Typing DNA profiles from previously enhanced fingerprints using direct PCR

Fingermarks are a source of human identification both through the ridge patterns and DNA profiling. Typing nuclear STR DNA markers from previously enhanced fingermarks provides an alternative method of utilising the limited fingermark deposit that can be left behind during a criminal act. Dusting with fingerprint powders is a standard method used in classical fingermark enhancement and can affect DNA data. The ability to generate informative DNA profiles from powdered fingerprints using direct PCR swabs was investigated. Direct PCR was used as the opportunity to generate usable DNA profiles after performing any of the standard DNA extraction processes is minimal. Omitting the extraction step will, for many samples, be the key to success if there is limited sample DNA. DNA profiles were generated by direct PCR from 160 fingermarks after treatment with one of the following dactyloscopic fingerprint powders: white hadonite; silver aluminium; HiFi Volcano silk black; or black magnetic fingerprint powder. This was achieved by a combination of an optimised double-swabbing technique and swab media, omission of the extraction step to minimise loss of critical low-template DNA, and additional AmpliTaq Gold^® DNA polymerase to boost the PCR. Ninety eight out of 160 samples (61%) were considered ‘up-loadable’ to the Australian National Criminal Investigation DNA Database (NCIDD). The method described required a minimum of working steps, equipment and reagents, and was completed within 4 h. Direct PCR allows the generation of DNA profiles from enhanced prints without the need to increase PCR cycle numbers beyond manufacturer's recommendations. Particular emphasis was placed on preventing contamination by applying strict protocols and avoiding the use of previously used fingerprint brushes. Based on this extensive survey, the data provided indicate minimal effects of any of these four powders on the chance of obtaining DNA profiles from enhanced fingermarks.     

DNA profiles from clothing fibers using direct PCR

We report on the successful use of direct PCR amplification of single fibers from items of worn clothing. Items of clothing were worn throughout the course of a day, with the individual commencing regular activities. Single fibers were taken from the cuff of the clothing at regular intervals and amplified directly. The same areas were subjected to tape-lifting, and also amplified directly for comparison. The NGM? kit that amplifies 15 STR loci plus amelogenin was used. A total of 35 single fiber samples were processed and analyzed from five items of clothing, with 81 % of samples returning a profile of 14 alleles or more. All tape-lift samples amplified directly produced DNA profiles of 15 alleles or more. The aim was to develop a simple, operational method that could be used routinely in forensic science casework and that has the potential to generate more complete profiles, which would not be detected using standard extraction methods on this type of sample. For ease of implementation, the process also adheres to standard methods with no increase in the cycle number.     

Detection of latent DNA on tape-lifts using fluorescent in situ detection

Tape-lifts can be used for the removal of a range of evidence types without damaging the substrate. In addition to loosely adhering material such as hairs and fibres, tape-lifts can be used to remove latent cellular material on clothing that had originated from the wearer’s body or mouth. Common forensic practice is to examine the entire tape-lift in a non-targeted approach. An innovative field of research uses DNA binding dyes for the detection of latent DNA. This research describes the use of these DNA binding dyes as a DNA-targeted screening tool for tape-lifts. The individual cells can be visualized allowing the relevant area of the tape to be removed for subsequent DNA typing. Full single source DNA profiles were generated that matched the expected control sample after staining tape-lifts with two different dyes. Direct amplification was undertaken from tape-lifts of samples where biological material had been deposited over 12 months previously, from which a DNA profile matching the donor was obtained. The process outlines an effective means to visualize the presence of cellular material from which STR profiles can be generated allowing a targeted approach to be performed.     

