Forensic Science International: Genetics - Articles in Press

Wildlife forensics: “Supervised” assignment testing can complicate the association of suspect cases to source populations - Corrected Proof

M.C. Ball, L.A. Finnegan, T. Nette, H.G. Broders, P.J. Wilson — 2010-03-08

Abstract: Forensic science techniques are an important component of investigations for wildlife-related offences. In particular, DNA analyses can be used to characterize several attributes of biological evidence including sex, individual and species identification. Additionally, genetic assignment testing has enabled forensic biologists to identify the local population from which an individual may have originated. This technique has proved useful in situations where animals have been illegally harvested from areas/populations where hunting is prohibited. For this report, we used individual-based clustering (IBC), in the program Structure 2.2, under both “supervised” and “unsupervised” approaches to assess whether three suspected, illegally harvested moose originated from an endangered population. Atypical circumstances, with Nova Scotia having two moose sub-species in its jurisdiction, enabled strong IBC assignment testing results to determine the source population of the suspected samples. We found differences between the “unsupervised” and “supervised” modeling approaches to define genetic structure among the a priori characterized populations in our data set. Our findings illustrate the fact that individual clustering assignment tests can assist wildlife forensic cases to identify the source population of illegally harvested animals. However, the accuracy of results are highly dependant on the model choice used to define genetic clusters, as well as on the availability of a thorough database of samples throughout the managed area to accurately identify all genetic populations. Further, it is clear from our analyses that political jurisdictions do not accurately reflect isolated populations and we recommend using unsupervised IBC modeling for biological accuracy.

Analysis of global variability in 15 established and 5 new European Standard Set (ESS) STRs using the CEPH human genome diversity panel - Corrected Proof

2010-03-08

Abstract: The CEPH human genome diversity cell line panel (CEPH-HGDP) of 51 globally distributed populations was used to analyze patterns of variability in 20 core human identification STRs. The markers typed comprised the 15 STRs of Identifiler, one of the most widely used forensic STR multiplexes, plus five recently introduced European Standard Set (ESS) STRs: D1S1656, D2S441, D10S1248, D12S391 and D22S1045. From the genotypes obtained for the ESS STRs we identified rare, intermediate or off-ladder alleles that had not been previously reported for these loci. Examples of novel ESS STR alleles found were characterized by sequence analysis. This revealed extensive repeat structure variation in three ESS STRs, with D12S391 showing particularly high variability for tandem runs of AGAT and AGAC repeat units. The global geographic distribution of the CEPH panel samples gave an opportunity to study in detail the extent of substructure shown by the 20 STRs amongst populations and between their parent population groups. An assessment was made of the forensic informativeness of the new ESS STRs compared to the loci they will replace: CSF1PO, D5S818, D7S820, D13S317 and TPOX, with results showing a clear enhancement of discrimination power using multiplexes that genotype the new ESS loci. We also measured the ability of Identifiler and ESS STRs to infer the ancestry of the CEPH-HGDP samples and demonstrate that forensic STRs in large multiplexes have the potential to differentiate the major population groups but only with sufficient reliability when used with other ancestry-informative markers such as single nucleotide polymorphisms. Finally we checked for possible association by linkage between the two ESS multiplex STRs closely positioned on chromosome-12: vWA and D12S391 by examining paired genotypes from the complete CEPH data set.

A comparison of mini-STRs versus standard STRs—Results of a collaborative European (EDNAP) exercise - Corrected Proof

2010-03-02

There is a general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new DNA markers is to increase the chance of obtaining DNA profiles from highly degraded DNA rather than to increase the discriminating power gained from full DNA profiles. Previous collaborative exercises have demonstrated that smaller amplicons tend to provide improved amplification when the DNA template has been degraded . These exercises have led to the commercial development of ‘mini-STR’ multiplex kits.

Typing of 49 autosomal SNPs by SNaPshot® in the Slovenian population - Corrected Proof

Katja Drobnič, Claus Børsting, Eszter Rockenbauer, Carmen Tomas, Niels Morling — 2010-03-02

Abstract: A total of 157 unrelated individuals residing in Slovenia were typed for 49 of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex with the SNaPshot® assay. We obtained full SNP profiles in all but one individual and perfect concordance was obtained in duplicated analyses. Allele frequencies are presented for the 49 SNPs. No deviation from HWE was observed for any SNP. FIS and FST were estimated. A principal coordinate analysis performed on six populations (Slovenian, Danish, Somali, Greenland, Turkish and Chinese) showed that the Slovenian population grouped with the Danish population. The mean power of discrimination for the Slovenian population was 1.1×10−19, and the mean exclusion probability for trios was 99.96%.

Allele frequencies of six non-CODIS miniSTR loci (D1S1627, D3S4529, D5S2500, D6S1017, D8S1115 and D9S2157) in three South African populations - Corrected Proof

Zainonesa Abrahams, Maria Eugenia D’Amato, Sean Davison, Mongi Benjeddou — 2010-03-02

Abstract: The allele frequency of six non-CODIS miniSTR loci: D1S1627, D3S4529, D5S2500, D6S1017, D8S1115 and D9S2157 were investigated in three South African populations (Afrikaner, Asian Indian and Mixed Ancestry). Deviation from Hardy–Weinberg equilibrium was observed in the Mixed Ancestry population for the locus D9S2157. All loci showed a moderate degree of polymorphism with heterozygosity values >0.6 for all populations. The combined power of discrimination was: 0.999997723, 0.999997163 and 0.99999961 for Afrikaner, Asian Indian and Mixed Ancestry populations, respectively. The respective values for the combined power of exclusion in these populations were: 0.99, 0.99 and 0.98.

