Forensic Science International: Genetics - Articles in Press

Using forensic microsatellites to decipher the genetic structure of linguistic and geographic isolates: A survey in the eastern Italian Alps - Corrected Proof

2012-05-17

Abstract: The study of geographically and/or linguistically isolated populations could represent a potential area of interaction between population and forensic genetics. These investigations may be useful to evaluate the suitability of loci which have been selected using forensic criteria for bio-anthropological studies. At the same time, they give us an opportunity to evaluate the efficiency of forensic tools for parentage testing in groups with peculiar allele frequency profiles. Within the frame of a long-term project concerning Italian linguistic isolates, we studied 15 microsatellite loci (Identifiler kit) comprising the CODIS panel in 11 populations from the north-eastern Italian Alps (Veneto, Trentino and Friuli Venezia Giulia regions). All our analyses of inter-population differentiation highlight the genetic distinctiveness of most Alpine populations comparing them either to each other or with large and non-isolated Italian populations. Interestingly, we brought to light some aspects of population genetic structure which cannot be detected using unilinear polymorphisms. In fact, the analysis of genotypic disequilibrium between loci detected signals of population substructure when all the individuals of Alpine populations are pooled in a single group. Furthermore, despite the relatively low number of loci analyzed, genetic differentiation among Alpine populations was detected at individual level using a Bayesian method to cluster multilocus genotypes. Among the various populations studied, the four linguistic minorities (Fassa Valley, Luserna, Sappada and Sauris) showed the most pronounced diversity and signatures of a peculiar genetic ancestry. Finally, we show that database replacement may affect estimates of probability of paternity even when the local database is replaced by another based on populations which share a common genetic background but which differ in their demographic history. These findings point to the importance of considering the demographic and cultural profile of populations in forensic applications, even in a context of substantial genetic homogeneity such as that of European populations.

DNA identification of Salvia divinorum samples - Corrected Proof

Terence M. Murphy, Gurpreet Bola — 2012-05-11

Abstract: Salvia divinorum (diviner's sage) is a plant in the mint family that produces an hallucinogenic compound, salvinorin A. The plant is used, often by chewing or smoking, as a “recreational” drug source and is regulated or banned in several states and countries. We describe a simple DNA technique, polymerase chain reaction of the ribulose bisphosphate carboxylase large subunit (rbcL) gene, that can distinguish S. divinorum leaf pieces from pieces of tobacco or cannabis. We have also found DNA sequences adjacent to the chloroplast leucine transfer RNA (trnL) gene that are specific to S. divinorum and distinguish it from other horticulturally popular Salvia species. We report some significant differences between the S. divinorum trnL sequences we determined and those now published in GenBank.

Potential use of DNA barcoding for the identification of tobacco seized from waterpipes - Corrected Proof

Cindy Carrier, François Cholette, Camilo Quintero, Chris Fulcher — 2012-05-11

Abstract: DNA barcoding was adopted in our laboratory for the identification of tobacco (Nicotiana spp.) in moassel samples seized from “hookah bars”. As recommended by the CBOL Plant Working Group, we used a 2-locus combination of rbcL and matK as the plant barcode. As previously reported rbcL routinely produced high quality bi-directional reads but had a lower discriminating power than matK. It was much more difficult obtaining high quality bi-directional reads with matK possibly because of poor sample quality. DNA barcoding successfully identified tobacco in over 60 commercial tobacco moassel products. On the other hand, negative results (no amplification) or the identification of non-tobacco species were obtained from herbal moassel products. Our study clearly demonstrates the practical utility of DNA barcoding beyond taxonomy.

Italian data of 23 STR loci amplified in a single multiplex reaction - Corrected Proof

2012-05-07

A protocol for the simultaneous amplification of 25 STR loci plus amelogenin has been recently published . We applied a slightly modified method to a sample of 100 unrelated subjects living in Tuscany, Italy; 23 loci (plus amelogenin) were successfully genotyped in a single multiplex.

Population genetic investigation of eight X-chromosomal short tandem repeat loci from a northeast German sample - Corrected Proof

Sylvio Tetzlaff, Rudolf Wegener, Iris Lindner — 2012-04-30

Especially in complex cases, typing of X-linked short tandem repeats has increasingly become an important role in kinship testing. A set of 318 participants of Caucasoid origin, 159 females and 159 males, from northeast Germany were analyzed using the X-STR loci DXS8378, HPRTB, DXS7423, DXS7132, DXS10134, DXS10074, DXS10101 and DXS10135 as well as Amelogenin. Peripheral blood samples and buccal swabs, respectively, were obtained from unrelated individuals, who signed an informed statement of compliance according to the approval of the Rostock university Ethics committee (reg. no. II HV 16/2003). DNA was purified using NucleoMag 96 Blood Kit (Macherey-Nagel, Düren, Germany) and DNA IQ System (Promega, Madison, USA) by manufacturers’ instructions. Eight X-STR loci were amplified in a multiplex PCR amplification reaction using the Mentype Argus X-8 PCR amplification kit (Biotype, Dresden, Germany) according to the manufacturer's recommendations. Capillary electrophoreses of PCR amplicons were conducted in an ABI Prism 3100-Avant genetic analyzer (Applied Biosystems, Foster City, USA), and data were analyzed using the GeneMapper ID software v.3.2.1 (Applied Biosystems). As DNA control reference samples, XY1 and XX28 (Biotype) were used. Allele designation was based on the allelic ladder included in the test kit. Data analysis was carried out with usage of the ChrX-STR.org 2.0 Web site (http://www.chrx-str.org) and the Arlequin software v.3.5.1.2 . The statistical analysis of this study included: polymorphic information content (PIC), heterozygosity (HET), power of discrimination (PD), mean paternity exclusion chance (MEC), allele frequency distribution, mutation rate estimates and the Hardy–Weinberg equilibrium (HWE) calculations.

Effect of linkage between vWA and D12S391 in kinship analysis - Corrected Proof

Kristen L. O’Connor, Andreas O. Tillmar — 2012-04-30

Abstract: Ideally for use in forensic analyses, genetic markers on the same chromosome should be more than 50 Mb in physical distance to ensure full recombination and thus independent inheritance. The forensic community has given attention to two STR markers, D12S391 and vWA, that are 6.3 megabases (Mb) apart on chromosome 12. Recent studies have shown no significant linkage disequilibrium between vWA and D12S391 in U.S. and worldwide populations, although genetic linkage has been identified. It is important to evaluate the impact of linkage effects on kinship analysis. In this study, we aimed to determine a more precise measurement of the recombination frequency between vWA and D12S391 based on a larger number of informative meiosis than has been studied previously. We estimated the recombination frequency (θ) to 0.089 (95% CI 0.044–0.158). Using pedigrees simulated under specific kinship scenarios where recombination was expected to affect the likelihood ratio (LR), we evaluated the impact on LR values of including or ignoring linkage between vWA and D12S391. For all pedigree scenarios considered, on average, LR values ignoring linkage were slightly underestimated than when linkage was considered. However, in the incest scenario considered, LR values could be overestimated up to 25–30 times when linkage was ignored. We demonstrate that the effect of ignoring linkage in the likelihood ratio calculation can be considerable. These results suggest that linkage should be considered during kinship analysis when vWA and D12S391 are tested for pedigrees where a recombination could impact the LR value.

