Reference DNA Profiles Research Articles

Discordance of Penta D x.4 microvariant alleles results between three capillary electrophoresis and one massively parallel sequencing short tandem repeat kits

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5’ flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3’ region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings.

Presence of Human DNA on Household Dogs and Its Bi-Directional Transfer.

Awareness of the factors surrounding the transfer of DNA from a person, item, or surface to another person, item, or surface is highly relevant during investigations of alleged criminal activity. Animals in domestic environments could be a victim, offender, or innocent party associated with a crime. There is, however, very limited knowledge of human DNA transfer, persistence, prevalence, and recovery (DNA TPPR) associated with domestic animals. This pilot study aimed to improve our understanding of DNA TPPR associated with domestic dogs by collecting and analysing samples from various external areas of dogs of various breeds, interactions with humans, and living arrangements, and conducting a series of tests to investigate the possibility of dogs being vectors for the indirect transfer of human DNA. Reference DNA profiles from the dog owners and others living in the same residence were acquired to assist interpretation of the findings. The findings show that human DNA is prevalent on dogs, and in the majority of samples, two-person mixtures are present. Dogs were also found to be vectors for the transfer of human DNA, with DNA transferred from the dog to a gloved hand during patting and a sheet while walking.

DNA transfer when using gloves in burglary simulations

Several studies have demonstrated that DNA can be indirectly transferred from an individual onto a surface. Therefore, the presence of DNA that is compatible with a given person does not necessarily mean that this person has touched the surface on which the DNA was recovered. The present work simulates cases, where DNA is recovered on a door handle and compared to several reference DNA profiles. The DNA profile of the trace shares DNA components with a person of interest (POI). When asked about the DNA results, the POI says he has nothing to do with the incident and has never been at the scene. However, a possibility would be that the DNA came from his recently stolen gloves. Someone else, the alternative offender (AO), could have opened the door wearing his gloves (POI’s gloves), and transferred his DNA (POI’s DNA). Based on the above-mentioned scenario, 60 burglary simulations experiments were carried out to generate data to assess DNA results given these allegations. The quantity and quality of DNA profiles (NGM SElect) recovered when the POI opened/closed the door bare-handed or when someone else performed the same activity but using POI’s gloves, were compared. The gloves were regularly worn during at least three months by their owner during the winter. On the contrary, the AO wore them only for two minutes. Among the traces collected on the door handles, less than 50% of the traces led to interpretable DNA profiles. In 30% of the cases (3/10), when the door was opened/closed with bare hands, the DNA found on the door handle led to a mixed DNA profile with the POI’s DNA aligning with the major contributor. For the experiments where the AO opened/closed the door with the POI’s gloves, the POI’s DNA was compatible with 22% (11/50) of the mixed DNA profile, aligning with the major in 8% of the cases (4/50). The DNA profiles of the offices’ occupants were observed on the door handles, but not the AO’s. In addition to the results of the experiments, we show two examples of how one can assess results observed in casework. Given the possibility of indirect transfer of minute DNA quantities, this research emphasizes the need to evaluate DNA results given the activities when the POI has a legitimate reason that can explain the presence of their DNA.

Heating Up Cold Cases: An Interview with Bruce Budowle on Human Identification

Heating Up Cold Cases: An Interview with Bruce Budowle on Human Identification

Human trafficking – Multinational challenge for forensic science

Human trafficking is one of the fastest growing and profitable types of transnational crime. This form of modern-day slavery encompasses diverse practices of human exploitation such as sex trafficking, forced labor, marriage, begging, child soldiers, and illegal organ transplantation. The consequences of human trafficking affect all countries, are devastating for victims and society while impacting social, economic, and health costs.The project led by the University of North Texas Center for Human Identification (UNTCHI) is assisting Central American countries to identify the missing and address human trafficking by developing model legislation and policy, effective DNA forensic science capabilities and creating database systems. The focus for human identification is on at-risk individuals who are the most vulnerable part of society. This program engages the public and government to establish legislative models for DNA identification of missing persons that protect the privacy of individuals.The State of Texas recently provided support for UNTCHI to develop programs that address the human trafficking problem in Texas. The program will concentrate on training law enforcement agencies, healthcare professionals, students, and the public to recognize the signs of human trafficking and provide avenues of interdiction for victims. Additionally, as part of this effort a standalone humanitarian DNA database, which is established and managed by UNTCHI, has been implemented to enhance identification of missing persons, especially those who have crossed the border or may have been trafficked and died during their journey. As a standalone database, the system is not connected to the State DNA Index System (SDIS) or the National DNA Index System (NDIS) and thus not accessible by law enforcement. With this database, reference DNA profiles can be obtained from Central American countries and Mexico to identify human remains found in Texas while maintaining confidentiality and human rights.This work is an example of an effective means of transferring forensics, technology, and database practices, establishing policy and legislation, engaging the public, and developing strategies for sustainability for high incident countries. Ultimately the approach can promote a multinational exchange of DNA data to identify the missing.