Feline non-repetitive mitochondrial DNA control region database for forensic evidence - Corrected Proof

2010-02-26

Abstract: The domestic cat is the one of the most popular pets throughout the world. A by-product of owning, interacting with, or being in a household with a cat is the transfer of shed fur to clothing or personal objects. As trace evidence, transferred cat fur is a relatively untapped resource for forensic scientists. Both phenotypic and genotypic characteristics can be obtained from cat fur, but databases for neither aspect exist. Because cats incessantly groom, cat fur may have nucleated cells, not only in the hair bulb, but also as epithelial cells on the hair shaft deposited during the grooming process, thereby generally providing material for DNA profiling. To effectively exploit cat hair as a resource, representative databases must be established. The current study evaluates 402bp of the mtDNA control region (CR) from 1394 cats, including cats from 25 distinct worldwide populations and 26 breeds. Eighty-three percent of the cats are represented by 12 major mitotypes. An additional 8.0% are clearly derived from the major mitotypes. Unique sequences are found in 7.5% of the cats. The overall genetic diversity for this data set is 0.8813±0.0046 with a random match probability of 11.8%. This region of the cat mtDNA has discriminatory power suitable for forensic application worldwide.

Y-chromosome and autosomal STR diversity in four proximate settlements in Central Anatolia - Corrected Proof

2010-02-24

Abstract: Due to the longstanding human presence in the region and the influence of social traditions, the genetic make-up of populations currently inhabiting Turkey (Anatolia) is quite complex. To characterize the patterns of genetic diversity in rural Central Anatolian villages, we analyzed samples collected at four local settlements for variation at 17 Y-chromosome STR and 15 autosomal STR loci. The resulting data reveal considerable diversity within these settlements, as well as some structure in the paternal genetic variation, with a limited number of haplotypes being shared between the communities. These findings have important implications for forensic studies of Turkish populations.

Population genetics of 17 Y-chromosomal STR loci in Yakutia - Corrected Proof

2010-02-24

Abstract: Haplotype and allele frequencies of 17 Y-chromosomal short tandem repeat (Y-STR) markers in a population sample of 133 Yakut male volunteers from two regions: Central (n=41) and Western Yakutia (n=92) were determined using the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems). A total of 65 haplotypes were identified in the Yakut population, with 15 haplotypes in Central sample and 54 haplotypes in Western sample. Haplotype diversity values of 0.79 and 0.96, and average gene diversity values of 0.14 and 0.41 were calculated for Central and Western samples, respectively. The Fst distances between both our Yakut populations with other Russian, Siberian and Chinese populations were represented by MDS plot. The graphical view demonstrated close distances between most Yakut populations and differences with other Siberian populations.

Frequency assessment of 25 SNPs in five different populations - Corrected Proof

2010-02-22

Abstract: Allele and genotype frequencies of 25 SNPs previously selected and validated for forensic purposes were assessed in 250 unrelated individuals originating from five different countries of Europe (Spain, Croatia, Bulgaria, Turkey and Serbia). All the SNPs generated extremely low Fst values confirming our previous results on Italian, African (Benin) and Asian (Mongolian) populations. As a consequence of such Fst values we observed similar values of random match probability across the populations: 2.26×10−10 in the Spanish population, 2.13×10−10 in the Croatian population, 4.21×10−10 in the Bulgarian population, 2.52×10−10 in the Serbian population and 1.46×10−10 in the Turkish population.

X-chromosome markers in kinship testing: A generalisation of the IBD approach identifying situations where their contribution is crucial - Corrected Proof

Nádia Pinto, Leonor Gusmão, António Amorim — 2010-02-19

Abstract: The standard practice of forensic kinship evaluation uses unlinked autosomal markers. However, X-chromosome markers have recently gained recognition as a powerful tool to complement the information provided by autosomes, particularly in complex cases.In this paper, the X-chromosome mode of transmission is addressed in the theoretical identity-by-descent framework. Formulas for the joint genotypic probabilities considering various pedigrees relating two inbred and/or non-inbred individuals are derived.Finally, the importance of X-chromosome markers is highlighted by the fact that, in addition to complementing the autosomal information, X-chromosome transmission allows differential weighting of certain hypotheses regarding pedigrees belonging to the same autosomal class, i.e., pedigrees that are indistinguishable by the use of unlinked autosomal markers. Illustrative examples of common kinship testing are shown.

Application of mtDNA SNP analysis in forensic casework - Corrected Proof

Stephan Köhnemann, Heidi Pfeiffer — 2010-02-19

Abstract: In this study six forensic cases are presented where the routine analysis of samples for short tandem repeats (STRs) failed. The sequencing of the mitochondrial hypervariable region I (HVR I) also failed. Nevertheless, it was possible to analyse the samples with mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) via SNaPshot technique. The age of the analysed samples ranged from 2 months to 1400 years. Saliva-, blood-, sperm-, hair-, tooth- and bone-samples were investigated. Furthermore the mtDNA SNP analysis of a forensic case sample showing a mixed stain profile is presented. It was possible to discriminate two different haplogroups in this mixed-person stain. If compared to another mtDNA SNP profile that was found in a hair, the discriminating SNPs of the hair were as well found in the mixed-person stain.To disburden the SNP analysis in forensic casework, haplogroup assignment criteria and quality criteria for mtDNA SNaPshot analysis are announced.