Speed of accumulation of DNA in a fingermark

ABSTRACT

Variation has been reported in the amount of DNA accumulating on the skin of individuals. A shedder status is the propensity of a person to transfer DNA to a substrate by touch. In previous tests of shedders, individuals washed their hands and after 15 minutes made contact with substrates at time points up to 180 minutes after handwashing. No examination has looked at the accumulation of cellular material between time zero and this 15 minute time point. Here we present the results of an examination of cellular material within thumbprints at time points 0, 2, 5, 15 and 60 minutes after handwashing using donors who are representative of heavy, intermediate and light shedders. The rate of accumulation of cellular material and the total amount detected in thumbprints showed a difference between these donors, but for all donors the initial rate of accumulation is surprisingly fast. Informative STR profiles can be generated only 2 minutes after handwashing from 100%, 33% and none of the heavy, intermediate and light shedders, respectively. These results confirmed that there was a correlation between the cellular material present on the thumbprint and the percentage success of an STR profile for each individual and time point.     

A comparison between direct PCR and extraction to generate DNA profiles from samples retrieved from various substrates

Direct PCR generates DNA profiles from samples without using the extraction process. During sample extraction, DNA may be lost due to the methods used, which can affect the quality of the DNA profile obtained. This is not the case with direct PCR, where the sample is transferred directly into the PCR tube. Here, we report on the ability of direct PCR to generate DNA profiles from low amounts of control DNA retrieved from various surfaces using PowerPlex 16 HS. A comparison is made with samples undergoing a preliminary extraction stage using QiaAmp DNA Micro kits. Samples subjected to direct PCR generated DNA profiles with higher peak heights and lower allele dropout on all the different substrates tested when compared to the samples subjected to extraction. The amount of DNA retrieved from each substrate also varied even though the same amount of starting material was deposited, proving that the type of substrate can affect the retrieval of DNA.     

Evaluating the prevalence of DNA mixtures found in fingernail samples from victims and suspects in homicide cases

An important aspect of homicide investigations is the identification of the persons that had the last contact with the victim prior to death. Violent crimes are frequently characterized by a struggle between the victim and the perpetrator where biological material can be expected to be exchanged between them.Forensic DNA typing enables the generation of genetic profiles by extraction and amplification of cellular material found under fingernails. The evidential value of these samples may be critical if the secondary contributor found in a DNA mixture, can be matched with a potential suspect, or through a DNA database search.The amount of biological material transferred under the fingernails during “casual” activities is not sufficient to genotype reportable mixtures. This may not be the case with homicide victims that may have struggled and died under violent circumstances.The aim of this study was to evaluate the prevalence of DNA mixtures found under the fingernails of both victims and suspected perpetrators of violent deaths.We present a retrospective study of 137 DNA profiles genotyped from fingernail samples of homicide victims and suspects, collected at the Israeli National Center of Forensic Medicine. The majority of the samples produced single source profiles (n = 107, 78%) that matched those of the donor's. DNA mixtures (n = 30, 22%) were found in increased frequency among victims (n = 25/100, 25%) compared to suspects (n = 5/37, 13.5%). Mixtures were sub-divided into high level (n = 15, 50%), low level (n = 9, 30%) and residual (n = 6, 20%), according to the number of the foreign contributors’ alleles. Thus, this distinctive group of homicide victims was found to express both elevated frequency of DNA mixtures together with highly informative value of the secondary foreign profiles, as compared to other studied populations. These findings support an important aspect for the criminal investigation in murder cases, where a struggle may have ensued and the identification of an additional profile found in a mixture from a fingernail sample may point to a possible perpetrator of the crime.     

