Mitochondrial DNA diversity of present-day Aboriginal Australians and implications for human evolution in Oceania.

Aboriginal Australians are one of the more poorly studied populations from the standpoint of human evolution and genetic diversity. Thus, to investigate their genetic diversity, the possible date of their ancestors' arrival and their relationships with neighboring populations, we analyzed mitochondrial DNA (mtDNA) diversity in a large sample of Aboriginal Australians. Selected mtDNA single-nucleotide polymorphisms and the hypervariable segment haplotypes were analyzed in 594 Aboriginal Australians drawn from locations across the continent, chiefly from regions not previously sampled. Most (~78%) samples could be assigned to mtDNA haplogroups indigenous to Australia. The indigenous haplogroups were all ancient (with estimated ages >40 000 years) and geographically widespread across the continent. The most common haplogroup was P (44%) followed by S (23%) and M42a (9%). There was some geographic structure at the haplotype level. The estimated ages of the indigenous haplogroups range from 39 000 to 55 000 years, dates that fit well with the estimated date of colonization of Australia based on archeological evidence (~47 000 years ago). The distribution of mtDNA haplogroups in Australia and New Guinea supports the hypothesis that the ancestors of Aboriginal Australians entered Sahul through at least two entry points. The mtDNA data give no support to the hypothesis of secondary gene flow into Australia during the Holocene, but instead suggest long-term isolation of the continent.

Antiquity and diversity of aboriginal Australian Y-chromosomes.

OBJECTIVE: Understanding the origins of Aboriginal Australians is crucial in reconstructing the evolution and spread of Homo sapiens as evidence suggests they represent the descendants of the earliest group to leave Africa. This study analyzed a large sample of Y-chromosomes to answer questions relating to the migration routes of their ancestors, the age of Y-haplogroups, date of colonization, as well as the extent of male-specific variation. METHODS: Knowledge of Y-chromosome variation among Aboriginal Australians is extremely limited. This study examined Y-SNP and Y-STR variation among 657 self-declared Aboriginal males from locations across the continent. 17 Y-STR loci and 47 Y-SNPs spanning the Y-chromosome phylogeny were typed in total. RESULTS: The proportion of non-indigenous Y-chromosomes of assumed Eurasian origin was high, at 56%. Y lineages of indigenous Sahul origin belonged to haplogroups C-M130*(xM8,M38,M217,M347) (1%), C-M347 (19%), K-M526*(xM147,P308,P79,P261,P256,M231,M175,M45,P202) (12%), S-P308 (12%), and M-M186 (0.9%). Haplogroups C-M347, K-M526*, and S-P308 are Aboriginal Australian-specific. Dating of C-M347, K-M526*, and S-P308 indicates that all are at least 40,000 years old, confirming their long-term presence in Australia. Haplogroup C-M347 comprised at least three sub-haplogroups: C-DYS390.1del, C-M210, and the unresolved paragroup C-M347*(xDYS390.1del,M210). CONCLUSIONS: There was some geographic structure to the Y-haplogroup variation, but most haplogroups were present throughout Australia. The age of the Australian-specific Y-haplogroups suggests New Guineans and Aboriginal Australians have been isolated for over 30,000 years, supporting findings based on mitochondrial DNA data. Our data support the hypothesis of more than one route (via New Guinea) for males entering Sahul some 50,000 years ago and give no support for colonization events during the Holocene, from either India or elsewhere.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Characterisation of novel and rare Y-chromosome short tandem repeat alleles in self-declared South Australian Aboriginal database.

Y-chromosome short tandem repeats (Y-STRs) are used in forensic science laboratories all over the world, as their application is wide and often vital in solving casework. Analysis of an in-house database of South Australian self-declared Aboriginal males held by Forensic Science South Australia (FSSA) using the Applied Biosystem's AmpFℓSTR® Yfiler™ PCR Amplification Kit revealed 43 variant Y-STR alleles at 6 of the 17 loci. All variant alleles were sequenced to determine the exact repeat structure for each. As a high level of admixture has previously been found within the SA Aboriginal database, samples were haplogrouped using Y-SNPs to determine their likely geographical origin. Although a number of variant alleles were associated with non-Aboriginal Y-haplogroups, a high frequency was observed within the Australian K-M9 lineage. Detailed knowledge of these variant alleles may have further application in the development of new DNA markers for identification purposes, and in population and evolutionary studies of Australian Aborigines.