Inference about the number of contributors to a DNA mixture: Comparative analyses of a Bayesian network approach and the maximum allele count method - Corrected Proof

A. Biedermann, S. Bozza, K. Konis, F. Taroni — 2012-04-25

Abstract: In the forensic examination of DNA mixtures, the question of how to set the total number of contributors (N) presents a topic of ongoing interest. Part of the discussion gravitates around issues of bias, in particular when assessments of the number of contributors are not made prior to considering the genotypic configuration of potential donors. Further complication may stem from the observation that, in some cases, there may be numbers of contributors that are incompatible with the set of alleles seen in the profile of a mixed crime stain, given the genotype of a potential contributor. In such situations, procedures that take a single and fixed number contributors as their output can lead to inferential impasses. Assessing the number of contributors within a probabilistic framework can help avoiding such complication. Using elements of decision theory, this paper analyses two strategies for inference on the number of contributors. One procedure is deterministic and focuses on the minimum number of contributors required to ‘explain’ an observed set of alleles. The other procedure is probabilistic using Bayes’ theorem and provides a probability distribution for a set of numbers of contributors, based on the set of observed alleles as well as their respective rates of occurrence. The discussion concentrates on mixed stains of varying quality (i.e., different numbers of loci for which genotyping information is available). A so-called qualitative interpretation is pursued since quantitative information such as peak area and height data are not taken into account. The competing procedures are compared using a standard scoring rule that penalizes the degree of divergence between a given agreed value for N, that is the number of contributors, and the actual value taken by N. Using only modest assumptions and a discussion with reference to a casework example, this paper reports on analyses using simulation techniques and graphical models (i.e., Bayesian networks) to point out that setting the number of contributors to a mixed crime stain in probabilistic terms is, for the conditions assumed in this study, preferable to a decision policy that uses categoric assumptions about N.

An integrated system of ABO typing and multiplex STR testing for forensic DNA analysis - Corrected Proof

2012-04-19

Abstract: A new amplification system for ABO and STR genotyping in a single reaction has been successfully developed. Two types of information can be obtained from a biological sample at one time. One is the classical information of ABO blood group typing for screening suspects and the other is STR information for individual identification. The system allows for the simultaneous detection of 15 autosomal STR loci (containing all CODIS STR loci as well as Penta D and Penta E), six ABO genotypes (O/O, B/B, A/A, A/O, A/B, and B/O) and the gender-determining locus Amelogenin. Primers are designed so that the amplicons are distributed ranging from 75bp to 500bp within a four-dye fluorescent design, leaving a fourth dye for the internal size standard. With 30 cycles, the results showed that the optimal amount of DNA template for this multiplex ranges from 250pg to 2ng and the lowest detection threshold is 125pg (as low as 63pg for ABO loci). For the DNA template outside the optimal detection range, we could adjust the number of cycles to obtain the robust profiles. Mixture studies showed that over 83% of minor alleles were detected at 1:9 ratios. The full profiles were still observed when 4ng of degraded DNA was digested by DNase I and 1ng undegraded DNA was added to 40μM haematin. Polymerase chain reaction (PCR)-based conditions including the concentrations of primers, magnesium and the Taq polymerase as well as volume, cycle numbers and annealing temperature were examined and optimised. In addition, the system was validated by 364 bloodstain samples and 32 common casework samples. According to the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, our system demonstrates good detection performance and is an ideal tool for forensic DNA typing with potential application.

Forensic STR analysis using massive parallel sequencing - Corrected Proof

Christophe Van Neste, Filip Van Nieuwerburgh, David Van Hoofstat, Dieter Deforce — 2012-04-16

Abstract: We explore the applicability of second generation sequencing (SGS) to sequence multiplexed forensic STR amplicons, both in a single contributor sample as in multiple-person mixtures with different ratios. We compare the results of a commercial STR profiling kit (Applied Biosystems AmpFlSTR® Profiler Plus®), analyzed both with capillary electrophoresis and with Roche GS FLX sequencing. An easy to use open-source software pipeline is provided, chaining together the different steps needed to start the analysis from a GS FLX FASTA file, resulting in a FASTA file containing the called and quantified alleles present in the data. Sequencing of multiplexed STR amplicons using Roche GS FLX titanium technology is technically feasible but the technology is not ideal for this purpose. The fraction of full length reads is small and the homopolymer sequencing error rate is high. The pipeline compresses the homopolymers to a single base to avoid false results caused by these homopolymers. The qualitative and quantitative results from the SGS STR analysis pipeline are comparable to the electrophoresis method. Additionally, the SGS method provides extra information and is able to call allele subtypes based on STR sequences in a database. In mixed samples, all alleles were reported from individuals that contributed at least 10% to the mixture.

A forensic DNA profiling system for Northern European brown bears (Ursus arctos) - Corrected Proof

2012-04-09

Abstract: A set of 13 dinucleotide STR loci (G1A, G10B, G1D, G10L, MU05, MU09, MU10, MU15, MU23, MU26, MU50, MU51, MU59) were selected as candidate markers for a DNA forensic profiling system for Northern European brown bear (Ursus arctos). We present results from validation of the markers with respect to their sensitivity, species specificity and performance (precision, heterozygote balance and stutter ratios). All STRs were amplified with 0.6ng template input, and there were no false bear genotypes in the cross-species amplification tests. The validation experiments showed that stutter ratios and heterozygote balance was more pronounced than in the tetranucleotide loci used in human forensics. The elevated ratios of stutter and heterozygote balance at the loci validated indicate that these dinucleotide STRs are not well suited for interpretation of individual genotypes in mixtures. Based on the results from the experimental validations we discuss the challenges related to genotyping dinucleotide STRs in single source samples. Sequence studies of common alleles showed that, in general, the size variation of alleles corresponded with the variation in number of repeats. The samples characterized by sequence analysis may serve as standard DNA samples for inter laboratory calibration. A total of 479 individuals from eight Northern European brown bear populations were analyzed in the 13 candidate STRs. Locus MU26 was excluded as a putative forensic marker after revealing large deviations from expected heterozygosity likely to be caused by null-alleles at this locus. The remaining STRs did not reveal significant deviations from Hardy–Weinberg equilibrium expectations except for loci G10B and MU10 that showed significant deviations in one population each, respectively. There were 9 pairwise locus comparisons that showed significant deviation from linkage equilibrium in one or two out of the eight populations. Substantial genetic differentiation was detected in some of the pairwise population comparisons and the average estimate of population substructure (FST) was 0.09. The average estimate of inbreeding (FIS) was 0.005. Accounting for population substructure and inbreeding the total average probability of identity in each of the eight populations was lower than 1.1×10−9 and the total average probability of sibling identity was lower than 1.3×10−4. The magnitude of these measurements indicates that if applying these twelve STRs in a DNA profiling system this would provide individual specific evidence.

