Single Source Profiles Research Articles

Carrying out common DNA donor analysis using DBLR™ on two or five-cell mini-mixture subsamples for improved discrimination power in complex DNA mixtures

Probabilistic genotyping systems are able to analyse complex mixed DNA profiles and show good power to discriminate contributors from non-contributors. However, the abilities of the statistical analyses are still unavoidably bound by the quality of information being analysed. If a profile has a high number of contributors, or a contributor that is present in trace amounts, then the amount of information about those individuals in the DNA profile is limited. Recent work has shown the ability to gain better resolution of the genotypes of contributors to complex profiles using cell subsampling. This is the process of taking many sets of a limited number of cells and individually profiling each set. These ‘mini-mixtures’ can provide greater information about the genotypes of underlying contributors. In our work we take the resulting profiles from multiple subsamplings of complex DNA profiles in equal amounts and show how testing for, and then assuming, a common DNA donor can further improve the ability to resolve the genotypes of contributors. Using direct cell sub-sampling and statistical analysis software DBLR™, we were able to recover single source profiles of uploadable quality from five out of the six contributors of an equally proportioned mixture. Through the analysis of mixtures in this work we provide a template for carrying out common donor analysis for maximum effect.

Evaluating DNA Mixtures with Contributors from Different Populations Using Probabilistic Genotyping.

It is common practice to evaluate DNA profiling evidence with likelihood ratios using allele frequency estimates from a relevant population. When multiple populations may be relevant, a choice has to be made. For two-person mixtures without dropout, it has been reported that conservative estimates can be obtained by using the Person of Interest's population with a θ value of 3%. More accurate estimates can be obtained by explicitly modelling different populations. One option is to present a minimum likelihood ratio across populations; another is to present a stratified likelihood ratio that incorporates a weighted average of likelihoods across multiple populations. For high template single source profiles, any difference between the methods is immaterial as far as conclusions are concerned. We revisit this issue in the context of potentially low-level and mixed samples where the contributors may originate from different populations and study likelihood ratio behaviour. We first present a method for evaluating DNA profiling evidence using probabilistic genotyping when the contributors may originate from different ethnic groups. In this method, likelihoods are weighted across a prior distribution that assigns sample donors to ethnic groups. The prior distribution can be constrained such that all sample donors are from the same ethnic group, or all permutations can be considered. A simulation study is used to determine the effect of either assumption on the likelihood ratio. The likelihood ratios are also compared to the minimum likelihood ratio across populations. We demonstrate that the common practise of taking a minimum likelihood ratio across populations is not always conservative when FST=0. Population stratification methods may also be non-conservative in some cases. When FST>0 is used in the likelihood ratio calculations, as is recommended, all compared approaches become conservative on average to varying degrees.

A method to enable forensic genetic genealogy investigations from DNA mixtures

The presence of more than one DNA contributor in an evidentiary sample may preclude attempts to use forensic genetic genealogy to develop an investigative lead. To address this issue, we developed a workflow for deconvolution of SNP mixtures into single source profiles that are suitable for matching against a genealogical database. Using the method, two-contributor DNA mixtures assayed using a commercial SNP typing kit can produce informative match results for both major and minor contributors.

The extent of STR chimerism in different biological samples following bone marrow transplantation: A case report