The efficacy of DNA mixture to mixture matching

Standard practice in forensic science is to compare a person of interest’s (POI) reference DNA profile with an evidence DNA profile and calculate a likelihood ratio that considers propositions including and excluding the POI as a DNA donor. A method has recently been published that provides the ability to compare two evidence profiles (of any number of contributors and of any level of resolution) comparing propositions that consider the profiles either have a common contributor, or do not have any common contributors. Using this method, forensic analysts can provide intelligence to law enforcement by linking crime scenes when no suspects may be available. The method could also be used as a quality assurance measure to identify potential sample to sample contamination. In this work we analyse a number of constructed mixtures, ranging from two to five contributors, and with known numbers of common contributors, in order to investigate the performance of using likelihood ratios for mixture to mixture comparisons. Our findings demonstrate the ability to identify common donors in DNA mixtures with the power of discrimination depending largely on the least informative mixture of the pair being considered. The ability to match mixtures to mixtures may provide intelligence information to investigators by identifying possible links between cases which otherwise may not have been considered connected.

Prevalence of DNA in vehicles: linking clothing of a suspect to car occupancy

ABSTRACTVehicles are commonly involved in crime-related incidents. To gain insight into the distribution (prevalence) of biological traces in vehicles, vehicles were studied under three different occupant scenarios; namely (1) a regular driver without and (2) with a regular passenger, and (3) an incidental driver introduced to scenario (1). One aspect of this study is elaborated in more detail in this paper, namely in respect to samples collected from the clothing of an incidental driver of a vehicle. This individual wore his own, clean clothing while driving the vehicle of another person for 30 minutes. After this activity samples were taken from the back of the upper garment and from the seat of the pants. The resulting DNA profiles were compared with reference DNA profiles from the wearer (incidental driver) and from the regular driver of the vehicle. Results of this study demonstrate that the clothing of the incidental driver can be related to the regular driver of the vehicle in a high percentage of instances. Our findings highlight a potential avenue for forensic investigation with high success rates when the relation between an individual and a vehicle is questioned.

Sex Determination Based on Raman Spectroscopy of Saliva Traces for Forensic Purposes.

The forensic analysis of body fluids has made great strides in recent years. Body fluids can easily be identified, and DNA analysis can be used to link a stain found at a crime scene to a specific person. When no reference DNA profile is available and the recovered DNA does not yield a match in a database, it would be incredibly useful if the evidence could still provide investigators with useful information. Biocatalytic and immunoassays can be used to determine a donor's sex, race, and other phenotypic characteristics. However, these tests depend on chemical reactions and are destructive to the sample. Here, we used Raman spectroscopy and multivariate data analysis to develop a nondestructive technique that could be used at a crime scene to determine the sex of a saliva donor. Our internally cross-validated classification model correctly identified 44 (92%) of the 48 donors used for model training. Subsequent external validation correctly identified 11 (92%) of the 12 donors saved for testing. This proof-of-concept study demonstrates the value of Raman spectroscopy as a forensic tool, and indicates that it can be used to elucidate phenotypic information about a body fluid donor. Future studies will expand to other body fluids and additional donor characteristics, such as race and age.

Generating Human DNA Profile(s) from Cell Phones for Forensic Investigation

The goals of this study were to investigate whether sufficient amount of human DNA can be recovered from a cell phone and if the identity of the single and/or multiple user(s) can be established by generating genetic profile(s). Cell phones were used to collect human DNA. Two Buccal swabs were used to collect Buccal cells as a DNA reference samples. Four soaked sterile cotton swabs were used to collect biological materials from each user’s cell phone. Sufficient quantity and quality of human genomic DNA was obtained from the cell phones using Quantifiler® Human DNA Quantitation Kit. More human DNA was collected from volunteers’ Buccal swabs (=74.56 ng) than from the surface of their cell phones (=0.23 ng). One nanogram (1 ng) of template DNA was used for buccal swabs and cell phones swabs, if available. However, when the DNA recovered from cell phone swabs was 0.00 to 500 pg, 10 μL of template DNA was used. In addition, the number of PCR cycles was increased from 28 to 32 in anticipation of Low Copy Number DNA. Genetic profiles were developed from the DNA on the cell phones and were compared with the reference DNA profiles from the buccal cells of the cell phone owners. Full Short Tandem Repeats (STR) DNA profiles for all 15 STR loci plus Amelogenin locus were typed for 36 Buccal swabs, reference DNA of cell phone owners. Complete STR profiles were generated from 33% (N=36) cell phones and were matched participants’. In addition, 28% of cell phones had partial DNA profiles matched with the cell phone owners. While, 33% of the cell phones had unknown partial or complete profiles. Four of the unknown partial profiles had more than two alleles at the STR loci indicating multiple unknown cell phone users. The Y chromosome was detected at the Amelogenin locus from the DNA profiles generated from two females’ cell phones identifying unknown male contributor(s). Our study demonstrates that DNA collected from the cell phones can be used as reliable physical evidence in forensic investigations.