Population genetic analysis of Moroccans residing in Belgium using 16 autosomal STRs of the PowerPlex ESI 17 multiplex - Corrected Proof

2010-02-12

As a consequence of immigration starting in the 1960s, about 265,000 people of Moroccan origin presently reside in Belgium, thus constituting the most numerous group of non-indigenous inhabitants . Most Moroccan immigrants in Belgium as well as the Netherlands and Germany belong to the Berber (Imazighen) ethnic group and originate from the Rif area, a mountainous region of northern Morocco. Because of socio-economic issues beyond the scope of this journal, they are over-represented in forensic as well as paternity testing statistics. We tested a sample of 239 apparently unrelated Moroccans (134 males, 105 females), representing the mothers and alleged fathers from paternity cases. All had consented to the anonymous use of their data for a population genetic study.

Population database of 17 autosomal STR loci from the four predominant Eastern Slovakia regions - Corrected Proof

2010-02-12

We investigated four sample sets of 1373 unrelated healthy randomly selected individuals from the eastern Slovakia regions: Šariš (424), Zemplín (279), Abov-Gemer (522) and Spiš (148) irrespective of sex and ethnic background. Historically, all four regions were relatively isolated with their own distinct dialects.

Allele frequencies of the new European Standard Set (ESS) loci in the Italian population - Corrected Proof

2010-02-10

Allele frequencies of five new STR loci (D22S1045, D10S1248, D1S1656, D12S391, D2S441) included in the new European Standard Set (ESS) were calculated in a sample of 209 unrelated Italians with the Powerplex ESI® 17 system (Promega Corporation, Madison, WI). Forensic and population indices were estimated.

Population study of fourteen X chromosomal short tandem repeat loci in a population from Bosnia and Herzegovina - Corrected Proof

2010-02-10

X chromosomal short tandem repeat (STR) loci can be used in combination with autosomal STRs to solve cases of complex kinship. In order to properly apply STR results to forensic cases, population databases must be established to which comparisons can be made and the rarity of profiles determined. Here, a population from Bosnia and Herzegovina was studied to determine the appropriate forensic efficiency parameters with 14 widely used X chromosomal STR loci.

Response to letter to the editor by Prinz et al. - Corrected Proof

Dan Frumkin, Adam Wasserstrom — 2010-02-08

We appreciate the letter by Prinz et al. in response to our paper , and view this as the beginning of a serious discussion on the issue of DNA authentication, which is critical for maintaining the credibility of DNA profiling in the judiciary system and the general public. Prinz et al. recognize the feasibility of creating fake DNA evidence which goes beyond “traditional” planting of items in crime scenes. However, they raised several concerns, mainly that (i) we provide technical instructions for faking DNA, (ii) we are creating a problem for the forensic community, and yet (iii) we do not provide a feasible solution. In response to this we point out that (i) our “technical instructions” in demonstrating the problem are merely commercial kits and online protocols that are trivial to any biologist with basic training, (ii) we do not create a problem, but rather expose an issue that needs to be addressed, and most importantly (iii) although our first generation authentication assay is indeed based on a relatively labor-intensive and time consuming technique, it does provide an immediate solution. Moreover, we are currently at late stages of development of an improved, quick, and non-labor-intensive second generation assay that meets all the criteria suggested by Prinz et al. and can be integrated into the standard forensic procedure.

Response to letter to the editor by Mr. Mark Barash - Corrected Proof

Dan Frumkin, Adam Wasserstrom — 2010-02-08

We are happy for the opportunity to respond to the letter by Mr. Barash in response to our paper , and see this as another part of the growing debate on the important subject of forensic DNA authentication. Mr. Barash recognizes the importance of our research, but claims it suffers from several shortcomings. We present the claims raised by Mr. Barash, followed by our response to each point:

mRNA profiling for the identification of blood—Results of a collaborative EDNAP exercise - Corrected Proof

2010-02-08

Abstract: A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests.

Genetic diversity of autosomal STRs in eleven populations of India - Corrected Proof

2010-02-08

Indian sub-continent is rich in people belonging to different ethnic, cultural and linguistic groups. It owes biological diversity to its location, being placed at the tri-junction of the African, the northern-Eurasian and the Oriental realm . Indian population can be sub-structured on the basis of ethnicity as well as linguistic lineages. Linguistically, Indian populations are classified into four major families: Indo-European (IE), Dravidian (DR), Tibeto-Burman (TB) and Austro-Asiatic (AA). The IE languages are mainly spoken all over the country except southern region and along the Himalayas. Dravidian populations are mostly found in southern and central part of India. Tibeto-Burman populations are concentrated along the Himalayas in the north-eastern region whereas Austro-Asiatic speakers are dispersed mostly in the central and eastern India. All Austric speakers are exclusively tribal and may be the oldest inhabitants of India. The different religious communities, hierarchical castes and sub-castes, and isolated tribal groups have strict social rules governing mating patterns and are largely endogamous . In the present study, genetic polymorphism of the 15 autosomal STR loci was studied in eleven endogamous populations of four major linguistic groups of India. On the socio-cultural level, five of the selected populations were caste and six were tribal as detailed in . The populations were selected from different geographical regions to have an idea about genetic diversity at pan-India level. These populations have fascinated researchers because of diversity in culture, language and degree of endogamy.

Authentication of forensic DNA samples [Forensic Sci. Int. Genet. (2009)] - Corrected Proof

Mark Barash — 2010-02-03

Frumkin et al. in their article entitled “Authentication of forensic DNA samples” Forensic Sci. Int. Genet. (2009) doi:10.1016/j.fsigen.2009.06.009, describe the possibility of faking the results of forensic DNA analysis by applying artificial DNA on objects at crime scenes. In addition, they propose an authentification procedure for distinguishing between natural and artificial DNA.