Biological samples recovered from recipients of allogeneic haematopoietic stem cell transplant (HSCT) contain genetic material from both donor and him/herself. This chimeric condition can greatly complicate analysis of a DNA evidence and undermine its power of discrimination, as the specimens could be mistakenly identified as a mixed sample when in fact it originated from a single person. Profiling reference samples could help clarify the profile anomalies, however, the degree of mixture between host’s and donor’s genetic materials has been reported to vary depending on the tissue from which DNA were obtained. As a result, knowledge to select appropriate sources of reference samples that would most likely reveal the person’s pre- and post-transplant alleles becomes necessary to minimise any possible misunderstanding during the subsequent DNA profile interpretation and comparison. This work investigated the extent of chimerism present in different types of biological samples collected from an individual who had undergone a bone marrow transplant as a child (post-transplant interval, PTI >28 years). DNA profiles from buccal cells, saliva, hair roots, and fingernail clippings were generated using AmpFℓSTR™ Identifiler™ Plus/Direct PCR Amplification Kit. The donor’s STR profile was used to identify donor- and host-specific alleles (DSA and HSA, respectively), and relative donor chimerism (%Ch) of each marker was calculated. Results showed that the saliva sample contained the highest level of chimerism, with complete profile of donor being detected and the mean %Ch was 34.0 ± 4.5. Four out of eight DSA dropped out from the buccal cell profile, and the mean %Ch was 24.2 ± 1.7. Hair roots and fingernail clippings yielded single source profiles and only host’s original alleles were present. Except for the fingernail results, these observations agreed with many of the previous studies, which further demonstrated the need for raising awareness among forensic genetic laboratories regarding the type of reference samples that should be collected in future cases.

Identification of missing persons through kinship analysis by microhaplotype sequencing of single-source DNA and two-person DNA mixtures

In forensic applications, there is an increasing demand for the analysis of DNA profiles arising from missing person identification (MPI) cases. A specific DNA profile may originate from a single source or more than one contributor (i.e., a DNA mixture). When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. As a novel kind of multiallelic marker, microhaplotypes have proven promising for relatedness determination and mixture deconvolution. Herein, we developed a large panel of 185 microhaplotype markers and demonstrated its application in different scenarios of relationship inference through a simulation study and real pedigree analysis, combined with probabilistic genotyping models for data interpretation. Based on single-source profiles, it was shown that the present microhaplotype panel was sufficient for pairwise close relative testing (parent/child, full-sibling and 2nd-degree relative). For more distant relatives (3rd-degree relatives), there was a clear improvement when data from one well-chosen extra relative were available. We further sought to evaluate the theoretical systematic effectiveness and actual performance of microhaplotype markers in identifying the contribution of a missing pedigree member to a two-person mixture (as a minor donor). It was observed that 100% correct assignments were made in the balanced mixtures (with no dropout) when referenced to close relatives. When the mixture profiles suffered from dropout, incorrect assignments of minor donors were markedly associated with relatedness and the dropout level. Meanwhile, the studied scenarios generally exhibited zero or very low false-positive rates, indicating a low probability of incorrectly assigning an unrelated contributor as a close relative of the reference. Our results indicate that microhaplotype data can be reliably interpreted for identifying missing persons through kinship analysis based on DNA profiles of single-source samples or two-person mixtures. Furthermore, this study could be extended to more complex scenarios, such as determining the relatedness of contributors in (or among) mixed DNA profiles, if combined with different statistical frameworks.

Probabilistic genotyping of single cell replicates from complex DNA mixtures recovers higher contributor LRs than standard analysis

DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micro-manipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2 to 6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmix™) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2–6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (≥1020) from donors that had returned non-probative LRs (<103) by standard methods.

Can a reference ‘match’ an evidence profile if these have no loci in common?

Cold case reinvestigations are a common occurrence. Occasionally some of the original work was conducted up to 30 years ago using profiling systems of the early 1990s, which targeted HLA-DQA1, ApoB, D1S80 and D17S5. When contemporary work is carried out, if a suspect is identified they will be profiled in contemporary profiling kits such as GlobalFiler. It would be common to then also attempt to profile the evidence profiles in the same contemporary profiling kit. Imagine a scenario where two evidence samples, E1 and E2, had previously produced single-source profiles, but only E2 had any DNA extract left to re-profile with GlobalFiler. At the old loci E1 matched E2, and at the new loci E2 matched the suspect reference. Of interest to the investigation was whether anything could be said about the suspect being a donor of DNA to E1 even though the reference of the suspect and the profile from E1 had no loci in common, by using the information from the profile of E2. This paper explores that possibility.