Patterns of Y-STR variation in Italy - Corrected Proof

2012-04-09

Abstract: The 17 Y-chromosomal short tandem repeats (STRs) included in the AmpFlSTR Yfiler Amplification Kit (AB Applied Biosystems) (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and GATA H4.1) were typed in 292 samples from seven Italian regions. Population comparisons with other European samples were undertaken; for this purpose, two databases were collated from the literature: (a) 19 population samples including >2900 Yfiler profiles, and (b) 67 population samples including >15,000 minimum haplotype profiles. A total of 276 different Yfiler haplotypes were observed in Italy, and only one of them was shared among our seven population samples. The overall haplotype diversity (0.9996) was comparable to other European samples. AMOVA indicates that among population variance depends on the amount of Y-STRs used, being higher when using minimal haplotypes. This is probably due to the fact that Yfiler profiles are represented by singleton haplotypes in all the population samples raising the diversity values to the maximum theoretical value. AMOVA results seems to depend even more strongly on the amount of population samples used, the among population variance in Italy ranging from 2.82% to 11.03% (using 15 and 32 Italian populations samples, respectively). Variance is not as strongly stratified geographically within Italy, although it is notorious that latitude is more important than longitude in the distribution of variance. The results also indicated that Italy is less stratified than other European samples. The present study contributes to enrich the Y-chromosome databases regarding high-resolution Y-chromosome data sets and demonstrates that extended Y-STR profiles substantially increases the discriminatory capacity in individual identification for forensic purposes.

Population genetic data for 10 X-STR loci in autochthonous Basques from Navarre (Spain) - Corrected Proof

2012-04-02

Abstract: Ten X chromosome markers (DXS6789, DXS6809, DXS7132, DXS7133, DXS7423, DXS8378, DXS9898, DXS9902, GATA172D05, and GATA31E08) were analyzed in a sample of 185 unrelated autochthonous Basques from Navarre. Deviations from Hardy–Weinberg equilibrium and linkage disequilibrium between markers were not observed at any loci. Combined power of discrimination was 0.999999999 (females) and 0.999998764 (males). Mean exclusion chance was 0.99999463 (trios) and 0.999761591 (duos). Pairwise genetic distances (Fst) of X-STR frequencies indicate significant differences in the allele frequency distribution between the autochthonous Basques from Navarre and American and Iberian populations except with the Basque Country.

An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains - Corrected Proof

2012-03-29

Abstract: Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases . Following a 20min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60s), leading to a ≥1h reduction in DNA preparation time.

Analysis of the SNPforID 52-plex markers in four Native American populations from Venezuela - Corrected Proof

Y. Ruiz, M.A. Chiurillo, L. Borjas, C. Phillips, M.V. Lareu, Á. Carracedo — 2012-03-29

Abstract: The SNPforID 52-plex single nucleotide polymorphisms (SNPs) were analyzed in four native Venezuelan populations: Bari, Pemon, Panare and Warao. None of the population-locus combinations showed significant departure from Hardy–Weinberg equilibrium. Calculation of forensic and statistical parameters showed lower values of genetic diversity in comparison with African and European populations, as well as other, admixed populations of neighboring regions of Caribbean, Central and South America. Significant levels of divergence were observed between the four Native Venezuelan populations as well as with other previously studied populations. Analysis of the 52-plex SNP loci with Structure provided an optimum number of population clusters of three, corresponding to Africans, Europeans and Native Americans. Analysis of admixed populations indicated a range of membership proportions for ancestral populations consisting of Native American, African and European components. The genetic differences observed in the Native American groups suggested by the 52 SNPs typed in our study are in agreement with current knowledge of the demographic history of the Americas.

Erratum: Specific and sensitive mRNA biomarkers for the identification of skin in ‘touch DNA’ evidence - Corrected Proof

E. Hanson, C. Haas, R. Jucker, J. Ballantyne — 2012-03-29

During a review of our recently published article entitled “Specific and sensitive mRNA biomarkers for the identification of skin in ‘touch DNA’ evidence” (Hanson et al., doi:10.1016/j.fsigen.2012.01.004), we have identified an error that we wish to identify and correct. In the introduction, we report our preliminary evaluation of CDSN and KRT9. However, our preliminary work included an evaluation of LOR, not KRT9, and the incorrect gene name was reported by mistake. Our preliminary work on CDSN and LOR was presented to the forensic community at the 24th ISFG Congress in Vienna, Austria, and the article included a brief discussion of these same results. This was truly a genuine mistake on our part and was in no way intended to misrepresent data or to inaccurately critique any of the important work conducted by Visser et al. We deeply apologize to the readers for any confusion this may have caused.

Collaborative genetic mapping of 12 forensic short tandem repeat (STR) loci on the human X chromosome - Corrected Proof

2012-03-29

Abstract: A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.

Genetic polymorphism of 17 STR loci in Chinese population from Hunan province in Central south China - Corrected Proof

2012-03-28

We selected 586 unrelated healthy individuals (338 males and 248 females) whose ancestors lived in Hunan Province for at least three generations. After informed consent in compliance with the ethical norms set by Chinese legislation was acquired, we obtained bloodstains from each of the subjects. The study was approved by the ethics committee of the Third Xiangya Hospital of Central South University, and was carried out following the guidelines for publication of population data required by the journal and according to the International Society of Forensic Genetics (ISFG) recommendations . The laboratory has passed through proficiency testing in the field of forensic science by the Institute of Forensic Science of Chinese Ministry of Justice.

Expanding data and resources for forensic use of SNPs in individual identification - Corrected Proof

2012-03-26

Abstract: The potential value of SNPs for individual identification has been recognized by many researchers and different panels have been proposed. Here we present a new interface in the ALFRED database to access compendia of allele frequencies for several published panels of markers for forensic uses. One of those is our panel of individual identification SNPs (IISNPs) based on samples of 44 populations originating from many parts of the world. Here we also present additional data and additional statistical analyses that continue to support the value of our panel of IISNPs as a universal panel. We also describe initial developments of multiplex methods and various robustness analyses for our 45 marker IISNP panel.