A transparent approach: Openness in forensic science reporting.

There have been numerous calls for increased transparency and disclosure in forensic science. However, there is a paucity of guidance on how to achieve this transparency in reports, and the impacts it may have on criminal justice proceedings. We describe one multi-disciplinary forensic laboratory's journey to fully transparent reporting, disclosing matters of scientific relevance and importance. All expert reports across 17 disciplines now contain information regarding the fundamental principles and methodology, validity and error, assumptions and limitations, competency testing and quality assurance, cognitive factors, and areas of scientific controversy. Staff support for transparent reporting increased following introduction, with most reporting largely positive impacts. A slight increase in questioning in court has been experienced, with increased legal attention paid to the indicia of scientific validity. Transparency in expert forensic science reports is possible, and can improve the use of scientific evidence in courts without compromising the timeliness of service.

Understanding 'error' in the forensic sciences: A primer.

This paper distils seven key lessons about 'error' from a collaborative webinar series between practitioners at Victoria Police Forensic Services Department and academics. It aims to provide the common understanding of error necessary to foster interdisciplinary dialogue, collaboration and research. The lessons underscore the inevitability, complexity and subjectivity of error, as well as opportunities for learning and growth. Ultimately, we argue that error can be a potent tool for continuous improvement and accountability, enhancing the reliability of forensic sciences and public trust. It is hoped the shared understanding provided by this paper will support future initiatives and funding for collaborative developments in this vital domain.

Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications.

Nonrecombining Y-chromosomal microsatellites (Y-STRs) are widely used to infer population histories, discover genealogical relationships, and identify males for criminal justice purposes. Although a key requirement for their application is reliable mutability knowledge, empirical data are only available for a small number of Y-STRs thus far. To rectify this, we analyzed a large number of 186 Y-STR markers in nearly 2000 DNA-confirmed father-son pairs, covering an overall number of 352,999 meiotic transfers. Following confirmation by DNA sequence analysis, the retrieved mutation data were modeled via a Bayesian approach, resulting in mutation rates from 3.78 × 10(-4) (95% credible interval [CI], 1.38 × 10(-5) - 2.02 × 10(-3)) to 7.44 × 10(-2) (95% CI, 6.51 × 10(-2) - 9.09 × 10(-2)) per marker per generation. With the 924 mutations at 120 Y-STR markers, a nonsignificant excess of repeat losses versus gains (1.16:1), as well as a strong and significant excess of single-repeat versus multirepeat changes (25.23:1), was observed. Although the total repeat number influenced Y-STR locus mutability most strongly, repeat complexity, the length in base pairs of the repeated motif, and the father's age also contributed to Y-STR mutability. To exemplify how to practically utilize this knowledge, we analyzed the 13 most mutable Y-STRs in an independent sample set and empirically proved their suitability for distinguishing close and distantly related males. This finding is expected to revolutionize Y-chromosomal applications in forensic biology, from previous male lineage differentiation toward future male individual identification.

Sex and gender issues in competitive sports: investigation of a historical case leads to a new viewpoint.

Based on DNA analysis of a historical case, the authors describe how a female athlete can be unknowingly confronted with the consequences of a disorder of sex development resulting in hyperandrogenism emerging early in her sports career. In such a situation, it is harmful and confusing to question sex and gender. Exposure to either a low or high level of endogenous testosterone from puberty is a decisive factor with respect to sexual dimorphism of physical performance. Yet, measurement of testosterone is not the means by which questions of an athlete's eligibility to compete with either women or men are resolved. The authors discuss that it might be justifiable to use the circulating testosterone level as an endocrinological parameter, to try to arrive at an objective criterion in evaluating what separates women and men in sports competitions, which could prevent the initiation of complicated, lengthy and damaging sex and gender verification procedures.

mRNA-based skin identification for forensic applications.

