Forensic Science International: Genetics

Editorial Board

2012-01-01

Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence

William R. Hudlow, Martin R. Buoncristiani — 2011-02-03

Abstract: We present a rapid alkaline lysis procedure for the extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic/dithiothreitol differential extraction. Specifically, a sodium hydroxide based differential extraction method has been developed in a single-tube format and further optimized in a 96-well format. The method yields purified extracts from a small sample set (∼2–6 swabs) in approximately 2h and from a larger sample set (up to 96 swabs) in approximately 4h. While conventional differential extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs.

A stochastic model of the processes in PCR based amplification of STR DNA in forensic applications

Jos Weusten, Jos Herbergs — 2011-02-08

Abstract: In forensic DNA profiling use is made of the well-known technique of PCR. When the amount of DNA is high, generally unambiguous profiles can be obtained, but for low copy number DNA stochastic effects can play a major role. In order to shed light on these stochastic effects, we present a simple model for the amplification process. According to the model, three possible things can happen to an individual single DNA strand in each complete cycle: successful amplification, no amplification, or amplification with the introduction of stutter. The model is developed in mathematical terms using a recursive approach: given the numbers of chains at a given cycle, the numbers in the next can be described using a multinomial probability distribution. A full set of recursive relations is derived for the expectations and (co)variances of the number of amplicon chains with no, 1 or 2 stutters. The exact mathematical solutions of this set are given, revealing the development of the expectations and (co)variances as function of the cycle number. The equations reveal that the expected number of amplicon chains without stutter grows exponentially with the cycle number, but for the chains with stutter the relation is more complex. The relative standard deviation on the numbers of chains (coefficient of variation) is inversely proportional to the square root of the expected number of DNA strands entering the amplification. As such, for high copy number DNA the stochastic effects can be ignored, but they play an important role at low concentrations. For the allelic peak, the coefficient of variation rapidly stabilizes after a few cycles, but for the chains with stutter the decrease is more slowly. Further, the ratio of the expected intensity of the stutter peak over that of the allelic peak increases linearly with the number of cycles. Stochastic models, like the one developed in the current paper, can be important in further developing interpretation rules in a Bayesian context.

Cell free DNA as a component of forensic evidence recovered from touched surfaces

Ignacio Quinones, Barbara Daniel — 2011-02-03

Abstract: In the course of a criminal investigation, DNA is often recovered from items that have been handled by an individual. Whilst there have been studies investigating the propensity of different individuals to deposit DNA, little is known about the factors involved in the transference of DNA through touch. This investigation seeks to clarify some of the underlying processes involved in DNA transfer, as to better understand the significance of so-called “touch DNA” evidence (tDNA). It was shown that an average yield of 11.5ng of DNA could be recovered from 1mL cell-free sweat samples leading to the hypothesis that cell-free nucleic acids (CNAs) of a suitable length for standard DNA profiling are transferred during handling/touching items. A method of standardization of tDNA deposition was developed to overcome the significant sample to sample variability in DNA levels characteristic of tDNA samples. The glass bead method allowed the creation of identical tDNA sample sets, thus permitting direct comparisons to be made in the efficiency of various extraction methods. Extraction methods designed to optimize CNA recovery from touched articles resulted in comparable yields in a general population study, however the methods resulted in a twofold increase in DNA yields from touched items touched by individuals with sweaty hands. These results suggest that the CNA component of touched surfaces should be included to maximize profiling success of tDNA.

Assessing a novel room temperature DNA storage medium for forensic biological samples

2011-02-16

Abstract: The ability to properly collect, analyze and preserve biological stains is important to preserving the integrity of forensic evidence. Stabilization of intact biological evidence in cells and the DNA extracts from them is particularly important since testing is generally not performed immediately following collection. Furthermore, retesting of stored DNA samples may be needed in casework for replicate testing, confirmation of results, and to accommodate future testing with new technologies.A novel room temperature DNA storage medium, SampleMatrix™ (SM; Biomatrica, Inc., San Diego, CA), was evaluated for stabilizing and protecting samples. Human genomic DNA samples at varying amounts (0.0625–200ng) were stored dry in SM for 1 day to 1 year under varying conditions that included a typical ambient laboratory environment and also through successive freeze–thaw cycles (3 cycles). In addition, spiking of 1–4× SM into samples prior to analysis was performed to determine any inhibitory effects of SM. Quantification of recovered DNA following storage was determined by quantitative PCR or by agarose gel electrophoresis, and evaluation of quantitative peak height results from multiplex short tandem repeat (STR) analyses were performed to assess the efficacy of SM for preserving DNA.Results indicate no substantial differences between the quality of samples stored frozen in liquid and those samples maintained dry at ambient temperatures protected in SM. For long-term storage and the storage of low concentration samples, SM provided a significant advantage over freezer storage through higher DNA recovery. No detectable inhibition of amplification was observed at the recommended SM concentration and complete profiles were obtained from genomic DNA samples even in the presence of higher than recommended concentrations of the SM storage medium. The ability to stabilize and protect DNA from degradation at ambient temperatures for extended time periods could have tremendous impact in simplifying and improving sample storage conditions and requirements. The current work focuses on forensics analysis; however this technology is applicable to all endeavors requiring storage of DNA.