Identification of single nucleotide polymorphisms within the mtDNA genome of the domestic dog to discriminate individuals with common HVI haplotypes - Corrected Proof

Donna L. Imes, Elizabeth J. Wictum, Marc W. Allard, Benjamin N. Sacks — 2012-03-21

Abstract: We sequenced the entire ∼16kb canine mitochondrial genome (mtGenome) of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. We then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. Haplotype frequencies in our data set were highly correlated with previous ones (e.g., FST=0.02, r=0.90), suggesting the total data set reasonably reflected the broader dog population. A total of 128 HVI haplotypes was represented. The 10 most common HVI haplotypes (n=184 dogs) represented 53.3% of the sample. We identified a total 71 SNPs in the mtGenomes (external to the control region) that resolved the 10 most common HVI haplotypes into 63 mtGenome subhaplotypes. The random match probability of the dataset based solely on the HVI sequence was 4%, whereas the random match probability of the mtGenome subhaplotypes was <1%. Thus, the panel of 71 SNPs identified in this study represents a useful forensic tool to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).

Genetic analysis of the 11 X-STR loci in Uigur population from China - Corrected Proof

2012-03-21

X-chromosomal short tandem repeats (X-STRs) loci which play important roles in some complex kinship cases are increasingly used for forensic practice in recent years. In our study, peripheral blood samples were collected from 309 unrelated healthy individuals from Uigur population (78 males and 231 females) in the Hetian city which is located in the south of Xinjiang Uygur Autonomous Region of China after obtaining written informed consents. Genomic DNA was extracted using the Chelex-100 method as described by Walsh et al. . 11 X-STRs loci including DXS8378, DXS6795, DXS7132, DXS6803, DXS9898, DXS6801, DXS7133, GATA165B12, HPRTB, DXS8377 and DXS7423 according to our previous study were coamplified in a multiplex PCR amplification reaction. PCR amplification fragments were detected by capillary electrophoresis in 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and genotyped with GeneMapper ID v3.2 software according to the manufacture's recommended protocols. As DNA control reference sample, 9947A cell line was used for calibrating homemade allelic ladder. The practices of DNA typing and nomenclatures are in agreement with the ISFG recommendations .

Mitochondrial DNA control region data from indigenous Angolan Khoe-San lineages - Corrected Proof

2012-03-21

Abstract: Here we provide 129 complete mitochondrial control region sequences of indigenous Khoe-San individuals from Angola to contribute to the still underrepresented pool of data from Africa. The dataset consists of exclusively African lineages with a majority of Sub-Saharan haplogroups. The probability of a random match was calculated as 0.09. The data set comprises 21 haplotypes occurring more than once and 17 unique haplotypes. Upon publication, haplotypes were incorporated in the EMPOP database (www.empop.org; EMP00069) .

Distribution of allele frequencies of 20 STRs loci in a population sample from Calabria, Southern Italy - Corrected Proof

2012-03-19

Allele frequencies of 20 STRs including the 5 new loci (D10S1248, D2S441, D1S1656, D12S391, D22S1045) approved by the European Union Council for the expansion of the European Standard Set (ESS) were calculated from a population sample from Calabria in southern Italy using the Applied Biosystems (AB) AmpflSTR Identifiler™ kit plus a next-generation 5-plex we previously developed as a supplementary assay to Identifiler™ .

Allele frequencies of 20 STRs from Northwest Spain (Galicia) - Corrected Proof

2012-03-19

Allele frequencies and forensic informativeness parameters for 15 established autosomal STRs and 5 new ESS autosomal STRs were obtained from 204 unrelated individuals of Northwest Spain (Galicia) with Identifiler® Plus kit of Applied Biosystems (typing D2S1338, D3S1358, D8S1179, D16S539, D18S51, D19S433, D21S11, FGA, TH01, vWA and Amelogenin) and an in-house designed pentaplex typing the five STRs (D1S1656, D2S441, D10S1248, D12S391 and D22S1045) adopted for the European Standard Set (ESS) in 2009 . This study followed the guidelines for publication of forensic population data as well as the recommendations of the ISFG with particular reference to the characterization of new forensic STR markers .

Evaluation and comparative analysis of direct amplification of STRs using PowerPlex® 18D and Identifiler® Direct systems - Corrected Proof

Blake A. Myers, Jonathan L. King, Bruce Budowle — 2012-03-14

Abstract: Short tandem repeat (STR) analysis remains the primary forensic tool for DNA identification. Because of the success of forensic DNA typing and the use of database searches to develop investigative leads, there is an increased demand for populating forensic DNA databases. Reference samples tend to be of high quantity and quality and are somewhat standardized in format. Being more predictable in quality than unknown forensic casework samples, reference samples lend themselves to alternate methods of analysis such as direct amplification. Two commercially available direct amplification kits for processing reference samples were evaluated. The kits are PowerPlex® 18D (Promega Corp., Madison, Wisconsin) and Identifiler® Direct (Life Technologies, Carlsbad, CA). Both kits offer the core CODIS loci plus amelogenin, and the loci D2S1338, D19S433. The PP18D kit offers two additional loci, Penta E and Penta D. To determine the robustness and reliability of the PP18D and ID Direct amplification systems, buccal cell samples (deposited on FTA paper using the EasiCollect™ device (Florham Park, NJ)) from 400 anonymous donors were analyzed under conditions to achieve a high rate of successful typing. First-pass success rates were 96.25%, 96.25%, and 95% for PP18D with a 5s injection, ID Direct with a 10s injection, and ID Direct with a 5s injection, respectively. Profiles that could not be typed were not a result of the kits’ performance but were a result of the inherent variation in the amount of DNA obtained with the collection device and buccal cells. Low signal profiles can be re-analyzed by either re-injecting for a longer time or by re-amplification with an additional PCR cycle. Overloaded profiles can be re-analyzed by re-injecting for a shorter time or by re-amplification with one less cycle. All called typing results, when interpretable, were consistent under the prescribed conditions, different injection times, and 26-28 PCR cycles for both chemistries. Peak height ratios for both kits were well balanced with peaks ranging in height >2000RFUs to those with one or more peaks with heights <100RFUs. A change in the ILS morphology sloping downward to the right relative to a normal ILS profile for PP18D and ID Direct was an indication of a poor injection. Re-injection effectively overcame the effect manifested by a sloping ILS phenomenon. A subset of samples were subjected to direct amplification using the reagents in Identifiler® Plus kit and successful typing results were obtained for the majority of samples. However, the profiles displayed increased amounts of non-adenylated products. The results of this study demonstrate that PP18D and ID Direct are both robust kits for direct amplification. The interpretation guidelines used for this study can form a basis for internal validation studies by databasing laboratories.