Improvements,factors, and influences on DNA recovery from firearms

Touch DNA recovery from firearms can be central to many criminal investigations, yet the generation of DNA profiles from these items remains poor. Currently in Australia, published casework data highlights extremely poor DNA success from samples recovered from firearms. Only between 5% and 25% of samples result in useful DNA data and therefore increasing the success of DNA recovered from firearms is highly important but has not yet been explored in-depth. This study focused on increasing the recovery of DNA from ten firearm components that were held for 15 s. Multiple recovery methods were used, and the resulting genetic data compared. DNA evidence may be deliberately removed from firearms after discharge to hamper forensic investigations, therefore this study examined the effect of wiping down the components or handling them with gloves. A standard double swab and rinse swab recovery method resulted in an average of 73% cellular recovery. A cumulative swab process had the highest average recovery at 86%, although it was found that increasing the DNA yield led to an increase in mixture complexity. Wiping over the components was observed to remove on average 69% of cellular material, compared with 33% when handed with gloves. However, the size and texture of the components affected the efficiency of cellular material removal. The results from this study allow for prioritisation of areas to sample on firearms, as well as suggesting techniques that can be applied for the optimum process of cellular recovery and subsequent generation of STR DNA data.     

Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory’s data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.     

Exploring tapelifts as a method for dual workflow STR amplification

Although a version of direct PCR is implemented in forensic laboratories for reference material, its incorporation into workflow for the analysis of touch DNA, as a form of latent DNA, from casework exhibits is not. In addition to concerns about increased sensitivity causing more complex mixtures or the generation of more genetic data implicating an individual superfluous to the context of the alleged event, the complete use of the collected sample in the PCR as template has meant that there is no possibility for data reproducibility when needed. Here it is proposed that the use of tapelifts in touch DNA collection can facilitate replicate direct PCR analysis from a single sample allowing the sample to be re-tested. If all portions of the tapelift result in profiles with allelic and likelihood ratio concordance, these sub-samples may be accepted as technical replicates, thus meeting any accreditation guideline requirements. Furthermore, we assess the use of a single tapelift for both direct PCR and extraction-based PCR workflows to illustrate the potential for benefits of both systems to be facilitated. DNA was deposited by three donors onto six substrates with five sample replicates of each condition. Separation of each tapelift into three portions for three direct PCRs ensued using VeriFiler™ Plus. Separation of single tapelifts into three direct PCRs showed no statistical difference in donor allele calls or RFU, or subsequent LRs associated with their profiles. Comparison of profiles within the single tapelift showed more similarity, with high mixture-to-mixture match likelihoods, than when these sub-samples were compared with profiles generated from other samples. This allows each sub-sample taken from the tapelift to be considered as technical replicates. For dual workflow facilitation assessment, one donor deposited DNA through touch onto six substrates with five research replicates of each. Separation of single tapelifts into two portions, one for direct PCR and the retention and use of the remaining portion for extraction and subsequent PCR, showed no significant difference in allelic yield and subsequent donor comparison LRs. Comparison of deconvoluted profiles produced from a single tapelift showed high mixture-to-mixture match likelihoods, supporting DNA donor concordance. This indicates that removing a portion of a tapelift for direct PCR amplification, while processing the remainder through standard processes, allows increased sensitivity through direct PCR while offering the preparation of an eluate suitable for repeated analyses.     

Development of a SNP set for human identification: A set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population

This study describes the development of a SNP typing system for human identification in the Thai population, in particular for extremely degraded DNA samples. A highly informative SNP marker set for forensic identification was identified, and a multiplex PCR-based Invader assay was developed. Fifty-one highly informative autosomal SNP markers and three sex determination SNP markers were amplified in two multiplex PCR reactions and then detected using Invader assay reactions. The average PCR product size was 71 base pairs. The match probability of the 54-SNP marker set in 124 Thai individuals was 1.48 × 10⁻²¹, higher than that of STR typing, suggesting that this 54-SNP marker set is beneficial for forensic identification in the Thai population. The selected SNP marker set was also evaluated in 90 artificially degraded samples, and in 128 naturally degraded DNA samples from real forensic casework which had shown no profiles or incomplete profiles when examined using a commercial STR typing system. A total of 56 degraded samples (44%) achieved the matching probability (PM) equivalent to STR gold standard analysis (successful genotyping of 44 SNP markers) for human identification. These data indicated that our novel 54-SNP marker set provides a very useful and valuable approach for forensic identification in the Thai population, especially in the case of highly to extremely degraded DNA.In summary, we have developed a set of 54 Thai-specific SNPs for human identification which have higher discrimination power than STR genotyping. The PCRs for these 54 SNP markers were successfully combined into two multiplex reactions and detected with an Invader assay. This novel SNP genotyping system also yields high levels of genetic information from naturally degraded samples, even though there are much more difficult to recover than artificially degraded samples.     