What is she doing here? Klinefelter syndrome in forensic casework

In this case report, we describe a sexual assault incident in which the male victim’s seminal fluid contained no sperm cells, as indicated by sperm cell staining and microscopic screening, and DNA profiling results from the non-sperm cell fraction showed a major/minor DNA mixture that could be interpreted as female and male. DNA profiling of a sample from a disposable drinking cup used by the victim at the crime scene provided a single source profile, and showed a 2:1 imbalance between the heights of the X and Y chromosomes, respectively. The victim’s DNA reference sample showed a similar imbalance of the X and Y chromosomes. These observations suggested that the victim might suffer from Klinefelter syndrome, a genetic disorder related to the sex chromosomes.Here, we describe the first reported use of the QIAGEN Investigator® Argus X-12 kit for characterization of X-chromosomal STR loci to potentially identify a case of Klinefelter syndrome. This commercially available kit is primarily used in forensic laboratories to investigate kinship relations and for paternity testing in alleged father/daughter cases. Results of the X chromosome DNA profiling from the victim’s disposable drinking cup and reference samples revealed two alleles at various X-chromosomal STR loci. Moreover, this kit can also amplify a Y chromosome specific sequence (AMEL-Y), and the results indicated that this sample actually originated from a male. Evidence of two X chromosomes in the victim's DNA suggested that he was likely to have Klinefelter syndrome. In this case report, we propose the use of the QIAGEN Investigator® Argus X-12 kit as a practical forensic tool for the detection of potential genetic syndromes related to the sex chromosomes, which can affect test results and, at times, make them difficult to interpret. We also aim to increase awareness within the forensic science community regarding the existence of genetic anomalies, which should be considered when analyzing DNA profiles.

RETRACTED ARTICLE: Uncertainty in estimating the number of contributors from simulated DNA mixture profiles, with and without allele dropout, from Chinese, Malay, Indian, and Caucasian ethnic populations

Determining the number of contributors (NOC) accurately in a forensic DNA mixture profile can be challenging. To address this issue, there have been various studies that examined the uncertainty in estimating the NOC in a DNA mixture profile. However, the focus of these studies lies primarily on dominant populations residing within Europe and North America. Thus, there is limited representation of Asian populations in these studies. Further, the effects of allele dropout on the NOC estimation has not been explored. As such, this study assesses the uncertainty of NOC in simulated DNA mixture profiles of Chinese, Malay, and Indian populations, which are the predominant ethnic populations in Asia. The Caucasian ethnic population was also included to provide a basis of comparison with other similar studies. Our results showed that without considering allele dropout, the NOC from DNA mixture profiles derived from up to four contributors of the same ethnic population could be estimated with confidence in the Chinese, Malay, Indian and Caucasian populations. The same results can be observed on DNA mixture profiles originating from a combination of differing ethnic populations. The inclusion of an overall 30% allele dropout rate increased the probability (risk) of underestimating the NOC in a DNA mixture profile; even a 3-person DNA mixture profile has a > 99% risk of underestimating the NOC as two or fewer contributors. However, such risks could be mitigated when the highly polymorphic SE33 locus was included in the dataset. Lastly there was a negligible level of risk in misinterpreting the NOC in a mixture profile as deriving from a single source profile. In summary, our studies showcased novel results representative of the Chinese, Malay, and Indian ethnic populations when examining the uncertainty in NOC estimation in a DNA mixture profile. Our results would be useful in the estimation of NOC in a DNA mixture profile in the Asian context.

Recovery of single source DNA profiles from mixtures by direct single cell subsampling and simplified micromanipulation

