells were fixed, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with automatic Metafer system. Endogenous and radiation-induced γH2AX and 53BP1 foci were enumerated using JCountPro. This analysis was performed from the same cell galleries by the researchers from two laboratories. Yield of foci was analyzed by either arithmetic mean (AM) value (foci/cell) or principal average (PA) derived from the approximation of foci distribution with Poisson statistics. Statistical analysis was performed using factorial ANOVA.

Results: Enumeration of 53BP1, γH2AX and co-localized 53BP1/γH2AX foci by JCountPro was essentially the same between laboratories. IRIF were detected at all doses and linear dose response was obtained in the studied dose range. PA values from Poisson distribution fitted the data better as compared to AM values and were more powerful and sensitive for IRIF analysis than the AM values. All JCountPro data were confirmed by visual focus enumeration.

Conclusions: We concluded that the JCountPro software was efficient in objectively enumerating IRIF regardless of an individual researcher’s bias and has a potential for usage in clinics and molecular epidemiology.     

Forensic DNA phenotyping: Inferring phenotypic traits from crime scene DNA

IntroductionForensic DNA Phenotyping (FDP) has provided better understanding of various phenotypic features (e.g., height, skin colour, eye colour, structure and shape of scalp hair, baldness, facial features etc.) and associated genetic variations. The current study was designed to investigate the genetic variants and their potential contribution towards accurate phenotype prediction systems. Short Tandem Repeat (STR) based DNA typing method can be uninformative or with little potential to solve a crime in absence of suspect DNA profile in the database. Forensic DNA Phenotyping (FDP), prediction of externally visible characteristics (EVCs) from the crime scene DNA would certainly provide a new dimension to personal identification. The aim of this review paper is to highlight the significance and future prospects of FDP.ResultsA comprehensive literature review was conducted using PubMed and similar e-databases with keywords from two main components-phenotype and the associated genetic variants. To ensure a thorough literature review, searches were extended using the snowballing technique from reference lists. Key data extracted were type of study, sample characteristics (sample size, age, geographical location and ancestry), details of SNPs studied and prediction accuracies.ConclusionPhenotyping tools based on genotyping and statistical analysis for the prediction of human pigmentation are propitious in solving cold cases. This indicates the inevitability of future studies for the identification of new genetic markers for accurate prediction of phenotype or EVCs via genome-wide association study (GWAS) in diverse global populations.     

NOCIt: A computational method to infer the number of contributors to DNA samples analyzed by STR genotyping

Repetitive sequences in the human genome called short tandem repeats (STRs) are used in human identification for forensic purposes. Interpretation of DNA profiles generated using STRs is often problematic because of uncertainty in the number of contributors to the sample. Existing methods to identify the number of contributors work on the number of peaks observed and/or allele frequencies. We have developed a computational method called NOCIt that calculates the a posteriori probability (APP) on the number of contributors. NOCIt works on single source calibration data consisting of known genotypes to compute the APP for an unknown sample. The method takes into account signal peak heights, population allele frequencies, allele dropout and stutter—a commonly occurring PCR artifact. We tested the performance of NOCIt using 278 experimental and 40 simulated DNA mixtures consisting of one to five contributors with total DNA mass from 0.016 to 0.25 ng. NOCIt correctly identified the number of contributors in 83% of the experimental samples and in 85% of the simulated mixtures, while the accuracy of the best pre-existing method to determine the number of contributors was 72% for the experimental samples and 73% for the simulated mixtures. Moreover, NOCIt calculated the APP for the true number of contributors to be at least 1% in 95% of the experimental samples and in all the simulated mixtures.     

Characteristics of ethylene vinyl alcohol copolymer (EVAL) mixtures.

PURPOSETo determine physical characteristics of mixtures of ethylene vinyl alcohol copolymer (EVAL) and metrizamide dissolved in dimethyl sulfoxide, liquid materials developed for embolization of arteriovenous malformations.METHODSEVAL and dimethyl sulfoxide were mixed in various proportions and sterilized. The viscosity and density of each mixture was measured. Precipitation times were determined by dropping the mixtures into saline or human blood. The mixtures were filtered and the filtrates weighed.RESULTSDensities and viscosities of the various mixtures differed significantly, proportionally to the concentration of EVAL. Precipitation times also differed significantly, in inverse proportion to the concentration of EVAL. Temperature and aqueous solution did not affect precipitation times significantly. The weight of the filtrate significantly increased with time but was constant for each precipitation time. Temperature significantly affected filtrate weight; aqueous solution did not.CONCLUSIONSBecause of their different physical properties, the various EVAL mixtures are suited to embolizing different types of arteriovenous malformations.