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

Dyan J. Daly, Charlotte Murphy, Sean D. McDermott — 2011-02-17

Abstract: 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.

Optimization and validation of a fast amplification protocol for AmpFlSTR® Profiler Plus® for rapid forensic human identification

Nancy Laurin, Chantal Frégeau — 2011-03-14

Abstract: The goal of this work was to optimize and validate a fast amplification protocol for the multiplex amplification of the STR loci included in AmpFlSTR® Profiler Plus® to expedite human DNA identification. By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR™ HS) and a more efficient thermal cycler instrument (Bio-RAD C1000™), we were able to reduce the amplification process from 4h to 26min. No modification to the commercial AmpFlSTR® Profiler Plus® primer mix was required. When compared to the current Royal Canadian Mounted Police (RCMP) amplification protocol, no differences with regards to specificity, sensitivity, heterozygote peak height ratios and overall profile balance were noted. Moreover, complete concordance was obtained with profiles previously generated with the standard amplification protocol and minor alleles in mixture samples were reliably typed. An increase in n−4 stutter ratios (2.2% on average for all loci) was observed for profiles amplified with the fast protocol compared to the current procedure. Our results document the robustness of this rapid amplification protocol for STR profiling using the AmpFlSTR® Profiler Plus® primer set and demonstrate that comparable data can be obtained in substantially less time. This new approach could provide an alternative option to current multiplex STR typing amplification protocols in order to increase throughput or expedite time-sensitive cases.

Characterising stutter in forensic STR multiplexes

Clare Brookes, Jo-Anne Bright, SallyAnn Harbison, John Buckleton — 2011-03-09

Abstract: Stutter is an artefact seen when amplifying short tandem repeats and typically occurs at one repeat unit shorter in length than the parent allele. In forensic analysis, stutter complicates the analysis of DNA profiles from multiple contributors, known as mixed profiles, a common profile type. Consequently it is important to both understand and predict stutter behaviour in order to improve our understanding of the resolution and interpretation of these profiles. Whilst stutter is well recognised and documented, little information is available that identifies and quantifies what influences the formation of stutter. In this work we use a novel approach to examine this. We have used synthetic oligonucleotides comprising multiple repeat units to test; the influence of repeat number, the influence of repeat sequence and the impact of interruptions to the repeat sequence length. Using multiple replicates allows detailed statistical analysis. We have confirmed a linear relationship between stutter ratio and repeat number. We have shown that increased A–T content increases stutter ratio and that interruptions in repeating sequences decreased stutter ratios to levels similar to the longest uninterrupted repeat stretch. We also found that there was no relationship between stutter ratio and repeat number for a repeat unit with an A–T content of 1/4 and that half of the interrupted repeat sequences stuttered significantly less than their longest uninterrupted repeat stretches. We have applied the knowledge gained to examine specific features of the loci present in the AmpFlSTR® SGM Plus® multiplex kit used in our laboratory.

Complex mixtures: A critical examination of a paper by Homer et al.

Thore Egeland, A. Elida Fonneløp, Paul R. Berg, Matthew Kent, Sigbjørn Lien — 2011-03-23

Abstract: DNA evidence in criminal cases may be challenging to interpret if several individuals have contributed to a DNA-mixture. The genetic markers conventionally used for forensic applications may be insufficient to resolve cases where there is a small fraction of DNA (say less than 10%) from some contributors or where there are several (say more than 4) contributors. Recently methods have been proposed that claim to substantially improve on existing approaches . The basic idea is to use high-density single nucleotide polymorphism (SNP) genotyping arrays including as many as 500,000 markers or more and explicitly exploit raw allele intensity measures. It is claimed that trace fractions of less than 0.1% can be reliably detected in mixtures with a large number of contributors. Specific forensic issues pertaining to the amount and quality of DNA are not discussed in the paper and will not be addressed here. Rather our paper critically examines the statistical methods and the validity of the conclusions drawn in Homer et al. (2008) .We provide a mathematical argument showing that the suggested statistical approach will give misleading results for important cases. For instance, for a two person mixture an individual contributing less than 33% is expected to be declared a non-contributor. The quoted threshold 33% applies when all relative allele frequencies are 0.5. Simulations confirmed the mathematical findings and also provide results for more complex cases. We specified several scenarios for the number of contributors, the mixing proportions and allele frequencies and simulated as many as 500,000 SNPs.A controlled, blinded experiment was performed using the Illumina GoldenGate® 360 SNP test panel. Twenty-five mixtures were created from 2 to 5 contributors with proportions ranging from 0.01 to 0.99. The findings were consistent with the mathematical result and the simulations.We conclude that it is not possible to reliably infer the presence of minor contributors to mixtures following the approach suggested in Homer et al. (2008) . The basic problem is that the method fails to account for mixing proportions.