Deconvolution of forensic DNA mixtures into their individual component DNA (geno)types is of great investigative value, though often complex and difficult. Two-person mixtures comprising a major and minor contributor are often easily interpreted although, when the DNA ratio of the two individuals is approximately equal (~1:1), deconvolution and interpretation becomes much more difficult. To address this issue, a physical separation of individual-, two- or three- cell subsamples prior to autosomal STR analysis was performed using a simplified micromanipulation technique paired with a decreased reaction volume and increased cycle number PCR. Using this method, single and multiple buccal epithelial cells were collected from a 1:1 two-person mixture (i.e. from individual 'A' and 'B') and directly amplified, omitting standard DNA extraction and purification steps. Single cell subsamples resulted in partial single-source profiles for both contributors while, in accordance with expectations of a quasi-binomial sampling schema, two- and three-cell subsamples resulted in single source informative partial profiles of individual A and individual B as well as complete consensus profiles, and equally mixed 1:1 (2-cell subsamples) and 2:1 (3-cell subsamples) admixed profiles of individual A and B.This proof-of-concept approach shows promise in permitting the DNA deconvolution of mixed samples where the individual contributors are present in similar amounts that would otherwise be difficult to interpret, resulting in an increase in evidentiary value. The subsampling approach can be readily investigated for DNA casework applications without additional investment in costly, new equipment, requiring only a stereo microscope and a tungsten needle.

ForeStatistics: A windows-based feature-rich software program for performing statistics in forensic DNA analysis, paternity and relationship testing

Forensic science is one of the most modern and applied fields of science, today and comprises of various domains. These include Fingerprints analysis, Questioned document analysis, Forensic DNA and serology, Anthropometry, Cyber and Digital forensics, and many other fields. All these fields aid the process of decision making in the courts of law and legal settings; however, DNA profiling and its analyses are one of the most important aspects of forensic science today. In Forensic DNA analysis, the statistical calculations are very important to estimate the conclusiveness of DNA evidence in forensic cases; and to establish paternity and relatedness in civil and criminal matters. These statistics, when performed manually, leave a chance of error or ambiguity in the calculation, and are hectic and time-taking. Therefore, the computer-aided approaches are opted in forensics to perform DNA statistics calculations. Keeping its importance in mind, a highly accurate windows-based software program namely ForeStatistics is proposed in this study. ForeStatistics is rich in features such as DNA statistical calculations, DNA profile management and its matching. The software can estimate random match probabilities for single-source profiles, combined probability of inclusion for mixed profiles, paternity index of a disputed child in duo and trio cases, paternity of the disputed child when the alleged father is related to mother or biological father and relatedness in cases of grandparents/grandchild, avuncular relation and cousin. It is validated through different protocols and the validation of ForeStatistics depicts that it is highly accurate in terms of performing DNA statistics or DNA profile matching. Thus, it is concluded, that ForeStatistics has a great utility in the field of Forensic DNA analysis and can help DNA scientists, in performing various DNA related statistics, accurately and very efficiently. ForeStatistics can be downloaded freely from (http://zeetu.org/forestatistics.html).

Enhanced DNA mixture deconvolution of sexual offense samples using the DEPArray™ system

The interpretation of DNA mixtures remains a significant challenge in the analysis of forensic evidence. The ability to selectively identify, collect, and analyze single cells or groups of cells has wide implications in the analysis of forensic samples and the subsequent deconvolution of DNA mixtures, particularly in the processing and interpretation of sexual offense evidence where the deconvolution of heterogeneous sources is essential. Single cell separation technology can be used to address this mixture separation challenge, specifically using the DEPArray™ system from Menarini Silicon Biosystems. We propose that the DEPArray™ will enable enhancements to the standard workflow for forensic biology/DNA analytical laboratories. We have demonstrated that the DEPArray™ workflow will lead to fewer mixture samples, enable purification of sperm and epithelial cell fractions without the need for differential extraction, improve the amplification success rate of samples and improve the interpretation of low template DNA samples. Sperm profiles were identified in 27 of 32 DEPArray™ processed samples, with 26 of 27 (96.2%) yielding single source profiles. In contrast, single source profiles were obtained from 9 of 28 (32.1%) differentially extracted samples. The use of the DEPArray™ also eliminates the need for additional confirmatory tests for the presence of human sperm and permits direct identification of the type and number of cells being analyzed eliminating the need for qPCR-based DNA quantification.