Characterization of a modified amplification approach for improved STR recovery from severely degraded skeletal elements - Corrected Proof

Jodi A. Irwin, Rebecca S. Just, Odile M. Loreille, Thomas J. Parsons — 2012-03-09

Abstract: Degraded skeletal remains generally contain limited quantities of genetic material and thus DNA-based identification efforts often target the mitochondrial DNA (mtDNA) control region due to the relative abundance of intact mtDNA as compared to nuclear DNA. In many missing person cases, however, the discriminatory power of mtDNA is inadequate to permit identification when associated anthropological, odontological, or contextual evidence is also limited, and/or the event involves a large number of individuals. In situations such as these, more aggressive amplification protocols which can permit recovery of STR data are badly needed as they may represent the last hope for conclusive identification. We have previously demonstrated the potential of a modified Promega PowerPlex 16 amplification strategy for the recovery of autosomal STR data from severely degraded skeletal elements. Here, we further characterize the results obtained under these modified parameters on a variety of sample types including pristine control DNA and representative case work specimens. Not only is the amplification approach evaluated here sensitive to extremely low authentic DNA input quantities (6pg), but when the method was applied to thirty-one challenging casework specimens, nine or more alleles were reproducibly recovered from 69% of the samples tested. Moreover, when we independently considered bone samples extracted with a protocol that includes complete demineralization of the bone matrix, the percentage of samples yielding nine or more reproducible alleles increased to 95% with the modified amplification parameters. Overall, direct comparisons between the modified amplification protocol and the standard amplification protocol demonstrated that allele recovery was significantly greater using the aggressive parameters, with only a minimal associated increase in artifactual data.

Automated analysis of sequence polymorphism in STR alleles by PCR and direct electrospray ionization mass spectrometry - Corrected Proof

2012-03-09

Abstract: Short tandem repeats (STRs) are the primary genetic markers used for the analysis of biological samples in forensic and human identity testing. The discrimination power of a combination of STRs is sufficient in many human identity testing comparisons unless the evidence is substantially compromised and/or there are insufficient relatives or a potential mutation may have arisen in kinship analyses. An automated STR assay system that is based on electrospray ionization mass spectrometry (ESI-MS) has been developed that can increase the discrimination power of some of the CODIS core STR loci and thus provide more information in typical and challenged samples and cases. Data from the ESI-MS STR system is fully backwards compatible with existing STR typing results generated by capillary electrophoresis. In contrast, however, the ESI-MS analytical system also reveals nucleotide polymorphisms residing within the STR alleles. The presence of these polymorphisms expands the number of alleles at a locus. Population studies were performed on the 13 core CODIS STR loci from African Americans, Caucasians and Hispanics capturing both the length of the allele, as well as nucleotide variations contained within repeat motifs or flanking regions. Such additional polymorphisms were identified in 11 of the 13 loci examined whereby several nominal length alleles were subdivided. A substantial increase in heterozygosity was observed, with close to or greater than 5% of samples analyzed being heterozygous with equal-length alleles in at least one of five of the core CODIS loci. This additional polymorphism increases discrimination power significantly, whereby the seven most polymorphic STR loci have a discrimination power equivalent to the 10 most discriminating of the CODIS core loci. An analysis of substructure among the three population groups revealed a higher θ than would be observed compared with using alleles designated by nominal length, i.e., repeats solely. Two loci, D3S1358 and vWA produced θ estimates of 0.0477 and 0.0234, respectively, when the expanded allele complement (i.e., nominal allele and SNPs) was considered compared to 0.0145 and 0.01266, respectively when only nominal repeat number was considered. These differences may indicate underlying population specific allele distributions exist within these populations. A system of nomenclature has been developed that facilitates the databasing, searching and analyses of these combined data forms.

Performance of Identifiler Direct and PowerPlex 16 HS on the Applied Biosystems 3730 DNA Analyzer for processing biological samples archived on FTA cards - Corrected Proof

Nancy Laurin, Anick DeMoors, Chantal Frégeau — 2012-03-09

Abstract: Direct amplification of STR loci from biological samples collected on FTA cards without prior DNA purification was evaluated using Identifiler Direct and PowerPlex 16 HS in conjunction with the use of a high throughput Applied Biosystems 3730 DNA Analyzer. In order to reduce the overall sample processing cost, reduced PCR volumes combined with various FTA disk sizes were tested. Optimized STR profiles were obtained using a 0.53mm disk size in 10μL PCR volume for both STR systems. These protocols proved effective in generating high quality profiles on the 3730 DNA Analyzer from both blood and buccal FTA samples. Reproducibility, concordance, robustness, sample stability and profile quality were assessed using a collection of blood and buccal samples on FTA cards from volunteer donors as well as from convicted offenders. The new developed protocols offer enhanced throughput capability and cost effectiveness without compromising the robustness and quality of the STR profiles obtained. These results support the use of these protocols for processing convicted offender samples submitted to the National DNA Data Bank of Canada. Similar protocols could be applied to the processing of casework reference samples or in paternity or family relationship testing.

Automating a combined composite–consensus method to generate DNA profiles from low and high template mixture samples - Corrected Proof

2012-03-06

Abstract: We present an automated method to generate DNA profiles from replicate PCRs by combining advantages of the composite and consensus method by a system of brackets in which an allelic balance threshold is used as a variable to separate DNA-profiles of major from minor donors. Through the analysis of artificial low (125pg) and high (250pg) template three-person mixtures with low (1:1.5:3) and high (1:5:10) donor ratios we demonstrate the usefulness of a tool to determine the optimal allelic balance threshold within a locus. The automated extraction of dominant profiles saves considerable amounts of time when producing composite–consensus profiles. Drop-in/drop-out rates are produced and a comparison is made with an alternative open source script to evaluate the dominant profiles generated. By introducing this script into the forensic community we hope to increase awareness of much needed collaborative efforts with bioinformaticians and statisticians to develop forensic open source software scripts.

Allele frequencies of 15 STR loci using AmpF/STR Identifiler kit in the Maldivian population - Corrected Proof

2012-03-05

The allele frequencies and statistical forensic parameters were determined for the population of Maldives on the basis of the 15 STR loci contained in the AmpF/STR Identifiler PCR amplification kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2s1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA).

FamLink – A user friendly software for linkage calculations in family genetics - Corrected Proof

Daniel Kling, Thore Egeland, Andreas O. Tillmar — 2012-03-05

Abstract: The present number of STR loci adopted in relationship testing is chiefly limited to unlinked markers, in most cases residing on different chromosomes. In order to solve more complex cases of relatedness, e.g. deficient paternities and disputed sibships, the number of core loci can be extended. The inclusion of multiple loci on the same chromosome will, however, increase the risk of possible linkage between markers. We present a new software, FamLink, freely available from http://www.FamLink.se, that can perform statistical calculations based on pedigree structures and account for linkage between pairs of markers. In addition, FamLink can simulate genotype data in order to study the effect of accounting for linkage or not. We demonstrate the importance of taking linkage properly into account using examples and real cases.