Would the real allele please stand up? Compiling DNA artefacts for the GlobalFiler PCR amplification kit – a South Australian approach

ABSTRACT

This article presents a compendium of DNA artefacts observed using the GlobalFiler and GlobalFiler Express PCR kits (ThermoFisher Scientific) during DNA reference profile assessment at Forensic Science SA (FSSA). The data are currently used to assist with the interpretation of GlobalFiler DNA evidence profiles encountered in the course of routine case work at FSSA. Over 1000 reference profiles have passed observation by reference DNA run readers. An artefact was considered confirmed if it was observed in five or more individual reference samples and could be confirmed upon re-PCR. Artefacts were documented in the following two categories: (1) those with a location independent of true allelic products and (2) those with a location relative to a true allelic product. The artefacts observed were positioned within and outside of allelic designations and were typically less than 1% of the closest allelic product. Artefacts reported here are observed from reference samples only. Their causes are largely unknown and warrant further investigation. Post-developmental artefacts may be encountered due to storage and handling conditions. As such, continued monitoring of reference samples for low level artefacts is warranted.     

DNA Double Strand Breaks in Ehrlich Ascites Tumour Cells at Low Doses of X-rays. I. Determination of Induced Breaks by Centrifugation at Reduced Speed

Summary

DNA double strand breaks (dsb) were determined in Ehrlich ascites tumour cells at doses down to 5 Gy. The method is based on the separation of DNA from other components by heating in a solution of pronase and detergents held in wide-mouth syringes, which were also used to facilitate the application of the released high molecular weight DNA to sucrose gradients. Purified DNA was sedimented in neutral sucrose gradients at low speed to reduce speed artifacts. The sedimentation profiles were analysed using a computer program and the number of dsb was determined by simulation of random breaks in the mass distribution of the control sample and by comparison of this simulated profile with that of the irradiated one. The number of dsb formed was proportional to X-ray dose in the range of 5 to 2000 Gy. The induction per dose was found to be nm^?1r D^?1 = (11·7 ± 2) × 10^?12 Gy^?1.     

Genotyping diptera using amplified fragment length polymorphism (AFLP): development of a genetic marker system for species in the families Calliphoridae and Sarcophagidae

Amplified Fragment Length Polymorphism (AFLP) analysis of entomological material provides a means of obtaining a reproducible DNA profile in a relatively short period of time. AFLP profiles were obtained using larval samples from Cochliomyia macellaria, Phormia regina and Sarcophaga bullata. Genomic DNA was isolated using the DNeasy Tissue Kit (Qiagen, Valencia, CA), double-digested by two restriction endonucleases (EcoRI and MseI) and ligated to oligonucleotide adapters. Two consecutive PCR reactions (preamplification and selective amplification) were performed using a modification of the AFLP protocol described by Gibco (Invitrogen, Rockville, MD). The DNA fragments were separated by capillary electrophoresis using the CEQ 8000 DNA Fragment Analyzer. Species specific profiles were developed for all three species. The results indicate that the AFLP technique is a viable and valuable technique for identification of entomological material.     

Evaluation of the QIAGEN 140-SNP forensic identification multiplex from latent DNA using massively parallel sequencing

ABSTRACT

Direct PCR can be used to successfully generate full STR profiles from latent DNA. However, some substrates have been shown to be more problematic and in some cases only partial profiles are recovered. As latent DNA is present on the surface of objects in very low quantity, and potentially low quality, the fragment lengths targeted by STR typing may be too large to successfully amplify all markers. As an alternative, QIAGEN have developed a 140-SNP multiplex that targets much shorter amplicons and generates extremely low probabilities of any two unrelated individuals having identical genotypes. Here, we present the first forensic identification SNP data from latent DNA using massively parallel sequencing. We applied the QIAGEN 140-SNP forensic identification multiplex to swabs collected from multiple substrates (including mobile phone, fingerprint, wire, zip-lock bag and SIM card) and multiple donors.     