RNA/DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise

2011-04-04

Abstract: A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5–0.001μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.

Divergent genetic strata in five Bahamian islands

Tanya M. Simms, Dianne A. Barrett, Quinn McCartney, Rene J. Herrera — 2011-04-11

Abstract: Based on historical records, the genetic landscape of the Bahamian archipelago is presumed to be complex and to exhibit island-specific characteristics, yet the genetic composition of the island chain, which could corroborate or refute these past accounts, remains poorly defined. As such, the current investigation was undertaken to genetically characterize 5 Bahamian populations representing the Northwest (Grand Bahama and Abaco) and Central (Eleuthera, Exuma and Long Island) Bahamas across the 15 autosomal Identifiler loci routinely employed in forensic analyses. Altogether, our findings suggest that Bahamians are a genetically heterogeneous group, with each island sampled receiving differential contributions from African, European, East Asian and Native American sources. Even though the strongest genetic signal in all 5 collections emanates from continental Africa, inter-island differentiation is noted in both the Structure and admixture analyses. The presence of alleles not in common among the 5 insular populations also signals genetic heterogeneity among the islands of the archipelago. This is especially the case when considering the Long Island population, which exhibits statistically significant genetic differences in relation to the other Bahamian collections and the New World groups of African descent (Afro-American and Afro-Caribbean) in the G-test pair-wise comparisons, even after application of the Bonferroni adjustment.

Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Melanie Meredith, Jo-Anne Bright, Sarah Cockerton, Sue Vintiner — 2011-03-16

Abstract: Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.

Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

Torben Tvedebrink, Poul Svante Eriksen, Helle Smidt Mogensen, Niels Morling — 2011-04-04

Abstract: DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above a certain level or above a signal to noise threshold. Degradation implies that these peak intensities decrease in strength for longer short tandem repeat (STR) sequences. Consequently, long STR loci may fail to produce peak heights above the limit of detection resulting in allelic or locus drop-outs.In this paper, we present a method for measuring the degree of degradation of a sample and demonstrate how to incorporate this in estimating the probability of allelic drop-out. This is done by extending an existing method derived for non-degraded samples. The performance of the methodology is evaluated using data from degraded DNA, where cases with varying amounts of DNA and levels of degradation are investigated.

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Natalie E.C. Weiler, Anuska S. Matai, Titia Sijen — 2011-04-01

Abstract: Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpFℓSTR® Identifiler® amplification parameters are changed to an annealing time of 20min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpFℓSTR® Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.

Discordance at D3S1358 locus involving SGM Plus™ and the European new generation multiplex kits

Aliza Raziel, Carla Oz, Aviva Dell’Ariccia Carmon, Rafi Ilsar, Ashira Zamir — 2011-04-07

Abstract: During the course of routine database sample analysis in the Israel Police DNA database, an off-ladder D3S1358 allele, calculated to be >22.1, extending into the adjacent vWA locus was observed using Applied Biosystems SGM Plus™ kit.To verify the size of this D3S1358 long allele and to ensure it was not part of a trialle pattern in the neighboring locus, the sample was amplified using three of the European new generation STR multiplex kits: NGMTM (Applied Biosystem), Powerplex™ ESX and ESI (Promega). The results of these amplifications determined the variant to be a 22 allele. Subsequent sequencing confirmed this designation and revealed a nucleotide polymorphism. Ten additional SGM Plus™ profiled samples with D3S1358 alleles larger than 19, were re-analyzed using NGMTM and Powerplex™ ESX which also showed discordance in the calculated results between original SGM Plus™ designations and those obtained with the European new generation multiplexes.