Development and characterization of two mini-X chromosomal short tandem repeat multiplexes - Corrected Proof

Toni M. Diegoli, Michael D. Coble — 2010-02-01

Abstract: This study presents the development and characterization of two X chromosomal short tandem repeat (STR) multiplexes utilizing reduced-size amplicons (less than 200 base pairs) for identity and kinship testing with degraded DNA. Approximately 800 samples across 5 U.S. population groups were typed for 16 X chromosomal STR markers: DXS6789, DXS7130, DXS9902, GATA31E08, DXS7424, GATA165B12, DXS101, DXS6795, GATA172D05, DXS10147, DXS8378, DXS7132, DXS6803, HPRTB, DXS7423, and DXS7133. A high degree of polymorphism was observed for each marker and both multiplexes were sensitive down to 200pg of pristine DNA. The two proposed multiplexes are suitable for forensic use, and show potential for improved analysis of compromised bone samples.

Generating DNA profiles from immunochromatographic cards using LCN methodology - Corrected Proof

Thanh-Tam Ho, Reena Roy — 2010-01-27

Abstract: The aim of this research was to obtain DNA profiles from immunochromatographic test devices which have already yielded positive results with body fluids obtained from fourteen volunteers. Three different immunochromatographic cards for the identification of human blood and one for the identification of human saliva were used for this research. Each body fluid was detected using the appropriate immunochromatographic card. The used cards were kept at room temperature for various lengths of time. The membranes were removed at the end of the designated times and the entire strip was extracted using low copy number (LCN) extraction procedure. The extracted DNA was amplified using reduced amplification volume and higher PCR cycle numbers. Autosomal STR profiles were detected using AmpFℓSTR® Identifiler™ PCR Amplification Kit from Applied Biosystems (AB). Additionally, DNA extracted from the male volunteers was amplified using the AB AmpFℓSTR® Yfiler™ PCR Amplification Kit. Analysis of the amplified products was carried out by capillary electrophoresis injection on the AB 3130xl Genetic Analyzer. The generated DNA data was analyzed using the SoftGenetics GeneMarker® HID Version 1.7 software.Autosomal and Y-STR DNA profiles were obtained from most of the cards which were stored at room temperature for up to three months. DNA profile was obtained from all four types of the immunochromatographic cards used in this study. These profiles were concordant with the profiles obtained from the donors’ reference samples.

German standards for forensic molecular genetics investigations in cases of mass disaster victim identification (DVI) - Corrected Proof

2010-01-22

This year, several events (e.g. the air crashes in Lukla (Nepal) and of Air France flight 447 over the Atlantic Ocean) again demonstrated the necessity of standards in several areas of the Disaster Victim Identification (DVI) process.

The use of bacteria for the identification of vaginal secretions - Corrected Proof

Rachel I. Fleming, SallyAnn Harbison — 2010-01-20

Abstract: We have used the 16S–23S rRNA intergenic spacer region for identifying vaginal specific bacteria. Lactobacillus crispatus and Lactobacillus gasseri were detected in vaginal secretions but not in semen, blood or saliva. Our data indicated that both L. crispatus and L. gasseri were detected in vaginal secretions from women with different levels of expression of hormonal genes including pregnant, pre- and post-menopausal women, and a woman who has had a hysterectomy. Therefore, we have demonstrated that these Lactobacilli are promising new markers for the forensic identification of vaginal secretions. We have incorporated the Lactobacilli markers into a mRNA multiplex system to produce an 11-plex assay that can identify circulatory blood, menstrual blood, saliva, semen (in the presence and absence of spermatozoa) and vaginal secretions.

Y-STR haplotypes of Native American populations from the Brazilian Amazon region - Corrected Proof

2010-01-20

Abstract: The allele and haplotype frequencies of nine Y-STRs (DYS19, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393, DYS385 I/II) were determined in a sample of six native tribes from the Brazilian Amazon (Tiriyó, Awa-Guajá, Waiãpi, Urubu-Kaapor, Zoé and Parakanã). Forty-eight different haplotypes were identified, 28 of which unique. Five haplotypes are very frequent and were shared by over 10 individuals. The estimated haplotype diversity (0.9114) was very low compared to other geographic groups, including Africans, Europeans and Asians.

Population genetics of 17 Y-STR loci in a large Chinese Han population from Zhejiang Province, Eastern China - Corrected Proof

Weiwei Wu, Lipeng Pan, Honglei Hao, Xiaoting Zheng, Jinfeng Lin, Dejian Lu — 2010-01-20

Abstract: Seventeen Y-STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a, DYS385b, DYS438, DYS439, DYS437, DYS448, DYS456, DYS458, DYS635 and YGATAH4) were analyzed for 4451 Chinese Han unrelated males from Zhejiang Province, Eastern China, with the AmpFlSTR Yfiler™ PCR Amplification kit. A total of 3389 different haplotypes was identified, of which 2877 were unique and 512 repeatedly found among different individuals. The overall haplotype diversity (HD) and discrimination capacity (DC) were 0.999696 and 0.761402, respectively. Analysis of molecular variance (AMOVA) tests demonstrated that genetic distance between Zhejiang Han and most Chinese Han populations is closer than that between Zhejiang Han and non-Han populations. This study provides information for the application of Y-chromosomal STRs to forensic identification, indicating that the extended genotyping of Y-STRs is needed for forensic practice.

Authentication of forensic DNA samples - Corrected Proof

Niels Morling, Peter M. Schneider, Wolfgang Mayr, Leonor Gusmao, Mechthild Prinz — 2010-01-20

In their recent paper entitled “Authentication of Forensic DNA samples” Frumkin et al. describe not only how they are suggesting to authenticate DNA found at a crime scene but also explain in detail how to fabricate samples and plant DNA of specific individuals as false evidence. We believe that this type of information should have been handled more carefully and have several concerns with this publication.