Although the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in forensic analyses. We examined 11 candidate genes with skin-specific expression, as ascertained from expression databases and the literature, as well as five candidate reference genes ascertained from previous studies, in skin samples and in other forensically relevant tissues. We identified mRNA transcripts from three genes CDSN, LOR and KRT9, showing strong over-expression in skin samples relative to samples from forensic body fluids, making them suitable markers for skin identification. Out of the candidate reference genes tested, only ACTB showed similarly high expression in skin and body-fluid samples, providing a suitable reference marker for quantitative real-time PCR (qPCR) analysis of skin. Analyses of palmar and thumbprint skin samples indicate that our qPCR approach for the three skin-targeted mRNA markers, as well as the reference mRNA marker ACTB, is highly sensitive, allowing successful detection of minute amounts of skin material including full, half and quarter thumbprints, albeit with decreased success in decreasing print material. Furthermore, thumbprints stored for 6.5 months provided similar results relative to freshly analysed samples implying reasonable time-wise stability of the three skin-targeted mRNAs as well as the ACTB reference mRNA. Our study represents the first attempt towards reliable mRNA-based skin identification in forensic applications with particular relevance for future trace/touched object analyses in forensic case work. Although the approach for skin identification introduced here can be informative when applied on its own, we recommend for increased reliability the integration of (one or more of) the skin-targeted mRNA markers presented here into multiplex assays additionally including mRNA markers targeting alternative cell types expected in forensic samples.

The forensic examination of structural fires in Victoria, Australia: Decision-making processes and impact on judicial outcomes.

There is a body of published research that has evaluated the contribution of forensic science to the criminal justice system, but many disciplines of forensic science remain unexplored in this regard. The aim of this study was to examine the contribution that forensic fire examination services provide to criminal investigations and court processes in arson cases. Forensic fire examination services differ in a number of ways to the disciplines covered in previous research on the impact of forensic evidence on justice outcomes. Forensic fire examinations involve a combination of scene examination and laboratory analyses, and the results can provide critical evidence of whether an incident that has occurred is a criminal offence (i.e. whether a fire has occurred as the result of an act of arson). Forensic fire examination is also a discipline that has faced challenges and undergone development in recent decades regarding its scientific basis and the issue of contextual bias. In this study, data were collated for 273 structural fires that were examined by the forensic fire services in Victoria, Australia. In this jurisdiction, scene and laboratory forensic services are delivered within short time frames with a focus on providing impartial scientific and investigative services to assist criminal investigations conducted by police. The current dataset was highly skewed in terms of criminal justice outcomes and was not suitable for conducting the planned statistical analyses. Nonetheless, the pattern of findings obtained suggested that the inclusion of forensic evidence which supported the prosecution of arson may be associated with an increased likelihood of suspects being charged and defendants found guilty. Examination of the decision-making process of the forensic fire examiners has provided insight into the variety of evidence that is considered by forensic experts in reaching the important conclusion about the origin and cause of structural fires.

Estimating trace deposition time with circadian biomarkers: a prospective and versatile tool for crime scene reconstruction.

Linking biological samples found at a crime scene with the actual crime event represents the most important aspect of forensic investigation, together with the identification of the sample donor. While DNA profiling is well established for donor identification, no reliable methods exist for timing forensic samples. Here, we provide for the first time a biochemical approach for determining deposition time of human traces. Using commercial enzyme-linked immunosorbent assays we showed that the characteristic 24-h profiles of two circadian hormones, melatonin (concentration peak at late night) and cortisol (peak in the morning) can be reproduced from small samples of whole blood and saliva. We further demonstrated by analyzing small stains dried and stored up to 4 weeks the in vitro stability of melatonin, whereas for cortisol a statistically significant decay with storage time was observed, although the hormone was still reliably detectable in 4-week-old samples. Finally, we showed that the total protein concentration, also assessed using a commercial assay, can be used for normalization of hormone signals in blood, but less so in saliva. Our data thus demonstrate that estimating normalized concentrations of melatonin and cortisol represents a prospective approach for determining deposition time of biological trace samples, at least from blood, with promising expectations for forensic applications. In the broader context, our study opens up a new field of circadian biomarkers for deposition timing of forensic traces; future studies using other circadian biomarkers may reveal if the time range offered by the two hormones studied here can be specified more exactly.