Single source DNA profile recovery from single cells isolated from skin and fabric from touch DNA mixtures in mock physical assaults

The ability to obtain DNA profiles from trace biological evidence is routinely demonstrated with so-called ‘touch DNA evidence’, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from a donor's hands to an object or person during direct physical contact. Current methods for the recovery of trace DNA employ swabs or adhesive tape to sample an area of interest. While of practical utility, such ‘blind-swabbing’ approaches will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even though the individuals' cells are principally located in topographically dispersed, but distinct, locations on the item. Thus the act of swabbing itself artifactually creates some of the DNA mixtures encountered in touch DNA samples. In some instances involving transient contact between an assailant and victim, the victim's DNA may be found in such significant excess as to preclude the detection and typing of the perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods for touch DNA evidence, we reported previously the development of a ‘smart analysis’ single cell recovery and DNA analysis method that results in enhanced genetic analysis of touch DNA evidence. Here we use the smart single cell analysis method to recover probative single source profiles from individual and agglomerated cells from various touched objects and clothing items belonging to known donors. We then use the same approach for the detection of single source male donor DNA in simulated physical contact/assault mixture samples (i.e. male ‘assailant’ grabbing the wrist, neck or clothing from the female ‘victim’, or being in transient contact with bedding from the ‘victim’). DNA profiles attributable to the male or female known donors were obtained from 31% and 35% of the single and agglomerated bio-particles (putative cells) tested. The known male donor ‘assailant’ DNA profile was identified in the cell sampling from every mixture type tested. The results of this work demonstrate the efficacy of an alternative strategy to recover single source perpetrator DNA profiles in physical contact/assault cases involving trace perpetrator/victim cellular admixtures.

Flow cytometry dataset for cells collected from touched surfaces

‘Touch’ or trace cell mixtures submitted as evidence are a significant problem for forensic laboratories as they can render resulting genetic profiles difficult or even impossible to interpret. Optical signatures that distinguish epidermal cell populations from different contributors could facilitate the physical separation of mixture components prior to genetic analysis, and potentially the downstream production of single source profiles and/or simplified mixtures. This dataset comprises the results from antibody hybridization surveys using Human Leukocyte Antigen (HLA) and Cytokeratin (CK) probes, as well as surveys of optical properties of deposited cells, including forward scatter (FSC), side scatter (SSC), and fluorescence emissions in the Allophycocyanin (APC) channel. All analyses were performed on “touch” samples deposited by several different contributors on multiple days to assess inter- and intra-contributor variability.

Analysis of fingerprint samples, testing various conditions, for forensic DNA identification

Fingerprints can be of tremendous value for forensic biology, since they can be collected from a wide variety of evident types, such as handles of weapons, tools collected in criminal cases, and objects with no apparent staining. DNA obtained from fingerprints varies greatly in quality and quantity, which ultimately affects the quality of the resulting STR profiles. Additional difficulties can arise when fingerprint samples show mixed STR profiles due to the handling of multiple persons. After applying a tested protocol for sample collection (swabbing with 5% Triton X-100), DNA extraction (using an enzyme that works at elevated temperatures), and PCR amplification (AmpFlSTR® Identifiler® using 31cycles) extensive analysis was performed to better understand the challenges inherent to fingerprint samples, with the ultimate goal of developing valuable profiles (≥50% complete). The impact of time on deposited fingerprints was investigated, revealing that while the quality of profiles deteriorated, full STR profiles could still be obtained from samples after 40days of storage at room temperature. By comparing the STR profiles from fingerprints of the dominant versus the non-dominant hand, we found a slightly better quality from the non-dominant hand, which was not always significant. Substrates seem to have greater effects on fingerprints. Tests on glass, plastic, paper and metal (US Quarter dollar, made of Cu and Ni), common substrates in offices and homes, showed best results for glass, followed by plastic and paper, while almost no profiles were obtained from a Quarter dollar. Important for forensic casework, we also assessed three-person mixtures of touched fingerprint samples. Unlike routinely used approaches for sampling evidence, the surface of an object (bottle) was sectioned into six equal parts and separate samples were taken from each section. The samples were processed separately for DNA extraction and STR amplification. The results included a few single source profiles and distinguishable two person mixtures. On average, this approach led to two profiles ≥50% complete per touched object. Some STR profiles were obtained more than once thereby increasing the confidence.