Molecular identification of vaginal fluid by microbial signature - Corrected Proof

2012-02-27

Abstract: The discrimination of body fluids in forensic examinations can play an important role in crime scene reconstruction. Conventional methods rely on the detection of antigens or enzymatic activity, limiting detection sensitivity and specificity, particularly on old forensic samples. Methods based on human RNA analysis are not easily applicable to samples exposed to harsh and degrading environments. An alternative approach based on the identification of prokaryotic genomes was developed. Specific bacterial communities are characteristic typical of different human non-sterile body fluids: the molecular characterization of a microbial signature, and not the typing of single bacterial species, can effectively lead to univocal identification of these fluids.A multiplex real time PCR assay was developed using oligonucleotide mixtures targeting genomes specific for a selected group of bacteria. Microflora DNA (mfDNA) was extracted from vaginal, oral and fecal clinical swabs. In addition forensic samples were processed. Vaginal samples showed a strong specific signal for bacteria of the female genital tract. Oral samples clearly showed signal for bacteria present in saliva, and in fecal samples the main signal was from Enterococcaceae. Vaginal casework samples showed results comparable to freshly collected ones; moreover the DNA extracted was successfully used for STR typing. Also mixtures of body fluids were analyzed, providing a microbiological signature compatible with the presence of microbes of oral, fecal and vaginal origin. The presented method can be useful in identifying biological fluids, and it is based on DNA technologies already available in forensic laboratories and feasible for further high throughput automation.

Genetic portrait of Brazilian immigrant population living in Lisboa - Corrected Proof

2012-02-24

As well as it occurs all over Europe, in Portugal, and particularly in Lisboa, immigrant populations are increasing . According to Portuguese Foreign Affair Services and to the Instituto Nacional de Estatística of Portugal, Brazilian immigrants with fixed residence in Portugal increased from 11,000 up to 119,000 individuals, between 1990 and 2010. From those 119,000 Brazilian individuals, up to 63,000 live in Lisboa and near cities or villages.

A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces - Corrected Proof

2012-02-24

Abstract: In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA-profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using ≥0.05μL body fluid starting material whereas full DNA profiles were obtained with ≥0.1μL. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers in more detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA- and RNA-profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old).The described approach of combined DNA- and RNA-profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell types are present.

17 Y-STR haplotype data for a population sample of Residents in the Basque Country - Corrected Proof

2012-02-20

Abstract: Non autochthonous population is the most numerous group in the Basque Country. This group is named “Residents” to distinguish them from the “Autochthonous Basque” population. In this work, the 17 Y-STR loci distribution of Resident population was studied in a sample of 197 individuals, who were concretely genotyped for DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS439, DYS438, DYS437, DYS448, DYS456, DYS458, DYS635 and Y GATA H4. Resident population showed a high haplotype diversity and discrimination capacity. The distribution of Y-STRs haplotypes of the Resident population was statistically significant different to the one of the Autochthonous Basque population. The genetic substructure found between Resident and Autochthonous Basque 17 Y-STR haplotype distributions advises for the use of two different databases in the Basque Country, to ensure the most trustworthy frequency estimate in casework.

Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples - Corrected Proof

2012-02-10

Abstract: Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary.DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO® 150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch® Forensic DNA Purification Kit (Invitrogen), the PrepFiler™ Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™ 96 Trace (Macherey-Nagel).After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination.

Specific and sensitive mRNA biomarkers for the identification of skin in ‘touch DNA’ evidence - Corrected Proof

E. Hanson, C. Haas, R. Jucker, J. Ballantyne — 2012-02-10

Abstract: In forensic casework analysis it is often necessary to attempt to obtain DNA profiles from microscopic amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is routinely demonstrated with so-called ‘touch DNA’ evidence, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. Although a genetic profile from trace biological evidence is routinely obtained, the tissue source of the profile is rarely known. This merely perpetuates the ‘mystery’ of the nature of ‘touch DNA’ evidence allowing the significance or meaningfulness of genetic profiles obtained from these samples to be challenged. Numerous reports state that the tissue source of origin of ‘touch DNA’ evidence cannot be determined due to the small amount of biological material present, while others conclude that the DNA profiles are obtained from shed skin cells (as opposed to, say, buccal epithelial cells present in saliva traces) without any scientific basis for this assertion. Proper identification of the biological material present might be crucial to the investigation and prosecution of a criminal offense and a misrepresentation of the nature of the evidence can have undue influence on the perception of the circumstance of the crime. Thus far, research has failed to provide forensic scientists with feasible, definitive methods to identify the tissue origin of ‘touch DNA’.In the present work, we sought to identify novel highly specific and sensitive messenger RNA (mRNA) biomarkers for the identification of skin. Gene candidates were identified using both literature searches and whole transcriptome deep sequencing (RNA-Seq). Utilizing this dual approach, we identified and evaluated over 100 gene candidates. Five mRNA markers were identified that demonstrated a high degree of specificity for skin. Using these markers, we have been able to successfully detect and identify skin using as little as 5–25pg of input total RNA from skin and, significantly, in swabs of human skin and various touched objects. One of the markers, LCE1C, is particularly highly sensitive and was detected in the majority of skin samples tested including touched objects. We have been successful in incorporating the five skin biomarkers into two multiplex systems. Although further work is needed to optimize the assay for routine casework, the initial studies demonstrate that a molecular-based characterization of the biological material recovered from touch samples is possible.

An investigation of admixture in an Australian Aboriginal Y-chromosome STR database - Corrected Proof

2012-02-01

Abstract: Y-chromosome specific STR profiling is increasingly used in forensic casework. However, the strong geographic clustering of Y haplogroups can lead to large differences in Y-STR haplotype frequencies between different ethnicities, which may have an impact on database composition in admixed populations.Aboriginal people have inhabited Australia for over 40,000 years and until ∼300 years ago they lived in almost complete isolation. Since the late 18th century Australia has experienced massive immigration, mainly from Europe, although in recent times from more widespread origins. This colonisation resulted in highly asymmetrical admixture between the immigrants and the indigenes.A State jurisdiction within Australia has created an Aboriginal Y-STR database in which assignment of ethnicity was by self-declaration. This criterion means that some males who identify culturally as members of a particular ethnic group may have a Y haplogroup characteristic of another ethnic group, as a result of admixture in their paternal line. As this may be frequent in Australia, an examination of the extent of genetic admixture within the database was performed. A Y haplogroup predictor program was first used to identify Y haplotypes that could be assigned to a European haplogroup. Of the 757 males (589 unique haplotypes), 445 (58.8%) were identified as European (354 haplotypes). The 312 non-assigned males (235 haplotypes) were then typed, in a hierarchical fashion, with a Y-SNP panel that detected the major Y haplogroups, C–S, as well as the Aboriginal subgroup of C, C4. Among these 96 males were found to have non-Aboriginal haplogroups. In total, ∼70% of Y chromosomes in the Aboriginal database could be classed as non-indigenous, with only 169 (129 unique haplotypes) or 22% of the total being associated with haplogroups denoting Aboriginal ancestry, C4 and K* or more correctly K(xL,M,N,O,P,Q,R,S). The relative frequencies of these indigenous haplogroups in South Australia (S.A.) were significantly different to those seen in samples from the Northern Territory and Western Australia. In S.A., K* (∼60%) has a much higher frequency than C4 (∼40%), and the subgroup of C4, C4(DYS390.1del), comprised only 17%. Clearly admixture in the paternal line is at high levels among males who identify themselves as Australian Aboriginals and this knowledge may have implications for the compilation and use of Y-STR databases in frequency estimates.