Improved STR profiles from improvised explosive device (IED): fluorescence latent DNA detection and direct PCR

Bombing accounts for the largest share of terrorist incidents worldwide. Most involve an improvised explosive device (IED): a bomb made from household items. Touch DNA may be left on parts of an IED during assembly. However, an IED conflagration degrades DNA, and there has never been a way to locate where touch DNA may remain. To solve this problem, we combined the use of fluorescent dye to locate latent DNA and direct PCR to improve STR profiles of DNA obtained from IEDs. Six fluorescent DNA-binding dyes were evaluated at various concentrations for the purpose of staining latent DNA. SYBR® Green I and Diamond™ Nucleic Acid dye were able to visualize touch DNA on IED substrates. Inhibition studies with extracted DNA and touch DNA using both dyes revealed that Diamond™ dye inhibited direct STR amplification, while SYBR® Green I did not. Stability studies at three temperatures showed optimum performance of SYBR® Green I up to 24 h after formulation. As such, only SYBR® Green I was further used to develop a “visualized-direct PCR” method. Using the conventional approach and the novel “visualized-direct PCR” approach in a single-blind investigation of mock IED evidence, the “visualized-direct PCR” approach had a 98.6% chance of obtaining more alleles (95% highest density interval (HDI): 0.7 to 10.0 alleles). A decrease in non-donor’s alleles (mixed profiles) was also observed. The developed approach has the potential to revolutionize the process of STR typing from touch DNA.     

The prevalence of mixed DNA profiles in fingernail samples taken from individuals in the general population

The fingernail hyponychium is an isolated area where biological material may accumulate and can provide a valuable source of evidential material in police investigations. DNA transfer between the victim and suspect frequently occurs during violent crimes and in court there is often reasonable doubt that a mixed DNA profile in a fingernail sample has originated from the assault as the profile may be attributed to previous contact between the two individuals. The purpose of this study was to assess background levels of foreign DNA under the fingernails of individuals from the general population in order to provide data that may help to determine whether DNA transfer occurred during or prior to the assault. Fingernail swabs sampled from 100 volunteers were processed by Qiagen™ extraction and amplified using AMPFlSTR^® SGM Plus™ to obtain DNA profiles. Foreign DNA was detected in 13% of samples, with only 6% of these giving reportable mixed DNA profiles, suggesting the incidence of foreign DNA under the fingernails was low. A significant proportion of the mixed DNA profiles came from male donors; the majority had experienced physical contact within the 24 h time period prior to sampling.     

Locating DNA within fingermarks using fluorescent in situ detection; a collaboration between ESR and Flinders University

ABSTRACT

A pilot study on the detection of DNA in fingermarks using fluorescent in situ detection after different time periods since hand washing was undertaken by Flinders University. Collaboration was sought to show the ability to obtain similar results within different laboratories. The Institute of Environmental Science and Research (ESR) was involved in this inter-laboratory study in collaboration with Flinders University. The newly developed method involves the use of Diamond Nucleic Acid dye for the staining of fingermarks (20× concentration in 75% ethanol) using a hand-held microscope (Dino-Lite Edge Digital Microscope). Fingermarks were deposited by volunteers onto glass slides at varying time intervals after hand washing (2, 5, 15, 30, 60 and 180 minutes). The amount of cellular debris was calculated by counting the fluorescent dots present in three fields of view and estimating the amount of transferred cellular material for each fingermark. This article will outline the results obtained from ESR and how they compare with results already collated from Flinders University.     