Simple and highly effective DNA extraction methods from old skeletal remains using silica columns - Corrected Proof

2010-01-20

Abstract: The recovery of DNA data from old skeletal remains is often difficult due to degraded and very low yield of extracted DNA and the presence of PCR inhibitors. Herein, we compared several silica-based DNA extraction methods from artificially degraded DNA, DNA with PCR inhibitors and DNA from old skeletal remains using quantitative real-time PCR. We present a modified large-scale silica-based extraction combined with complete demineralization, that enables maximum DNA recovery and efficient elimination of PCR inhibitors. This is performed with high concentration of EDTA solution for demineralization of bone powder followed by QIAamp® spin columns and buffers from the QIAquick® PCR purification kit. We have successfully used this modified technique to perform STR analysis for 55-year-old skeletal remains. The results of this study will contribute to solve the forensic cases dealing with skeletal remains.

Evidence of partial and weak gametic disequilibrium across clusters of pericentromeric short tandem repeats loci on human X chromosome: Proceed with caution in forensic genetics - Corrected Proof

Enrique Medina-Acosta — 2010-01-14

Typing human DNA with clusters of X chromosome-linked polymorphic short tandem repeat (X-STRs) loci has been proposed as a useful approach to solve kinship deficiency cases, particularly, when autosomal polymorphic markers are insufficient to discriminate between close relationships . Markers closely linked in clusters are expected to segregate as stable haplotypes blocks. The problem is that that haplotype block reasoning is often applied in forensic analyses under the assumption of no recombination, a practice deemed inadequate .

Fundamental problem of forensic mathematics—The evidential value of a rare haplotype - Corrected Proof

Charles H. Brenner — 2010-01-13

Abstract: Y-chromosomal and mitochondrial haplotyping offer special advantages for criminal (and other) identification. For different reasons, each of them is sometimes detectable in a crime stain for which autosomal typing fails. But they also present special problems, including a fundamental mathematical one: When a rare haplotype is shared between suspect and crime scene, how strong is the evidence linking the two? Assume a reference population sample is available which contains n−1 haplotypes. The most interesting situation as well as the most common one is that the crime scene haplotype was never observed in the population sample. The traditional tools of product rule and sample frequency are not useful when there are no components to multiply and the sample frequency is zero. A useful statistic is the fraction κ of the population sample that consists of “singletons” – of once-observed types. A simple argument shows that the probability for a random innocent suspect to match a previously unobserved crime scene type is (1−κ)/n – distinctly less than 1/n, likely ten times less. The robust validity of this model is confirmed by testing it against a range of population models.This paper hinges above all on one key insight: probability is not frequency. The common but erroneous “frequency” approach adopts population frequency as a surrogate for matching probability and attempts the intractable problem of guessing how many instances exist of the specific haplotype at a certain crime. Probability, by contrast, depends by definition only on the available data. Hence if different haplotypes but with the same data occur in two different crimes, although the frequencies are different (and are hopelessly elusive), the matching probabilities are the same, and are not hard to find.

Human eye colour and HERC2, OCA2 and MATP - Corrected Proof

Jonas Mengel-From, Claus Børsting, Juan J. Sanchez, Hans Eiberg, Niels Morling — 2010-01-11

Abstract: Prediction of human eye colour by forensic genetic methods is of great value in certain crime investigations. Strong associations between blue/brown eye colour and the SNP loci rs1129038 and rs12913832 in the HERC2 gene were recently described. Weaker associations between eye colour and other genetic markers also exist. In 395 randomly selected Danes, we investigated the predictive values of various combinations of SNP alleles in the HERC2, OCA2 and MATP (SLC45A2) genes and compared the results to the eye colours as they were described by the individuals themselves. The highest predictive value of typing either the HERC2 SNPs rs1129038 and/or rs12913832 that are in strong linkage disequilibrium was observed when eye colour was divided into two groups, (1) blue, grey and green (light) and (2) brown and hazel (dark). Sequence variations in rs11636232 and rs7170852 in HERC2, rs1800407 in OCA2 and rs16891982 in MATP showed additional association with eye colours in addition to the effect of HERC2 rs1129038. Diplotype analysis of three sequence variations in HERC2 and one sequence variation in OCA2 showed the best discrimination between light and dark eye colours with a likelihood ratio of 29.3.

Population genetic data on 15 STR loci in the Hungarian population - Corrected Proof

Szilvia Ágnes Rak, Andrea Zalán, György Szabados, Horolma Pamjav — 2010-01-08

The use of autosomal STRs for paternity and forensic caseworks is an integral part of forensic studies. For this reason it is required to establish and expand the databases that reflect the actual allele distribution in the population applied. During the past few years we have collected a greater number of samples from different regions of Hungary and established our own database. So far we have used the allele frequencies of the Hungarian population reported before . The application of the allele frequencies of the population for statistics is recommended on published data regulated by the national guideline. Therefore the recently detected allele frequencies can only be used in the routine work if they are considered to be published data. The aim was to study the population genetic data on 15 STR loci, and to calculate the statistical and forensic efficiency parameters and afterwards to compare the received data to the earlier report.

A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ™ system - Corrected Proof

Anna Bowden, Rachel Fleming, SallyAnn Harbison — 2010-01-06

Abstract: The use of messenger RNA profiling to identify the origin of biological samples (e.g. blood, semen and saliva) from crime scenes is now at the stage of being implemented into routine forensic casework. We report on the successful modification of the Promega DNA IQ™ system to enable co-extraction of DNA and RNA from the same sample without compromising the potential DNA profile. Using the protocol in our laboratory for extracting DNA using the DNA IQ™ system combined with the Zymo Research Mini RNA Isolation Kit™ II we demonstrate the simultaneous co-extraction of DNA and RNA from the same sample for routine DNA and mRNA profiling for the identification of both the individual and the biological stain.