To trace or not to trace: A survey of how police use and perceive chemical trace evidence.

There is limited information available about the impact of chemical trace evidence and it has tended to be anecdotal and mostly pertaining to court outcomes. Very little is known about the use of chemical trace evidence by police investigators or the impact that this evidence form has on criminal investigations. This survey, which was conducted in Victoria, Australia, was aimed at addressing these inadequacies by capturing information from police investigators about: (i) the purpose of using chemical trace and other forensic services; (ii) the expectation of what value forensic services would provide; (iii) the actual impact of forensic evidence in specified cases; and (iv) the general perceptions of forensic science. Police officers who were the lead investigators in a sample of criminal investigations were selected as the subjects for this survey. Each of the sample cases included chemical trace evidence and many of the cases also included other forms of forensic evidence. The police investigators indicated that they use chemical trace evidence with the expectation that it will assist decision-making in their investigations and contribute to building a case for court. Survey responses indicated that chemical trace evidence can impact on multiple stages of a case and that this form of evidence can play a part in guiding police investigators in making decisions about how their cases progress through the criminal justice system. It was found that an important aspect of the impact of chemical trace evidence can involve connections with other forensic and non-forensic evidence in the cases. The provision of preliminary results, prior to the formal written reports that are issued for use in court, enables chemical trace evidence to contribute timely support to investigations. The findings of this survey study contradict prevailing perceptions that the contribution of chemical trace evidence is limited to the presentation of evidence in court.

Communicating forensic science opinion: An examination of expert reporting practices.

Forensic scientists endeavour to explain complex scientific principles to legal decision-makers with limited scientific training (e.g., police, lawyers, judges, and jurors). Much of the time this communication is limited to written opinions in expert reports. Notwithstanding considerable scientific research and debate about the best way to communicate forensic science opinions, it is unclear how much of the advice has translated into forensic science practice. In conducting this descriptive study, we examined the reporting practices adopted by forensic scientists across a range of forensic science disciplines. Specifically, we used a quantitative content analysis approach to identify the conclusion types and additional information submitted by forensic scientists in proficiency tests during 2016 ("What would be the wording of the Conclusions in your report?"). Our analysis of 500 randomly selected responses in eight disciplines indicated that the conclusion type which has received the most criticism in recent years (categorical statements) remains the preferred means of expression in a clear majority of responses. We also found that the provision of additional information often considered necessary for rational evaluation of the evidence (e.g., information about reliability and validity) was rarely reported. These results suggest limited engagement with recent recommendations and are concerning given the gravity of the legal decisions that hinge on accurate and transparent forensic science communication.

The impact of chemical trace evidence on justice outcomes: Exploring the additive value of forensic science disciplines.

The focus of this research was to examine the contribution chemical trace evidence makes to criminal justice outcomes. The aim of this work was to place the discipline of chemical trace evidence under the spotlight as there is a dearth of robust research on the impact of this discipline. In this study, data relating to the forensic examinations in a sample of 238 cases which included chemical trace evidence, was collated with data from police investigations and court processes. The findings show that chemical trace evidence is frequently used in combination with other forensic disciplines to support the progress of high-level criminal cases through the justice system. Due to characteristics of how the criminal cases in the dataset were investigated and prosecuted, in combination with the methodology applied in this study, the impact of forensic evidence on the decision to charge suspects could not be analysed quantitatively. However, the impact of forensic evidence on court outcomes in the sample of cases was analysed using methodology that considered the results of the examinations, and the ability of the evidence to provide support for the inclusion or exclusion of persons of interest. The possibility of chemical trace evidence having impact when applied in combination with other forensic disciplines was also examined. It was found that biological examination results was a significant standalone predictor of court outcomes. In contrast, chemical trace examinations did not predict court outcomes when considered as a standalone predictor but examination results of chemical trace evidence in combination with ballistics/tool marks was significantly associated with court outcomes. The findings of this research indicate that, to assess the full impact of any discipline of forensic evidence on the criminal justice system, the analysis must take into account the potential for important synergies that may exist with other forensic and non-forensic evidence.