Belgian canine population and purebred study for forensics by improved mitochondrial DNA sequencing

Stijn Desmyter, Leonie Gijsbers — 2011-04-14

Abstract: In canine population studies for forensics, the mitochondrial DNA is profiled by sequencing the two hyper variable regions, HV1 and HV2 of the control region.In a first effort to create a Belgian population database some samples showed partially poor sequence quality. We demonstrated that a nuclear pseudogene was co-amplified with the mtDNA control region. Using a new combination of primers this adverse result was no longer observed and sequencing quality was improved. All former samples with poor sequence data were reanalyzed. Furthermore, the forensic canine population study was extended to 208 breed and mixed dogs. In total, 58 haplotypes were identified, resulting in an exclusion capacity of 0.92. The profile distribution of the Belgian population sample was not significantly different from those observed in population studies of three other countries.In addition to the total population study 107 Belgian registered pedigree dogs of six breeds were profiled. Per breed, the obtained haplotypes were supplemented with those from population and purebred studies. The combined data revealed that some haplotypes were more or less prominent present in particular dog breeds. The statistically significant differences in haplotype distribution between breeds and population sample can have consequences on mtDNA databasing and matching probabilities in forensics.

An unusual case of identification by DNA analysis of siblings

2011-03-28

Abstract: A badly decomposed body required identification by means of DNA analysis. A brother and sister of the deceased were available as reference subjects. Although investigation of Y-chromosomal markers established an exclusion condition, autosomal markers suggested a positive identification. In order to increase the reliability of the tests, X-chromosomal markers were also investigated. This analysis showed the body to have an XXY genotype (Klinefelter's syndrome). A number of hypotheses were assessed using biostatistical methods, ultimately resulting in a definite identification. The special aspect of Klinefelter's syndrome proved highly useful for biostatistical analysis.

Developmental validation of the PowerPlex® ESX 16 and PowerPlex® ESX 17 Systems

2011-04-06

Abstract: We describe the developmental validation study performed on the PowerPlex® ESX 16 (European Standard Extended 16) and the PowerPlex® ESX 17 Systems, part of a suite of four new DNA profiling kits developed by Promega in response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. The PowerPlex® ESX 16 System combines the 11 loci compatible with the UK National DNA Database, contained within the AmpFlSTR® SGM Plus® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to incorporate these five new loci as mini- and midi-STRs while maintaining the loci found in the AmpFlSTR® SGM Plus® kit as standard size. The PowerPlex® ESX 17 System amplifies the same loci as the PowerPlex® ESX 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR® SGM Plus® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex® ESX 16 and ESX 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5pg of a fully heterozygous single source DNA template. In mixture analysis, a range of 52–95% of unique minor contributor alleles was observed at 19:1 mixture ratios where only 25pg of the minor component was present. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of information obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.

Allelic frequencies and statistical data obtained from 48 AIM INDEL loci in an admixed population from the Brazilian Amazon

2011-05-03

Abstract: Allelic frequencies of 48 informative insert-delete (INDEL) loci were obtained from a sample set of 130 unrelated individuals living in Macapá, a city located in the northern Amazon region, in Brazil. The values of heterozygosity (H), polymorphic information content (PIC), power of discrimination (PD), power of exclusion (PE), matching probability (MP) and typical paternity index (TPI) were calculated and showed the forensic efficiency of these genetic markers. Based on the allele frequency obtained for the population of Macapá, we estimated an interethnic admixture for the three parental groups (European, Native American and African) of, respectively, 50%, 21% and 29%. Comparing these allele frequencies with those of other Brazilian populations and the parental populations, statistically significant distances were found. The interpopulation genetic distance (FST coefficients) to the present database ranged from FST=0.0431 (p<0.00001) between Macapá and Belém to FST=0.266 (p<0.00001) between Macapá and the Native American group.

Distribution of Y chromosomal STRs loci in Mayan and Mestizo populations from Guatemala

2011-05-12

Abstract: In this study, a sample of 225 Guatemalan males, comprising 115 Mestizo-Guatemalan and 110 Mayan-Guatemalan, was typed for 17 Y-short tandem repeats (STRs) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, YGATA_H4.1 and DYS385a/b). The haplotype diversity (H=1) and discrimination capacity (96.86%) were calculated. Analysis of molecular variance (AMOVA) demonstrated a low but significant interpopulation differentiation when compared with the results obtained when we confront the Mestizo and Mayan populations with the European populations.Furthermore, the genetic variability and differences among the American, African, Asian, and European populations were analyzed with the software Statistica 9.1. In addition, the genetic distances were also calculated using other published data. Reynolds and Slatkińs genetic distance was visualized using the multidimensional scaling (MDS) analysis. All the analysis performed locates the Mayan population next to the Native American population, while Guatemalan-Mestizo population was found to be between these populations and the European population, similar to other Mestizo one.The implementation of the estimation of individual ancestry proportions of the whole population sample showed the presence of two well-differentiated population groups.