Genetic analysis of 10 X-STRs in Argentinian population - Corrected Proof

Cecilia Bobillo, Andrea Sala, Leonor Gusmão, Daniel Corach — 2010-01-04

Abstract: A set of 200 samples, 100 males and 100 females, obtained from unrelated Argentinean donors were analyzed using 10 X-STRs—DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902 and DXS6789—in order to obtain allele frequencies. Statistical analysis was performed using Powerstats and Arlequin software. The population showed no deviation from Hardy Weinberg equilibrium at all loci analyzed. Allele frequencies were compared with Spanish and Portuguese sample sets showing no statistical significant differences in four of the 10 markers analyzed. The most polymorphic marker was DXS6809 and the high values of combined power of exclusion (99.9993355% in trios and 99.9715450% in duos) reinforce the usefulness of this set of markers for kinship test and human identification.

Use of DNA profiles for investigation using a simulated national DNA database: Part II. Statistical and ethical considerations on familial searching - Corrected Proof

T. Hicks, F. Taroni, J. Curran, J. Buckleton, V. Castella, O. Ribaux — 2009-12-25

Abstract: Familial searching consists of searching for a full profile left at a crime scene in a National DNA Database (NDNAD). In this paper we are interested in the circumstance where no full match is returned, but a partial match is found between a database member's profile and the crime stain. Because close relatives share more of their DNA than unrelated persons, this partial match may indicate that the crime stain was left by a close relative of the person with whom the partial match was found. This approach has successfully solved important crimes in the UK and the USA. In a previous paper, a model, which takes into account substructure and siblings, was used to simulate a NDNAD . In this paper, we have used this model to test the usefulness of familial searching and offer guidelines for pre-assessment of the cases based on the likelihood ratio. Siblings of “persons” present in the simulated Swiss NDNAD were created. These profiles (N=10,000) were used as traces and were then compared to the whole database (N=100,000). The statistical results obtained show that the technique has great potential confirming the findings of previous studies. However, effectiveness of the technique is only one part of the story. Familial searching has juridical and ethical aspects that should not be ignored. In Switzerland for example, there are no specific guidelines to the legality or otherwise of familial searching. This article both presents statistical results, and addresses criminological and civil liberties aspects to take into account risks and benefits of familial searching.

Validation and development of interpretation guidelines for low copy number (LCN) DNA profiling in New Zealand using the AmpFlSTR® SGM Plus™ multiplex - Corrected Proof

2009-12-21

Abstract: The characteristics of STR profiles produced from approximately 1ng starting template using the AMPFlSTR® SGM Plus™ multiplex and 28 PCR cycles, are well documented.However, the analysis of samples perceived as low in starting template (less than 100pg), and referred to as low template DNA (LTDNA), can require a test of higher sensitivity in order to achieve successful results. One way of increasing this sensitivity is to increase the number of PCR amplification cycles from 28 to 34. This type of analysis has become known as low copy number, or LCN, DNA profiling.Amplification of LTDNA under LCN conditions can result in increased incidents of profile characteristics such as allelic ‘drop-in’ and allelic ‘drop-out’. Adopting a testing regime which includes duplicate analysis, and maintaining a laboratory environment of stringent and monitored cleanliness, enables the scientist to identify and control these phenomena for a reliable interpretation of the DNA profiling results.A recent court ruling has questioned the reliability of LCN analysis and commented on the paucity of publications surrounding the validation of the technique.We present data for the LCN validation undertaken in our laboratory, and describe the guidelines and working practices we have developed for the analysis and interpretation of profiles generated after LCN profiling. This study augments the published record relating to LCN validation and should act as a useful guide for other laboratories who are considering implementing LCN profiling.

The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids - Corrected Proof

Rachel I. Fleming, SallyAnn Harbison — 2009-12-14

Abstract: With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain.

Population database defined by 13 autosomal STR loci in a representative sample from Bahia, Northeast Brazil - Corrected Proof

Eugênio Nascimento, Eneida Cerqueira, Leonor Gusmão — 2009-12-14

This work aimed to build a database with the allele frequencies of 13 STRs loci (short tandem repeats) genotypes provided by the Instituto de Ciências da Saúde (ICS) – Universidade Federal da Bahia (UFBA) and the Coordenação de Genética Forense do Laboratório Central de Polícia Técnica (LCPT) to give the technique for human identification by DNA Bahia greater discrimination power of the individual author of a criminal event. Allele frequencies for 13 STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, vWA, TPOX, D18S51, D5S818, FGA) were calculated in a sample of 1000 unrelated individuals from Bahia. The analysis of DNA is a major technical advance in criminal investigation. Within the methodology used frequencies were calculated by the Hardy–Weinberg Law, which governs the allele frequencies in a population in equilibrium. The data obtained here are of fundamental importance for the statistical calculations used in criminal investigations, since from the allele frequencies is possible to estimate rates will reflect the likelihood that a suspect is the perpetrator, compared to the chances of any individual chosen randomly in the population.