Use of forensic investigations in anti-doping

The fight against doping is mainly focused on direct detection, using analytical methods for the detection of doping agents in biological samples. However, the World Anti-Doping Code also defines doping as possession, administration or attempted administration of prohibited substances or methods, trafficking or attempted trafficking in any prohibited substance or methods. As these issues correspond to criminal investigation, a forensic approach can help assessing potential violation of these rules. In the context of a rowing competition, genetic analyses were conducted on biological samples collected in infusion apparatus, bags and tubing in order to obtain DNA profiles. As no database of athletes' DNA profiles was available, the use of information from the location detection as well as contextual information were key to determine a population of suspected athletes and to obtain reference DNA profiles for comparison. Analysis of samples from infusion systems provided 8 different DNA profiles. The comparison between these profiles and 8 reference profiles from suspected athletes could not be distinguished. This case-study is one of the first where a forensic approach was applied for anti-doping purposes. Based on this investigation, the International Rowing Federation authorities decided to ban not only the incriminated athletes, but also the coaches and officials for 2 years.

Y-STR DNA analysis of 154 female child sexual assault cases in the Philippines

The laboratory evaluated 154 sexual assault cases from four Child Protection Units in the Philippines involving female child victims aged from 2years to 18years old. All child victims sought medical attention within 72h after sexual contact. In 130 cases, the child victim knew the alleged offender and identified them during the interview with the social worker. Penile ejaculation was reported by 68 child victims with varying reports of washing after contact. Overall, 84 child victims admitted having wiped their genitalia prior to the collection of biological samples for DNA testing. Laboratory personnel examined vaginal smears in only 109 cases using a light microscope and reported 23 samples to be positive for sperm cells. Using the PowerPlex® short tandem repeat of the Y chromosome (Y-STR) DNA multiplex system, male DNA was detected in vaginal swab samples from 63 child victims. In 39 cases, positive amplification at 11 Y-STR DNA markers consistent with a single male DNA profile was observed. Twenty-eight of these full single Y-STR DNA profiles were found to be unique when searched in worldwide Y-STR DNA population databases (~40,000 haplotypes), eight haplotypes matching Filipinos and/or Asian haplotypes and one Y-STR DNA profile only matching European, Caucasian, and Latin American haplotypes. Y-STR DNA profiles generated will be compared with reference DNA profiles of alleged offenders once reference samples are submitted to the laboratory.

The prevalence of mixed DNA profiles in fingernail samples taken from couples who co-habit using autosomal and Y-STRs

Physical contact can result in the transfer of DNA from one individual to another. In cases of sexual offence this can often be by means of an aggressive or sexual act. Biological material can accumulate under the fingernail hyponychium of both the victim and/or the suspect and has the potential to provide evidence and intelligence information to the police. The incidence of mixed DNA profiles obtained from fingernail samples of couples who co-habit was explored using AmpF lSTR ® SGM Plus ® (SGM Plus) (Applied Biosystems ®). It was predicted that the higher incidence of contact from co-habitation and therefore the greater opportunity for DNA transfer would result in an increased level of foreign DNA from the partner under the donor's fingernails. Fingernail swabs were taken from all fingers of the left and right hands of 12 couples on three separate occasions. DNA profiles were compared to both the donor and partner's reference DNA profiles. The number of reportable mixed DNA profiles obtained (17%) was higher than in previous reports. In this study, the majority of non-donor alleles matched the partner's reference DNA profile, indicating that co-habitation affected non-donor DNA obtained from fingernail swabs. The results demonstrated that as the couples spent increasing amounts of time together, the incidence of mixed DNA profiles increased. The large variation in the incidence of foreign alleles observed within and between couples suggested that a combination of lifestyle factors were having a significant effect on whether mixed DNA profiles were observed. Further DNA profiling using the AmpF lSTR ® Y-filer ® (Yfiler) PCR amplification (Applied Biosystems) was carried out to determine whether female fingernail samples that had previously given single donor profiles contained low levels of male DNA that may be detected by targeting the Y-chromosome. It was found that 63% of the samples analysed using Yfiler produced full or partial Y-STR profiles. The Y-STR profiles were compared to partners’ profiles and searched through two haplotype databases to determine the evidential value of such samples.