Current Next Generation Sequencing technology may not meet forensic standards

Hans-Jürgen Bandelt, Antonio Salas — 2011-05-12

Abstract: In a Nature paper of 2010, the concern was raised that intra-individual mtDNA variation may be more pronounced than previously believed, in that heteroplasmies are common and vary markedly from tissue to tissue. This claim taken at face value would have considerable impact on forensic casework. It turns out however that the employed technology detected the germ-line variation relative to the reference sequence only incompletely: on average at least five mutations were missed per sample, as an in silico reassessment of the data reveals. Before one can really set out to access to entire mtDNA genome data with relative ease for forensic purposes, one needs careful calibration studies under strict forensic conditions—or might have to wait for another generation.

The role of probabilities in graphical models

A. Biedermann, F. Taroni — 2010-11-09

We have read with great interest the paper “A universal strategy to interpret DNA profiles that does not require a definition of low-copy-number” by Peter Gill and John Buckleton (Forensic Science International: Genetics 4 (2010) 221–227). We are very happy to see many carefully prepared arguments in support of a likelihood ratio based assessment of DNA profiling results. The paper also mentions the use of graphical models. It is on this point that we would like to draw the reader's attention here, and add additional explanations. Specifically, on page 225, the authors write: “Probabilistic determinations can be made using graphical models (or Bayes nets) [44–47] but these require the utilisation of prior probabilities, which is problematic for scientists to use within the UK courts [48,49].” From a purely technical point of view, that is, concerning the requirement of prior probabilities, this view is not wrong. However, from a conceptual point of view, this does not necessarily imply a problem for scientists because Bayesian networks can be used in a way that renders actual values for prior probabilities irrelevant. Below, we would like to point this out in some further detail.

Allele frequencies of fifteen STR loci in U.S. immigrants from Haiti compared with African Americans and Afro-Caribbeans

Amie Branham, Robert Wenk, Francis Chiafari — 2010-12-06

About 20,000 Haitians emigrate to the United States each year. Many are beneficiaries of family-based petitions from naturalized U.S. citizens . Since Haitian birth and marriage records are often nonexistent, adjudicators of the U.S. Citizenship and Immigration Service have evaluated claimed relationships in light of genetic tests for many years . Tests are provided by non-governmental laboratories accredited by the AABB and meeting College of American Pathologists proficiency test requirements. The population derives from the European colonization of the Americas and the slave trade that flourished between the 15th and 19th centuries. The present derivative black populations of the Western hemisphere are admixtures of primarily West Africans and Europeans with small contributions from Amerindians and other groups. We report Haitian allele frequencies of 15 widely-used short tandem repeat (STR) loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, PentaD, PentaE, TH01, TPOX and vWA) and compare these frequencies with those of African Americans (AAs) and with four Afro-Caribbean populations from Trinidad, Jamaica and the Bahamas as well as from the adjacent Dominican Republic .

Genetic analysis of 17 Y-chromosomal STRs haplotypes of three ethnic groups residing in West Bengal, India

Soma Roy, Sabahat Noor, I. Haque — 2010-12-13

We have analyzed 17 Y-chromosomal STR loci (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS393, DYS391, DYS439, DYS635, DYS392, Y-GATA-H4, DYS437, DYS438, DYS448 and DYS385a/b) in 125 unrelated male individuals from three ethnic group of West Bengal, India. A total of 109 distinct haplotypes for the 17 Y-STR markers were identified and, of these, 33 from Rajbanshi, 38 from Paliya and 21 haplotypes from Dhimal were found to be unique. Paliya share one haplotype with Rajbanshi and one haplotype with Dhimal populations. Moreover, none of the identified distinct haplotypes revealed in the present study have been described in YHRD database till date.

The Israel Police DNA database: Recognizing the capability of databases in providing investigative leads

Carla Oz, Merav Amiel, Ashira Zamir — 2010-12-22

The Israel Police DNA database has been operative since February 2007. During this period of operation a number of criminal cases have been encountered and resolved not by direct cold hits, but by following up investigative leads provided by the DNA database. DNA databases contain a wealth of investigative information not yet routinely exploited. Partial matches obtained by DNA database searches, although presently not universally accepted, are rapidly becoming significant sources of investigative assistance for law enforcement agencies. In unsolved cases where all other investigative leads have been exhausted, a profile containing a large amount of allelic similarities to that from a crime scene may point the detectives toward a sibling or close relative who may be the actual perpetrator of the crime. In a number of global databases, law enforcement requests for such searches in the form of “familial searches” are becoming commonplace. In this letter we present a number of cases and scenarios encountered during the routine operation of the Israel DNA database illustrating the benefit that can be afforded to the local investigative process when databases are recognized and exploited for more than just as a source of direct hits.