Addendum to expanding the CODIS core loci in the United States - Corrected Proof

Douglas R. Hares — 2012-02-01

An important objective in proposing new CODIS core loci is to ensure that all loci would be available for all potential manufacturers. During the evaluation process, appropriate steps were taken to document access to all proposed core loci. Since publication of the proposed list of core loci, additional information has come to our attention indicating that there may be outstanding issues with respect to some of the proposed loci. Consequently, to ensure the availability for all interested manufacturers in accordance with our stated objective, we are withdrawing Penta D and Penta E as proposed CODIS core loci and recommending the revised listing of core loci in . Manufacturers are still encouraged to attempt loci in Section B, in ranked order of preference, for inclusion in potential kits provided the impact on the kit's sensitivity and overall performance is negligible. Please refer to the original letter to the editor for complete descriptions (D.R. Hares, Expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. 6 (2012) e52–e54).

Mitochondrial DNA control region variation in an autochthonous Basque population sample from the Basque Country - Corrected Proof

2012-01-25

Abstract: Mitochondrial control region (16024–576) sequences were generated from 106 samples from autochthonous Basques from the Autonomous Community of the Basque Country. It is especially important to generate mtDNA databases from isolated populations in order to maximize the power of discrimination of this molecular marker. It also represents a useful approach to carry out a more accurate haplogroup classification. This is the first database report of complete control region sequences in an autochthonous Basque population sample. Strict selection criteria of autochthonous individuals, automation of laboratory processing and independent reviews of the raw electropherograms ensure the high quality of these sequences and their utility as reference population data of the autochthonous Basque population.

Indel markers: Genetic diversity of 38 polymorphisms in Brazilian populations and application in a paternity investigation with post mortem material - Corrected Proof

2012-01-25

Abstract: Aiming to evaluate the usefulness of 38 non-coding bi-allelic autosomal indels in genetic identification and kinship testing, three Brazilian population samples were studied: two from Rio de Janeiro (including a sample of individuals with self-declared African ancestry) and one Native American population of Terena from Mato Grosso do Sul. Based on the observed allele frequencies, parameters of forensic relevance were calculated. The combined power of discrimination of the 38 indels was high in all studied groups (PD≥0.9999999999997), although slightly lower in Native Americans. Genetic distance analysis showed significant differences between the allele frequencies in the Rio de Janeiro population and those previously reported for Europeans, Africans and Asians explained by its intermediate position between Europeans and Africans. As expected, the Terena sample was significantly different from all the other populations: Brazilians from Rio de Janeiro general population and with self-declared African ancestry, Europeans, Africans and East Asians. Finally, the performance of the 38-indel multiplex assay was tested in post-mortem material with positive results, supporting the use of short amplicon bi-allelic markers as an additional tool to STR analysis when DNA molecules are degraded.

Human tissue preservation for disaster victim identification (DVI) in tropical climates - Corrected Proof

A. Allen-Hall, D. McNevin — 2012-01-24

Abstract: Disaster victim identification (DVI) poses unique challenges for forensic personnel. Typical scenarios may involve many bodies or body parts to identify in remote locations with limited access to laboratory facilities and in extreme temperatures. Transportation of tissue samples to a forensic laboratory for DNA profiling can take weeks without refrigeration. As well as protecting DNA for subsequent analysis, tissue preservation methods ideally should be safe, readily available and easy to transport to the scene at relatively low cost. We examined eight tissue preservatives (salt, DMSO, ethanol, ethanol with EDTA, TENT buffer, RNAlater®, DNA Genotek Tissue Stabilising Kit and DNAgard®) and compared the quantity and quality of DNA recovered from human tissue and preservative solution stored at 35°C. Salt, DMSO, ethanol solutions, DNA Genotek and DNAgard® produced full Identifiler® genotypes up to one month from DNA extracts. In addition, DMSO, DNA Genotek and DNAgard® produced full profiles from aliquots of the liquid preservative.

Performance of two 17 locus forensic identification STR kits—Applied Biosystems's AmpFℓSTR® NGMSElect™ and Promega's PowerPlex® ESI17 kits - Corrected Proof

Torben Tvedebrink, Helle Smidt Mogensen, Maria Charlotte Stene, Niels Morling — 2012-01-24

Abstract: We compared the performance of two recently released 17 loci STR multiplexes for human identification: Applied Biosystems's AmpFℓSTR® NGMSElect™ and Promega's PowerPlex® ESI17. The comparative parameters were chosen by their relevance in forensic identification and particularly in crime cases. The comparative analyses encompass: amplification ability, heterozygote balance, allelic drop-out, drop-in, stutter analysis and inter-locus balance.Four DNA profiles were analysed in various concentrations in a serial dilution experiment. The amounts of DNA in the PCR ranged from 3pg to 420pg and were analysed in triplicate using 28, 29 and 30 PCR cycles. In order to compare the kits, aliquots from each sample were analysed with both kits under identical conditions. Furthermore, DNA profiles from 200 reference profiles were analysed using both kits.The results from the statistical analyses did not indicate any substantial differences of practical relevance between the kits for forensic case work. For all parameters included in this comparative study, the two kits showed no departure from previously observed patterns relative to e.g. the amounts of DNA or amplicon lengths. Based on our analyses, both kits are considered applicable for forensic crime case work.

Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads - Corrected Proof

Chantal J. Frégeau, Anick De Moors — 2012-01-20

Abstract: The Promega DNA IQ™ system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ™-based protocol, the competition for binding sites on the DNA IQ™ magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ™ beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30min instead of the usual overnight at 56°C prevented competition from black denim dye/chemicals and increased DNA yields.

Population genetic data for 16 STR loci (PowerPlex ESX-17 kit) in El Salvador - Corrected Proof

J.C. Monterrosa, J. Morales, I. Yurrebaso, O. García — 2012-01-16

We determined the allele frequencies and forensic parameters for 16 STR autosomal loci (D3S1358, TH01, D21S11, D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1045, VWA, D8S1179, FGA, D2S441, D12S391, D19S433 and SE33). Blood samples were collected from 150 unrelated healthy El Salvadorean donors following informed consent. This sample represents to an urban resident population according to its distribution in the country: 83% mestizo (indigenous and European, mainly Spaniards), 16% white population of Spanish origin and 1% is of indigenous origin.