Validation of a DNA IQ™-based extraction method for TECAN robotic liquid handling workstations for processing casework - Corrected Proof

Chantal J. Frégeau, C. Marc Lett, Ron M. Fourney — 2009-12-07

Abstract: A semi-automated DNA extraction process for casework samples based on the Promega DNA IQ™ system was optimized and validated on TECAN Genesis 150/8 and Freedom EVO robotic liquid handling stations configured with fixed tips and a TECAN TE-Shake™ unit. The use of an orbital shaker during the extraction process promoted efficiency with respect to DNA capture, magnetic bead/DNA complex washes and DNA elution. Validation studies determined the reliability and limitations of this shaker-based process. Reproducibility with regards to DNA yields for the tested robotic workstations proved to be excellent and not significantly different than that offered by the manual phenol/chloroform extraction. DNA extraction of animal:human blood mixtures contaminated with soil demonstrated that a human profile was detectable even in the presence of abundant animal blood. For exhibits containing small amounts of biological material, concordance studies confirmed that DNA yields for this shaker-based extraction process are equivalent or greater to those observed with phenol/chloroform extraction as well as our original validated automated magnetic bead percolation-based extraction process. Our data further supports the increasing use of robotics for the processing of casework samples.

Powerplex® ES versus Powerplex® S5—Casework testing of the new screening kit - Corrected Proof

2009-12-04

Abstract: The new Powerplex® S5 Mini STR-System from Promega with the four provided STR loci D18S51, D8S1179, TH01 and FGA as well as the Amelogenin marker (PCR products ranging from 80 to 220bp not considering the longer FGA fragments) is designed as a screening tool especially in difficult casework samples. To test its suitability we amplified highly degraded DNA from casework samples, which had shown no or only poor results in analyses with the Powerplex® ES kit, as well as artificially degraded DNA or DNA samples containing PCR inhibitors. Despite a tendency for allelic drop-ins in the amplification of highly degraded DNA the Powerplex® S5 kit was a reliable tool for the analysis of casework samples with degraded DNA which gave better results than the Powerplex® ES kit in 64% of analysed swabs. Furthermore, it was especially suitable for the investigation of formalin fixed tissue, tissue samples showing advanced putrefaction or telogen hair samples. However, there was no strict relation between positive Powerplex® S5 results and amplification success with the Powerplex® ES kit.

Forensic DNA against wildlife poaching: Identification of a serial wolf killing in Italy - Corrected Proof

Romolo Caniglia, Elena Fabbri, Claudia Greco, Marco Galaverni, Ettore Randi — 2009-11-30

Abstract: The recent expansion of the Italian wolf population through the Apennine and western Alps, after centuries of contractions, is causing conflicts with human activities leading to a rise in poaching or illegal killings. Here we show how molecular population genetics has been used to identify a suspect serial wolf killer. We analysed DNA extracted from a necklace made of ten presumed wolf canine teeth, confiscated in 2008 to a man living in the northern Italian Apennine (Liguria Region). Individual genotypes were determined using 12 unlinked autosomal microsatellites (STRs), mtDNA control-region sequences, a male-specific ZFX/ZFY restriction-site and three Y-linked STRs. Results indicate that the teeth belonged to six different individuals (three males and three females), which were assigned to the Italian wolf population with p>0.90 by Bayesian procedures. One of these genotypes matched with the genetic profile of a male wolf previously found-dead and already non-invasively sampled in the same area. Another genotype matched with that of a female wolf non-invasively sampled twice in the same area 1 year before. These data are being used as forensic genetic evidence in the ongoing criminal trial against the suspect serial wolf killer.

Forensic analysis of polymorphism and regional stratification of Y-chromosomal microsatellites in Belarus - Corrected Proof

2009-11-26

Abstract: Nine loci defining minimal haplotypes and four other Y-chromosomal short tandem repeats (Y-STRs) DYS437, DYS438, DYS439 and GATA H4.1 were analysed in 414 unrelated males residing in four regions of Belarus. Haplotypes of 328 males were further extended by 7 additional Y-STRs: DYS388, DYS426, DYS448, DYS456, DYS458, DYS460 and DYS635. The 13-locus haplotype diversity was 0.9978 and discrimination capacity was 78.7%, indicating presence of identical haplotypes among unrelated males. Seven additional Y-STRs enabled almost complete discrimination of undifferentiated 13-locus haplotypes, increasing haplotype diversity to 0.9998 and discrimination capacity to 97.9%. Analysis of molecular variance of minimal haplotypes excluded the use of a Y-STR database for Belarusians residing in northeastern Poland as representative for the Belarusian population in forensic practice, and revealed regional stratification within the country. However, four additional markers (DYS437, DYS438, DYS439 and GATA H4.1) were shown to eliminate the observed geographical substructure among Belarusian males. The results imply that in case of minimal and PowerPlex Y haplotypes, a separate frequency database should be used for northern Belarus to estimate Y-STR profile frequencies in forensic casework. In case of Yfiler haplotypes, regional stratification within Belarus may be neglected.

Genetic polymorphism of eight X-linked STRs of Mentype® Argus X-8 Kit in Chinese population from Shanghai - Corrected Proof

Zhang Su-hua, Li Cheng-tao, Zhao Shu-min, Li Li — 2009-11-23

Abstract: X-chromosomal STR markers DXS10135 and DXS8378 in linkage group 1, DXS7132 and DXS10074 in linkage group 2, HPRTB and DXS10101 in linkage group 3, and DXS10134 and DXS7423 in linkage group 4 included in Mentype® Argus X-8 Kit were studied in Chinese Han population. After genotyping unrelated male (106) and female (92), haplotype frequencies and forensic parameters were calculated. Deviations form Hardy–Weinberg equilibrium could not be detected (p<0.05). The obtained haplotype frequency distributions were compared with other previously reported population data.