Allele frequencies of nine non-CODIS STR loci in Chinese Uyghur ethnic minority group

2011-01-27

We investigated the genetic polymorphisms of nine non-combined DNA index system (CODIS) short tandem repeat (STR) loci for 252 healthy unrelated autochthonous individuals of Chinese Uyghur ethnic minority group in Xingjiang. The 9 STR loci included in STRtyper10G/F™ kit (D18S1364, D12S391, D13S325, D6S1043, D2S1772, D11S2368, GATA198B05, D8S1132, and D7S3048) were highly polymorphic markers and reliable tools for forensic casework . These markers are probably unlinked to common STRs included in commercially available kits (e.g., PowerPlex 16 system) and powerful to obtain additional information in STR analysis for complicated cases .

Population data for 10 X-chromosome STRs from north-east of Spain

Belén García, Manuel Crespillo, Miguel Paredes, Juan L. Valverde — 2011-02-04

Autosomic and chromosome Y STRs markers have been widely applied in forensic identity and paternity test, however, the application of X chromosome STR markers plays a minor role in forensic casework. Increasingly, forensic laboratories have introduced the use of X-STR markers, especially in cases of complex kindship analysis. Population data of chromosome X can help to increase and to complete the information and knowledge about this chromosome .

Bracketing off population does not advance ethical reflection on EVCs: A reply to Kayser and Schneider

Amade M’charek, Victor Toom, Barbara Prainsack — 2011-02-01

In a recent contribution to this journal, Kayser and Schneider reviewed the relevance of external visible characteristics (EVCs) for criminal investigation . Their aim was to broaden the debate about the scientific, legal, and ethical dimensions of the use of EVCs for criminal investigation, which will help to achieve a firm legal basis for the application of EVCs eventually. While we applaud Kayser's and Schneider's overall very thoughtful and nuanced discussion of this topic, we were surprised to read that they suggest that a discussion of ‘the challenges of using problematic definitions of populations […] has to be kept separate from using EVCs’ (p. 158). In contrast to these authors, we contend that questions about defining populations – both at the level of scientific research, and the application of EVCs in criminal investigation – lie at the core of most social, ethical, and legal issues raised by the translation of EVCs into forensic and police practices.

Reply to “Bracketing off population does not advance ethical reflection on EVCs: A reply to Kayser and Schneider” by A. M’charek, V. Toom, and B. Prainsack

Manfred Kayser, Peter M. Schneider — 2011-02-14

We appreciate the letter by M’charek et al. regarding our publication on DNA-based predictions of human externally visible characteristics (EVCs) in forensics . It indeed was one of the intentions of our paper to stimulate a debate across disciplines by providing scientific facts about the state of the art in this area of biological research, and by developing a scenario for applying DNA-based EVC prediction in the context of future criminal investigations. That our decision to separate the discussion about DNA-based EVC prediction from DNA-based inferences of bio-geographic ancestry (“ethnic orgin”) having had triggered the concerns raised by M’charek and colleagues clearly indicates that the underlying scientific knowledge of both aspects is not yet widespread enough. Apart from that, we would like to note that in our previous article we had several reasons not to mix DNA-based bio-geographic ancestry inference with DNA-based EVC prediction intending to deliver the messages on EVCs in a clear and straightforward way. In the following we would like to provide some more explanations, while also including comments on some of the statements made by M’charek et al. in their letter .

Genetic variation of 15 autosomal STR loci in various populations from southern Africa

Carina M. Schlebusch, Himla Soodyall, Mattias Jakobsson — 2011-01-31

We determined the allele frequencies for the 15 autosomal STR loci included in the AmpFlSTR® Identifiler™ kit in 324 unrelated individuals from southern African, representing San, Khoe, Coloured and Bantu-speaking populations. All individuals in the study originated from southern African countries including South Africa, Botswana, Namibia and Angola (). The individuals represented different sub-groups of the San, Khoe, Coloured, southeastern Bantu-speaking and southwestern Bantu-speaking populations ().

Population genetics polymorphisms on 17 autosomal STRs from Chinese Bai ethnic minority group

2011-01-31

We studied the genetic polymorphisms and evaluated the forensic application values of 17 autosomal short tandem repeat (STR) loci. We have co-amplified and analyzed the 13 loci STRs included in the Combined DNA Index System (D3S1358, D13S317, D7S820, D16S539, TPOX, THO1, CSF1PO, vWA, D5S818, FGA, D8S1179, D21S11, D18S51), and also five additional loci Penta E, D19S433, D6S1043, D2S1338 plus Amelogenin using one multiplex PCR reaction system in a population sample of Chinese Bai ethnic minority group residing in Dali Bai Nationality Autonomous Prefecture in Yunnan province, China.