Conservation of endemic and threatened wildlife: Molecular forensic DNA against poaching of the Cypriot mouflon (Ovis orientalis ophion, Bovidae) - Corrected Proof

2012-01-09

Abstract: Molecular DNA techniques in combination with appropriate reference population database and statistical methods are fundamental tools to forensic wildlife investigations. This is even more relevant when taxa with uncertain systematics are involved, as is the case of the genus Ovis (Bovidae), whose evolution has been influenced by multiple events of domestication. The Cypriot mouflon, Ovis orientalis ophion, a protected subspecies endemic to Cyprus, is threatened by poaching. This study deals with a case of alleged poaching that occurred in Cyprus (September, 2010). A car did not stop at a checkpoint and when finally blocked by the police, several bloodstained exhibits (n=12) were recovered. Three recently deceased mouflons were found by game wardens at the roadside. The Cyprus Veterinary Services established that these animals had been killed by gunshot. As part of the investigation, DNA testing was performed to establish if there was a link between the dead mouflons and the bloodstained exhibits. The mitochondrial Cytochrome-b gene (Cyt-b) and 12 loci of microsatellite DNA were used as markers. The Cyt-b sequences were obtained from 11 exhibits. They were the same as each other and the same as the single haplotype obtained from the three dead mouflons and all the investigated wild Cypriot mouflons (20 individuals). A database of wild mouflons (47 individuals) from which the unknown samples may have originated was generated. The probability of identity (PID) of the microsatellite panel, computed by genotyping all 47 wild mouflons (10 selected loci, PID=10−5), allowed us to assign nine exhibits to two out of the three carcasses (seven with very strong support: Likelihood Ratio, LR>3000 and Random Match Probability, RMP, <10−3). This study represents the first genetic reference for the Cypriot mouflon and the first published material of forensic wildlife investigations in Cyprus.

Variants observed for STR locus SE33: A concordance study - Corrected Proof

2012-01-04

Abstract: Discordance of STR typing results can be expected between kits that employ different primers for amplification. The complex motif of the SE33 locus and its flanking regions can contribute to the degree of discordant results. Sequence-dependent conformational changes can manifest as length differences under certain electrophoretic conditions and/or use of different primers. The AmpFlSTR® NGM SElect™ PCR Amplification Kit (Life Technologies, Carlsbad, CA), PowerPlex® ESX 17 system (Promega Corporation, Madison, WI), and PowerPlex® ESI 17 system (Promega Corporation) were compared for concordance of allele calls for the SE33 marker in selected samples. A total of 16 samples were identified that were discordant at one of the SE33 alleles by an apparent one nucleotide in size. While the ESX 17 and NGM SElect™ kits yielded concordant results for these 16 samples, the ESI 17 kit generated alleles that differed. The discordant alleles were observed in individuals of African and European descent. Sequence analysis revealed that the one-base difference in size is not due to an indel but is instead the result of a single nucleotide polymorphism (SNP) in the flanking region of the SE33 repeat region. Three different SNPs were observed, one of which is novel.Although these migration anomalies were observed only with the ESI 17 kit, one cannot preclude that a similar phenomenon may occur with the other kits as data sets increase. The type and degree of discordance of STR allele calls among STR kits is an important issue when comparing STR profiles among laboratories and when determining search parameters for identifying candidate associations in national databases.

Sequence variation of mitochondrial DNA control region in North Central Venezuela - Corrected Proof

2011-12-21

Dear editor, The analysis of the complete mitochondrial DNA control region (mtDNA-CR) has become increasingly common in the last years, in part due to general interest in the greater discriminatory power and phylogenetic signal provided by entire control region data . In countries like those of the Americas, with known historical admixture between genetically different groups, characterization of the mtDNA diversity is critical for understanding regional heterogeneity and substructure. These types of population genetic features are, in turn, important for the proper application of statistics in mtDNA-based forensic casework.

How to distinguish genetically between an alleged father and his monozygotic twin: A thought experiment - Corrected Proof

Michael Krawczak, David N. Cooper, Fred Fändrich, Wolfgang Engel, Jörg Schmidtke — 2011-12-16

Abstract: Recently, a first direct estimate of the single base-pair substitution rate in the human germline was derived from genome-wide DNA sequence data. This result has shed new light upon the question of whether cutting-edge molecular genetic analysis could, in a paternity dispute, potentiate discrimination between two alleged fathers who are monozygotic (MZ) twins. Such paternity cases are not infrequent and usually receive a high level of public attention. We performed a ‘thought experiment’, the outcome of which strongly suggests that, by a combination of currently available laboratory techniques, paternity testing is indeed feasible in the context of MZ twins. Taking into consideration what is known about the biology of the human male germline, we would predict that >80% of the offspring of one twin brother would carry at least one germline mutation that would be detectable in the sperm of their father, but not in that of the other twin.

Low copy number DNA profiling from isolated sperm using the aureka®-micromanipulation system - Corrected Proof

C. Schneider, U. Müller, R. Kilper, B. Siebertz — 2011-12-12

Abstract: A new cell isolation technique linked to the aureka® micromanipulation system (aureka®) was used to pick sperm from mixed samples containing sperm and epithelial cells. Both cell types were stained using the HY-LITER™ high-resolution, fluorescent staining kit. To isolate a single sperm of interest under a fluorescent microscope, a specific microsphere picking technique was used.This sensitive and reliable cell identification and isolation technique enables low-copy-number (LCN) DNA profiling, as few as 20 sperm are sufficient for obtaining a full short tandem repeat (STR) profile without any allelic drop out.The presented protocol covers the whole workflow, from sample staining and cell pick up to STR analysis.

Use of DNA fingerprints to control the origin of sapelli timber (Entandrophragma cylindricum) at the forest concession level in Cameroon - Corrected Proof

C. Jolivet, B. Degen — 2011-12-12

Abstract: Illegal logging and associated trade are the cause of many economic and ecological problems both in producer and in consumer countries. There are an increasing number of national and international regulations in place that call for efficient timber tracking systems. We present results of a pilot study of a DNA-based method to control the geographical origin of timber in forest concessions in Cameroon. We addressed genetic differentiation at five nuclear microsatellite loci in seven sapelli (Entandrophragma cylindricum, Meliaceae) populations located in three forest concessions in Eastern Cameroon. In the framework of a blind test, seven anonymous timber sample sets were analysed at three microsatellite loci and compared to the genetic reference data of the forest concessions in Cameroon. Our results show that genetic differentiation was low within and among concessions. Combining the results of Bayesian genetic assignment method and exclusion test, we could determine that the timber stemmed or did not stem from the focus forest concession in six out of the seven blind sample sets. We further discuss the accuracy of the presented method and draw conclusions for a better sampling and genotyping strategy. Our work provides clear evidence that the use of genetic fingerprints is a useful tool to fight against illegal logging.