Genetic polymorphisms of 15 AmpFlSTR Identifiler loci in a Serbian population - Corrected Proof

2009-11-20

The highly polymorphic short tandem repeat (STR) loci in the human genome are widely used for forensic and paternity testing as well as for population genetic studies. During the last two decades, new states were formed in the territory of former Yugoslavia. Analyses of STR loci polymorphism for the Serbian population published so far included mixed samples of Serbians and ethnically close but different populations from the other countries . Here we have collected samples from a widespread ethnic Serbian population inhabiting the newly formed Republic of Serbia and analyzed them by 15 AmpFlSTR Identifiler loci (D3S1358, TH01, D21S11, D18S51, D2S1338, D5S818, D13S317, D7S820, D16S539, CSF1PO, D19S433, vWA, D8S1179, TPOX, FGA). The data obtained were compared with ethnically close neighboring nations in order to resolve confused earlier comparisons based on samples that included different nations living in the country of research .

Validation of a dual cycle ethylene oxide treatment technique to remove DNA from consumables used in forensic laboratories - Corrected Proof

Emily Archer, Heather Allen, Andy Hopwood, Diane Rowlands — 2009-11-16

Abstract: Validation of a dual cycle ethylene oxide (EO) approach to removing DNA contamination from consumables has been undertaken. The limits of the technique were investigated resulting in evidence that the DNA from up to 50μl of blood and saliva can be removed to a level where the consumable can be considered DNA free. DNA from semen was more resilient and some allelic peaks remained that would have prevented the consumable being classed as suitable for use in low template DNA analysis. No residual effect on consumables resulting in inhibition of subsequent DNA analysis was noted. However, if consumables had been previously treated with gamma or electron beam irradiation then slight inhibition of the downstream STR process was observed.Dual cycle EO treatment was effective at removing recoverable DNA from swabs and stain cards and consideration should be given to the suitability of EO treatment for use on critical consumables used in the forensic laboratory.

Polymorphism of 11 non-CODIS STRs in a population sample of Lithuanian minority residing in northeastern Poland - Corrected Proof

Witold Pepinski, Anna Niemcunowicz-Janica, Malgorzata Skawronska, Jerzy Janica — 2009-11-16

Lithuanians in northeastern Poland are an indigenous group residing along the border with the Republic of Lithuania since the extinction of the Yotvingians (Sudovians) around the 13th century. Nowadays, about 20,000 people of the Lithuanian ancestry compose one of the most emancipated, best organized and least assimilated ethnic minority communities in the country, the linguistic factor playing a crucial role in maintaining their regional and national identity.

Allele frequencies of fifteen STR loci in a population from Central Brazil - Corrected Proof

2009-11-12

Abstract: Allele frequencies for 15 short tandem repeats included in Powerplex 16 Kit (Penta E, D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818) were determined in a sample of 429 unrelated individuals from the population of Goiânia, Goias, Central Brazil. Determination of the allele frequencies as well as of several commonly used statistics in forensic and paternity testing were defined. The forensic parameters presented high values and the most polymorphic loci were Penta E, following FGA and D18S51. The exact test demonstrated that the fifteen loci analyzed in the population of Goiania have no deviation from Hardy–Weinberg equilibrium (P>0.05).

Haplotype data for 12 Y-chromosome STR loci of Sri Lankans - Corrected Proof

2009-11-09

Abstract: Haplotype data estimated from 12 Y-chromosomal STRs were obtained from a sample of 207 unrelated male individuals from Sri Lanka. A total of 195 different haplotypes were identified, of which 183 were unique. Haplotype diversity was found be high (0.9948±0.0012) indicating increased discriminating capacity of these 12 Y-STR loci in forensic identification of Sri Lankan individuals. DYS385, representing two loci, was the most diverse marker (0.853). The lowest diversity (0.351) was observed with DYS391.

Developmental validation of the PowerPlex® 16 HS System: An improved 16-locus fluorescent STR multiplex - Corrected Proof

2009-11-09

Abstract: STR multiplexes remain the cornerstone of genotyping forensic samples. The PowerPlex® 16 HS System contains the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. The PowerPlex® 16 HS System is an updated version of the PowerPlex 16® System; while the primers and dyes remain unchanged, it introduces an enhanced buffer system that includes hot-start Taq DNA polymerase and ensures robust performance. Due to the modification of the reaction mix, a multi-laboratory developmental validation study was completed to document performance capabilities and limitations for the revised assay. Data within this validation was generated by eight laboratories and served as the basis for the following conclusions: genotyping of single-source samples was consistent across a large range of template DNA concentrations with most laboratories obtaining complete profiles at 62.5pg. Mixture analyses showed that over 90% of minor alleles were detected at 1:9 ratios. Optimum amplification cycle number was ultimately dependent on the sensitivity of the detection instrument and could be adjusted to accommodate a range of DNA template concentrations. Reaction conditions including volume and annealing temperature as well as the concentrations of primers, Taq DNA polymerase, and magnesium were shown to be optimal and able to withstand moderate variations without affecting multiplexed STR amplification. Finally, data from non-probative samples and concordance studies showed consistent results when comparing the PowerPlex® 16 HS System with the PowerPlex® 16 System as well as other commercially available systems.

World War One Italian and Austrian soldier identification project: DNA results of the first case - Corrected Proof

2009-11-06

Abstract: We report the results of an attempt to identify the supposed remains of a famous World War I (WWI) Italian soldier who was killed in battle along the Italian front in 1915.Thanks to the availability of offspring from both paternal and maternal lineage Y-STRs and mtDNA were analysed and both showed a clear exclusion scenario: the remains did not belong to the supposed war hero.This is the first effort of identification of the remains of soldiers who perished during World War I within a multidisciplinary project aimed at the retrieval of historical and cultural aspects linked to WWI, and the systematic study of the remains of soldiers and ultimately their identification. This last step involves both Italian and Austrian laboratories.