X-chromosomal haplotype frequencies of four linkage groups using the Investigator Argus X-12 Kit

Jeanett Edelmann, Sabine Lutz-Bonengel, Jana Naue, Sandra Hering — 2011-02-03

The Investigator Argus X-12 Kit allows simultaneous amplification of 12 X-chromosomal STR loci which are highly informative for kinship and paternity testing, population genetics and anthropological studies. The markers of the Investigator Argus X-12 Kit are clustered into 4 linkage groups with 3 closely linked markers per group. For genotyping each set of markers should be handled as haplotype. Here we present first haplotype frequency data of a German population involving 1037 unrelated males.

Forensic genetic data of 6 Y-STR loci: An expanded Korean population database

2011-02-04

Applications of Y-chromosomal DNA analysis in forensic casework benefit from large population databases for estimating the probability of identity by chance. Thus, it is important that Y-STR databases continue to be expanded, and become more reliable to provide a better tool for forensic analysis. We analyzed the allelic frequencies of six Y-STR loci (DYS522, DYS533, DYS549, DYS570, DYS576, and DYS643) in 506 unrelated males from six major Korean provinces to provide an expanded and reliable Korean data base, showing individualizing male lineages by adding the 6 Y-STRs to 17 Y-STRs in the Yfiler kit. These 506 individuals have been genotyped previously for 12 Y-SNPs (M9, M45, M89, M119, M122, M174, M175, M214, RPS4Y, P31, SRY465, and 47z) and 17 Y-STRs (Yfiler) . Multiplex-PCR of six Y-STRs was performed as previously described . Amplified products were analyzed by capillary electrophoresis using the ABI3730 genetic analyzer system (Applied Biosystems, Foster City, CA, USA). The Y-STR allele nomenclatures were designated by the number of repeats, according to the DNA Commission of the International Society of Forensic Genetics . Proficiency testing of Collaborative Testing Service (CTS) and the YHRD (http://www.yhrd.org) trials was conducted . The same standard DNA (007) was included as an internal control for each genotype batch. Allele frequencies were estimated by direct counting from the observed phenotypes. Haplotype diversity and gene diversity were calculated as previously described .

Allele frequencies of the new European Standard Set (ESS) loci in a population of Southern Italy (Calabria)

Anna Barbaro, Patrizia Cormaci, Stefano Votano, Alessandro Agostino — 2011-03-14

Allele frequencies of 5 STRs loci (D10S1248, D2S441, D1S1656, D12S391, and D22S1045) included in the new European Standard Set (ESS) were calculated in Calabria (South of Italy) population using the next generation multiplex AmpFlSTR NGM by Applied Biosystems .

Genetic characterization of Western Iberia using Mentype® Argus X-8 kit

2011-04-18

In order to build a X-STR database in individuals from Western Iberia, 327 samples (83 females and 244 males) were genotyped by the use of Mentype® Argus X-8 kit. In particular, a total of 410 X chromosomes from unrelated individuals living in four regions from western Iberia: Northern, Central and Southern Portugal and Galicia (north-western Spain), were investigated. In the case of Galicians, 163 samples (83 females and 80 males) were taken in buccal swabs, whilst 164 blood samples were collected from Portuguese male individuals. All donors previously signed an informed consent. Then, DNA was extracted from saliva samples using the QIAamp® DNA Blood Mini Kit (Qiagen) following manufacturer's recommendations and that from blood samples was extracted using a standard Chelex based procedure.

Genetic data for D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045 miniSTR loci from the state of Rio Grande do Sul, Southern Brazil

2011-04-14

We determined the allelic frequencies for six miniSTR loci D10S1248, D14S1434, D22S1045 (miniplex NC01) and D1S1677, D2S441, D4S2364 (miniplex NC02) in a sample of 439 unrelated individuals from the Rio Grande do Sul State (RS), Southern Brazil. This paper followed the FSI Genetics guideline recommendations .

Genetic data of 15 STR loci in five populations from Afghanistan

Julie Di Cristofaro, Stéphane Buhler, Shah Aga Temori, Jacques Chiaroni — 2011-04-13

The variability of 15 hyper-variable autosomal STR loci included in the AmpFlSTR Identifiler™ PCR amplification kit (Applied Biosystems) was analyzed in Afghan individuals from five populations located at a major continental crossroad in the history of human migrations. A total of 456 blood samples from unrelated male individuals donors aged from 20 to 55 years old with ancestry traced back at least two generations and originating from five tribes: Hazara, Ouzbek, Tadjik, Turkmen and Pachtoun were collected and analyzed (see ).

A genetic study of 12 X-STR loci in the Hungarian population

Gergely Horváth, Andrea Zalán, Zoltán Kis, Horolma Pamjav — 2011-04-06

Males carry one X-chromosome, thus they transmit their X-chromosome to daughters as haplotypes. Females carry two X-chromosomes which are liable to recombination during meiosis, thus this must be taken into consideration for interpretation and calculation of probabilities in kinship studies.