Forensic science evidence: Naive estimates of false positive error rates and reliability.

We do not know how often false positive reports are made in a range of forensic science disciplines. In the absence of this information it is important to understand the naive beliefs held by potential jurors about forensic science evidence reliability. It is these beliefs that will shape evaluations at trial. This descriptive study adds to our knowledge about naive beliefs by: (1) measuring jury-eligible (lay) perceptions of reliability for the largest range of forensic science disciplines to date, over three waves of data collection between 2011 and 2016 (n=674); (2) calibrating reliability ratings with false positive report estimates; and (3) comparing lay reliability estimates with those of an opportunity sample of forensic practitioners (n=53). Overall the data suggest that both jury-eligible participants and practitioners consider forensic evidence highly reliable. When compared to best or plausible estimates of reliability and error in the forensic sciences these views appear to overestimate reliability and underestimate the frequency of false positive errors. This result highlights the importance of collecting and disseminating empirically derived estimates of false positive error rates to ensure that practitioners and potential jurors have a realistic impression of the value of forensic science evidence.

Determination of the maximum distance blood spatter travels from a vertical impact.

Bloodstain evidence can be very powerful evidence in assault related crimes. Determination of the distance that blood droplets may travel as a result of an impact into liquid blood may be of significance to corroborate or disprove a version of events, provide likely scenarios, or help determine the culpability of a person in determining their proximity to the blood shedding event. It was the aim of this research to determine the potential maximum distance blood droplets travel horizontally following a vertical impact into liquid blood. A custom apparatus was designed and constructed to replicate a vertical impact of a timber weapon, rotating on a fixed axis at one end, striking a pool of liquid blood. The device was positioned at three different levels of elevation to replicate an impact to the head of a person near ground level, a seated or kneeling height and standing height. Overall, the results indicated that the application of kinetic energy of between 1 and 5J at a height of 1780mm led to the blood droplets travelling a maximum horizontal distance of 5361mm (and average maximum distance of 4981mm). The horizontal distance blood droplets may travel upon impact does not appear to follow a linear trend with differing kinetic energy, but is affected by the applied force and release height in a curvilinear relationship. The results provide a valuable tool to bloodstain pattern analysts and investigators in determining search zones within a scene, as well as providing information about the proximity of an individual to an impact event.

Transfer and persistence of non-self DNA on hands over time: Using empirical data to evaluate DNA evidence given activity level propositions.

Questions relating to how DNA from an individual got to where it was recovered from and the activities associated with its pickup, retention and deposition are increasingly relevant to criminal investigations and judicial considerations. To address activity level propositions, investigators are typically required to assess the likelihood that DNA was transferred indirectly and not deposited through direct contact with an item or surface. By constructing a series of Bayesian networks, we demonstrate their use in assessing activity level propositions derived from a recent legal case involving the alleged secondary transfer of DNA to a surface following a handshaking event. In the absence of data required to perform the assessment, a set of handshaking simulations were performed to obtain probabilities on the persistence of non-self DNA on the hands following a 40min, 5h or 8h delay between the handshake and contact with the final surface (an axe handle). Variables such as time elapsed, and the activities performed and objects contacted between the handshake and contact with the axe handle, were also considered when assessing the DNA results. DNA from a known contributor was transferred to the right hand of an opposing hand-shaker (as a depositor), and could be subsequently transferred to, and detected on, a surface contacted by the depositor 40min to 5h post-handshake. No non-self DNA from the known contributor was detected in deposits made 8h post-handshake. DNA from the depositor was generally detected as the major or only contributor in the profiles generated. Contributions from the known contributor were minor, decreasing in presence and in the strength of support for inclusion as the time between the handshake and transfer event increased. The construction of a series of Bayesian networks based on the case circumstances provided empirical estimations of the likelihood of direct or indirect deposition. The analyses and conclusions presented demonstrate both the complexity of activity level assessments concerning DNA evidence, and the power of Bayesian networks to visualise and explore the issues of interest for a given case.

Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples.

The analysis of LCN or highly degraded DNA samples presents a challenge for forensic science. Improving the quantity and/or quality of samples would greatly increase the profiling success rate from LCN and degraded samples. Whole genome amplification (WGA) is one method that has such potential. Two commercially available WGA kits, GenomePlex and GenomiPhi, were investigated for use on LCN and degraded DNA samples. Both kits amplified genomic DNA, producing microgram quantities from sub-nanogram templates. Profiling success of LCN DNA samples was increased, with improvements of over 700% from 10pg template DNA compared to non-WGA-amplified control samples. The amplification success with degraded DNA was also improved by WGA. Degraded DNA was simulated using restriction enzymes to demonstrate that the application of WGA can result in the typing of STR loci that could not previously be amplified. An increase in artefacts, such as stutter alleles and amplification biases, were observed in many samples. Results show that WGA is capable of increasing both the quality and quantity of DNA, and has the potential to improve profiling success from difficult samples in forensic casework.

Peer review in forensic science.

Peer review features prominently in the forensic sciences. Drawing on recent research and studies, this article examines different types of peer review, specifically: editorial peer review; peer review by the scientific community; technical and administrative review; and verification (and replication). The article reviews the different meanings of these quite disparate activities and their utility in relation to enhancing performance and reducing error. It explains how forensic practitioners should approach and use peer review, as well as how it should be described in expert reports and oral testimony. While peer review has considerable potential, and is a key component of modern quality management systems, its actual value in most forensic science settings has yet to be determined. In consequence, forensic practitioners should reflect on why they use specific review procedures and endeavour to make their actual practices and their potential value transparent to consumers; whether investigators, lawyers, jurors or judges. Claims that review increases the validity of a scientific technique or accuracy of opinions within a particular case should be avoided until empirical evidence is available to support such assertions.

DNA decontamination of fingerprint brushes.

Genetic profiling of DNA collected from fingerprints that have been exposed to various enhancement techniques is routine in many forensic laboratories. As a result of direct contact with fingermark residues during treatment, there is concern around the DNA contamination risk of dusting fingermarks with fingerprint brushes. Previous studies have demonstrated the potential for cross-contamination between evidentiary items through various mechanisms, highlighting the risk of using the same fingerprint brush to powder multiple surfaces within and between crime-scenes. Experiments were performed to assess the contamination risk of reused fingerprint brushes through the transfer of dried saliva and skin deposits from and to glass surfaces with new unused squirrel hair and fiberglass brushes. Additional new unused brushes and brushes previously used in casework were also tested for their ability to contaminate samples. In addition, the ability to eradicate DNA from used squirrel hair and fiberglass fingerprint brushes was assessed using a 1% sodium hypochlorite solution and a 5% solution of a commercially available alternative, Virkon. DNA profiling results from surfaces contacted by treated and untreated brushes were compared to determine the effectiveness of the devised cleaning protocol. Brush durability was also assessed over multiple wash/rinse/dry cycles with both agents. Varying amounts of DNA-containing material were collected and transferred by squirrel hair and fiberglass brushes, with detectability on the secondary surface dependent on the biological nature of the material being transferred. The impact of DNA contamination from dirty fingerprint brushes was most apparent in simulations involving the transfer of dried saliva and brushes previously used in casework, while minimal transfer of touch DNA was observed. Alarmingly, large quantities of DNA were found to reside on new unused squirrel hair brushes, while no DNA was detected on new unused fiberglass brushes or brushes sold as DNA-free. Squirrel hair brushes were easily and effectively cleaned with both hypochlorite and Virkon, with no evidence of DNA transfer between exhibits by treated brushes. Brushes were still deemed useable after multiple cleaning cycles with either agent. In contrast, fiberglass bristles became tangled and matted when wet and could not be cleaned effectively using either method. It is recommended they are disposed of following use. Each laboratory should consider their current circumstances before adapting a cleaning method. The implementation of a program to monitor the effectiveness of the cleaning regime is also advised.