Automatic data processing of reference DNA-profiles from FTA and non-FTA samples

The new Swedish DNA legislation resulted in a huge increase in reference samples. In 2006 approximately 25,000 reference samples were received compared to 5000 in 2005. To meet this increase the reference samples had to be handled in a more automatic process than previously. A new module in the LIMS system automatically compares duplicate results and creates confirmed results if the DNA profiles meet the set requirements. Profiles without automatically confirmed results need to be manually investigated. Certain rules and settings in the LIMS sort these samples. Evaluators are able to combine the results to form confirmed results or chose to reanalyse the samples. At a rough estimate, 80% of all FTA samples are automatically assigned confirmed results, without any manual handling. Only 0.09% of the reference samples was terminated without results in every marker.

Short tandem repeat (STR) genotyping of keratinised hair Part 2. An optimised genomic DNA extraction procedure reveals donor dependence of STR profiles

A feasibility study of short tandem repeat (STR) genotyping of telogen phase hairs in particular, and hair shaft in general, is presented. A number of extraction procedures in common use were investigated and the quantities of nuclear DNA (nuDNA) delivered were quantified via a real-time PCR assay. The extracts were subjected to two variations on AmpFℓSTR ® Profiler Plus™ PCR amplification strategies (extended cycles, two rounds of PCR) and the genotypes compared. Nuclear DNA was found to persist in human hair shafts, albeit at very low levels. Full Profiler Plus™ profiles consistent with the hair donor were obtained from 100 mg hair shaft samples (bleached and unbleached). These were, however, mixed profiles, indicating low copy number (LCN) contamination in the extracts. Single telogen hair clubs and single hair shafts delivered partial profiles with usually only one allele of heterozygous loci. Telogen phase hairs yielded the same amount of nuDNA (and no more) as hair shafts (either anagen or telogen). Whether hair shafts dissolved or not in lysis buffer had no effect on either the quantitated yield of DNA or on the chance of obtaining a correct genotype. These results provide evidence that genomic DNA resides on the exterior of the hair shaft and we use this information to suggest an optimal procedure for nuDNA extraction from keratinised hair samples: soaking hairs in simple digestion buffers containing Tris–HCl, a salt and a chelating agent without prior cleaning of the hair shafts. It is proposed that cleaning removes most of the recoverable DNA. This procedure was applied to obtain genotypes from 3 cm hair shafts which matched reference profiles from the donors at up to 9 out of 10 AmpFℓSTR ® Profiler Plus™ STR loci. When the genotyping success was measured by counting the number of matches between the two dominant alleles at each locus for each extract with the reference DNA profile of the hair donor, the success was found to be highly dependent on the donor. The number of matching alleles varied between not less than 10 for one donor to no more than two for another donor. These results may well be linked to the environmental experience of the hairs from each donor prior to removal.

Issues and strategies in the DNA identification of World Trade Center victims

Identification of the nearly 3000 victims of the World Trade Center attack, represented by about 15,000 body parts, rests heavily on DNA. Reference DNA profiles are often from relatives rather than from the deceased themselves. With so large a set of victims, coincidental similarities between non-relatives abound. Therefore considerable care is necessary to succeed in correlating references with correct victims while avoiding spurious assignments. Typically multiple relatives are necessary to establish the identity of a victim. We describe a 3-stage paradigm—collapse, screen, test—to organize the work of sorting out the identities. Inter alia we present a simple and general formula for the likelihood ratio governing practically any potential relationship between two DNA profiles.

Recovery of DNA for Forensic Analysis from Lip Cosmetics

To obtain a reference DNA profile from a missing person, we analyzed a variety of personal effects, including two lip cosmetics, both of which gave full DNA profiles. Further investigations were undertaken to explore this previously unreported source of DNA. We have tested a range of brands and types of lip cosmetics. Our studies have revealed that lip cosmetics are an excellent source of DNA, with almost 80% of samples giving a result. However, artifacts are frequently observed in the DNA profiles when Chelex is used for the DNA extraction and additional DNA purification procedures are required to ensure that an accurate DNA profile is obtained.