Genetic diversity of 12 X-chromosomal short tandem repeats in the Moroccan population

2011-04-04

Morocco is a country located in northwest Africa. It has a long coastline on the Atlantic Ocean that reaches past the Strait of Gibraltar into the Mediterranean Sea. Morocco has been the home of the Berbers since the second millennium BC. It was annexed by Rome in the 1st century and invaded by Arabs in the 7th century. The country was later united (11th–13th century) under Berber-Muslim dynasties. Since several invasions have successively interspersed with the original population throughout history (e.g. Phoenicians, Romans, Arabs, Spanish, French and Sub-Saharan Africans) , actually, the Moroccan population is an admixture of Mediterranean, African and Oriental populations, as evidenced by the heterogeneous gene pool .

Allele frequencies and concordance study of 16 STR loci – including the new European Standard Set (ESS) loci – in an Austrian population sample

P. Hatzer-Grubwieser, B. Berger, D. Niederwieser, M. Steinlechner — 2011-05-02

National DNA databases have been established as an important crime prevention tool in many European countries. The increasing exchange of DNA-profiles, which is facilitated by the treaty of Prüm and the Interpol DNA-database, necessitates to enhance the power of standard DNA Profiles. As a consequence ENFSI (European Network of Forensic Sciences Institutes) and EDNAP (European DNA Profiling Group) have recommended that the European Standard Set of Loci (D3S1358, FGA, D8S1179, THO1, vWA, D18S51, D21S11) should be extended by 5 additional loci (D2S441, D10S1248, D22S1045, D1S1656, D12S391) . Several companies now provide kits containing these new ESS loci and meet therefore the new recommendations for DNA profile sharing across Europe.

Expanding the CODIS core loci in the United States

Douglas R. Hares — 2011-05-05

After over a decade of operation, the National DNA Index System (NDIS) continues to grow in importance and size . While the STR DNA technology has remained relatively consistent, other key aspects of the NDIS program have been reevaluated and revisions implemented. For example, based upon recommendations of the Scientific Working Group on DNA Analysis Methods, the Director of the Federal Bureau of Investigation (FBI) issued revised Quality Assurance Standards (QAS) for Forensic DNA Testing and Databasing Laboratories that took effect July 1, 2009 . The FBI Laboratory also embarked on an examination of the standards, procedures and policies governing NDIS to ensure that such processes were not impeding the efficient uploading of quality DNA data to NDIS. Consistent with these reviews, the FBI also launched a review of the current Combined DNA Index System (CODIS) core loci to determine if additional loci should be included in the CODIS core to facilitate greater discrimination, assist missing person investigations and promote compatibility for international data sharing efforts.

Allele frequencies of the five new generation forensic STR (D1S1656, D2S441, D10S1248, D12S391 and D22S1045) in the population from Rio Grande do Sul, Southern Brazil

2011-07-04

Allele frequencies of the five recently introduced European Standard Set (ESS) STRs markers (D1S1656, D2S441, D10S1248, D12S391 and D22S1045) were obtained from a sample of 353 individuals undergoing paternity testing in the population of Rio Grande do Sul, Southern Brazil. Rio Grande do Sul was essentially colonized by Portuguese, Germans, and Italians immigrants in the 18th century . All participants signed informed consent forms ().

Allele frequencies of 15 STRs in the Calchaqui Valleys population (North-Western Argentina)

A. Muñoz, M.V. Albeza, N. Acreche, J.A. Castro, M.M. Ramon, A. Picornell — 2011-06-06

Abstract: Allele frequencies for 15 short tandem repeat (STR) loci were obtained from a sample of 110 individuals from the Calchaqui Valleys population (North-Western Argentina). The combined power of exclusion and combined power of discriminating for the 15 tested STR loci were 0.999964 and 0.9999999999999998, respectively. Matching probability was 1 in 4.58×10(15). Therefore, it may be concluded that the set of 15 STRs included in the AmpF STR Identifiler kit, represents a powerful tool for forensic applications, paternity testing and population genetics studies in the Calchaqui Valleys population.

A paternity case with mutations at three CODIS core STR loci

Hong-yu Sun, Hai-xia Li, Xiang-pei Zeng, Zheng Ren, Wen-jing Chen — 2011-06-03

Short tandem repeat (STR) is the most widely used genetic marker in the paternity identification. Mutations of STR loci have been observed and reported during the paternity testing . However, most of these mutations occurred at 1–2 loci in a case. It was very rare that mutations occurred simultaneously at three STR loci, especially at 13 CODIS core loci . Here we show a non-exclusion paternity case with three CODIS core loci incompatibilities.