Evaluation of DNA profiles obtained from deceased individuals at Salt River Mortuary (South Africa)

ABSTRACT

DNA profiling is a valuable tool for human identification, particularly when deceased individuals cannot be identified by traditional methods. Annually, at Salt River Mortuary (SRM), ~320 bodies remain unidentified following post-mortem investigation. While much research has been conducted into DNA profiling on living individuals, relatively little has focused on suitability within the deceased population, and thus this formed the focus of this research. Buccal cells were collected from deceased infants (n = 38) and adults (n = 37) at SRM, with a time interval between death and sample collection of 1 – 887 days. Control blood samples were obtained from a subset of participants. Samples were processed using the PowerPlex ESI 16 System or Y23 System (Promega). Full DNA profiles were obtained from all blood samples. Following optimization, 74.7% and 18.7% of buccal swabs yielded full and partial profiles respectively, while 6.7% failed completely. Upon analysis, partial and failed profiles were significantly associated with the DNA degradation index from cotton swabs (p < 0.001), but not with time between death and sample collection (p = 0.16). These results indicate that full DNA profiles can be obtained long after death. It is hypothesized that factors influencing degradation (e.g. decomposition) may contribute to failure. Overall, further research is required to identify factors influencing DNA profile quality.     

Searching mixed DNA profiles directly against profile databases

DNA databases have revolutionised forensic science. They are a powerful investigative tool as they have the potential to identify persons of interest in criminal investigations. Routinely, a DNA profile generated from a crime sample could only be searched for in a database of individuals if the stain was from single contributor (single source) or if a contributor could unambiguously be determined from a mixed DNA profile. This meant that a significant number of samples were unsuitable for database searching. The advent of continuous methods for the interpretation of DNA profiles offers an advanced way to draw inferential power from the considerable investment made in DNA databases. Using these methods, each profile on the database may be considered a possible contributor to a mixture and a likelihood ratio (LR) can be formed. Those profiles which produce a sufficiently large LR can serve as an investigative lead.In this paper empirical studies are described to determine what constitutes a large LR. We investigate the effect on a database search of complex mixed DNA profiles with contributors in equal proportions with dropout as a consideration, and also the effect of an incorrect assignment of the number of contributors to a profile. In addition, we give, as a demonstration of the method, the results using two crime samples that were previously unsuitable for database comparison. We show that effective management of the selection of samples for searching and the interpretation of the output can be highly informative.     

The transfer of touch DNA from hands to glass, fabric and wood

The transfer of DNA from hands to objects by holding or touching has been examined in the past. The main purpose of this study was to examine the variation in the amount of DNA transferred from hands to glass, fabric and wood. The study involved 300 volunteers (100 for glass, 100 for fabric and 100 for wood) 50% of which were male and 50% female. The volunteers held the material for 60s. The DNA was recovered from the objects using a minitape lift, quantified using the Quantifiler kit assay, extracted using a 'Qiagen(?) QIAamp DNA mini kit' and amplified using the AmpFlSTR(?) SGM Plus? Amplification Kit at 28 cycles. The results show that using ANOVA there was a significant difference (F=8.2, p<0.05) between the three object types in the amount of DNA recovered. In terms of DNA transfer and recovery, wood gave the best yield, followed by fabric and then glass. The likelihood of success of obtaining a profile indicative of the holder was approximately 9% for glass samples, 23% for fabric and 36% for wood. There was no significant difference between the amount of DNA transferred by male or female volunteers. In this study good shedder status, as defined by obtaining useful profiles of 6 or more alleles, is estimated at approximately 22% of the population. The phenomenon of secondary transfer was observed when mixed DNA profiles were obtained but the incidence was low at approximately 10% of the total number of samples. DNA profiles corresponding to more than one person were found on objects which had been touched by only one volunteer. Although secondary transfer is possible the profiles obtained from touched objects are more likely to be as a result of primary transfer rather than a secondary source.