Flow cytometry analysis of epithelial cell populations from touch samples using the BD Influx flow cytometry platform

'Touch' or trace cell mixtures submitted as evidence are a significant problem for forensic laboratories as they can render resulting genetic profiles difficult or even impossible to interpret. Optical signatures that distinguish epidermal cell populations from different contributors could facilitate the physical separation of mixture components prior to genetic analysis, and potentially the downstream production of single source profiles and/or simplified mixtures. For this dataset, optical properties including forwards scatter (FSC), side scatter (SSC), and fluorescence emissions in the Allophycocyanin (APC) channel were measured in epithelial cell populations from touch samples collected from several different contributors on multiple days to assess inter- and intra-contributor variability.

Familial searching on DNA mixtures with dropout

Familial searching, the act of searching a database for a relative of an unknown individual whose DNA profile has been obtained, is usually restricted to cases where the DNA profile of that person has been unambiguously determined. Therefore, it is normally applied only with a good quality single source profile as starting point. In this article we investigate the performance of the method if applied to mixtures with and without allelic dropout, when likelihood ratios are computed with a semi-continuous (binary) model. We show that mixtures with dropout do not necessarily perform worse than mixtures without, especially if some separation between the donors is possible due to their different dropout probabilities. The familial searching true and false positive rates of mixed profiles on 15 loci are in some cases better than those of single source profiles on 10 loci. Thus, the information loss due to the fact that the person of interest's DNA has been mixed with that of other, and is affected by dropout, can be less than the loss of information corresponding to having 5 fewer loci available for a single source trace. Profiles typed on 10 autosomal loci are often involved in familial searching casework since many databases, including the Dutch one, in part consist of such profiles. Therefore, from this point of view, there seems to be no objection to extend familial searching to mixed or degraded profiles.

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &amp;#8216;Touch DNA&amp;#8217; Evidence

DNA profiles can be obtained from ‘touch DNA’ evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a ‘blind-swabbing’ approach will co-sample cellular material from the different individuals, even if the individuals’ cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim’s DNA may be found in significant excess thus masking any potential perpetrator’s DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, ‘smart analysis’ method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., “clumps”) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Enhanced genetic analysis of single human bioparticles recovered by simplified micromanipulation from forensic 'touch DNA' evidence.

DNA profiles can be obtained from ‘touch DNA’ evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a ‘blind-swabbing’ approach will co-sample cellular material from the different individuals, even if the individuals’ cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim’s DNA may be found in significant excess thus masking any potential perpetrator’s DNA.In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, ‘smart analysis’ method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., “clumps”) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Forensic DNA typing strategy of degraded DNA on discarded cigarette ends using the AmpFℓSTR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR amplification kits.

DNA left on a forensic sample is often prone to degradation, especially if left to the elements. To maximize the chance of retrieving the most information from such compromised DNA, an appropriate profiling scheme using the available technologies needs to be devised. In this study, a total of 62 cigarette ends collected under different conditions of environmental exposure were employed to test the effectiveness of three DNA amplification kits, namely the Applied Biosystems™ AmpFℓSTR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR Amplification Kits, in the profiling of such compromised DNA. We demonstrated that Identifiler® Plus could substitute Identifiler® to improve the effectiveness of profiling for those inhibited cigarette samples. MiniFiler™, on the other hand, could supplement Identifiler®/Identifiler® Plus profiles and provide additional genetic information to enhance the evidential value of the samples, especially for those that have suffered from DNA degradation to a greater extent. The findings in this work allowed us to propose a DNA profiling strategy as follow: 1) samples yielding complete Identifiler®/Identifiler® Plus profiles require no further testing with MiniFiler™; 2) samples yielding partial single-source profiles to be tested with MiniFiler™ to add genetic information; 3) samples yielding no results are unlikely to yield any results with MiniFiler™.