Forensic Science International: Genetics - Articles in Press

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.

An evaluation of potential allelic association between the STRs vWA and D12S391: Implications in criminal casework and applications to short pedigrees - Corrected Proof

Peter Gill, Chris Phillips, Catherine McGovern, Jo-Anne Bright, John Buckleton — 2011-12-09

Abstract: An evaluation was carried out to determine the effect on routine forensic calculations when incorporating STRs D12S391 and vWA. These loci are co-located on the same arm of chromosome 12. It has been suggested that allelic association could result in over-estimates of strength-of-evidence calculations. In the first place, we argue that is very unlikely that genotypes collected from typical cosmopolitan forensic databases can provide meaningful information about effects attributable to physical linkage. Since admixture is the most likely cause of allelic association in modern populations we specifically evaluate this effect. We use computer simulation as the preferred approach to generate populations with disequilibrium and observe the effect on match probability. Although we have specifically evaluated the linkage between D12S391 and vWA, the methods described in this paper can be extended and generalized to evaluate linkage effects between any pair of loci where the recombination rate is known.Many jurisdictions apply a subpopulation correction following the standard method of Balding and Nichols. Such corrections would appear to be more than adequate to compensate for any increase in match probability that we were able to create by this admixture.Linkage is likely to have an appreciable effect on relatedness calculations in short pedigrees in some but not all instances. We examined those circumstances where an effect is likely and give formulae for some common situations. The complexity of these calculations is a cause for concern in some laboratories. We discuss possible strategies that might be employed and plausible effects.

Y-chromosomal STR analysis in the Pashtun population of Southern Afghanistan - Corrected Proof

2011-12-05

Abstract: Afghanistan is a landlocked country in the heart of Asia and since the dawn of humankind Afghanistan has faced centuries of turmoil, strife, conflict, warfare, distress, social unrest, difficult climate, harsh terrain and due to its unique geostrategic position in Eurasia which has historically attracted commerce and conflict. It is an important stop along the Silk Road, connecting the far eastern civilizations to the western world. A 5000-year history of constant invasion. Afghanistan has been repeatedly invaded and conquered by rulers and super powers, neighboring interference in this conflict-tattered land for centuries yet rarely leading to the conquest of this rugged and challenging terrain nation. Afghans are not only shepherds, farmers and nomads but also intense fighters and fierce warriors. Currently very limited genetic studies have been performed in Afghan populations.17 Y chromosomal short tandem repeats (Y-STRs) were analyzed in 125 unrelated Pashtun (in hindi: Pathan) males residing in the Kandahar region of Southern Afghanistan. A total of 92 unique haplotypes were observed. The predominant haplotype reached a frequency of 9.6%. The haplotype diversity was 0.987 and the discrimination capacity 73.6%. Analysis of molecular variance (AMOVA) reveals a considerable regional stratification within the country as well as between different Pashtun (Pathan) groups from Afghanistan, Pakistan and India.

Typing short amplicon binary polymorphisms: Supplementary SNP and Indel genetic information in the analysis of highly degraded skeletal remains - Corrected Proof

2011-11-28

Abstract: Two sets of short amplicon binary markers (SABs): 50 single nucleotide polymorphisms (SNPs) and 38 insertion/deletion polymorphisms (Indels) were used to genotype bones of 35 years “post-mortem”. Typing results of these binary markers were compared with those obtained for standard commercial STR and mini-STR DNA typing kits. We observed SAB marker performance to be better compared with conventional STR and mini-STR genotyping in degraded bone sample analysis. Furthermore, additional genetic information provided by these 88 binary markers, 50 SNPs and 38 Indels, combined with classical markers gave very high discrimination power even in severely degraded specimens, with all tested bone samples showing Random Match Probabilities (RMPs) higher than 1019. Missing person and disaster victim identification by kinship analysis is considerably strengthened by the addition of SAB markers since they can be successfully typed on degraded bone samples while adding considerable extra genetic data when poor or incomplete information is available from conventional forensic markers for the analysis of family pedigrees.

Application of STR markers in wildlife forensic casework involving Australian black-cockatoos (Calyptorhynchus spp.) - Corrected Proof

2011-11-21

Abstract: Parrots and cockatoos are highly prized aviary birds and the demands for such species has fuelled their illegal trade and harvest from the wild. Here we report on three forensic case studies involving black-cockatoos (Calyptorhynchus spp.) endemic to Australia. These cases involve suspected poaching and illegal killing of endangered red- and white-tailed black-cockatoos. Through the prior development of 20 polymorphic microsatellite loci and population databases for white- and red-tailed black-cockatoos, the tools are available to conduct high-resolution paternity and individual identity testing. In one case, we matched a red-tailed black-cockatoo nestling to a tree hollow from which it was poached through the use of DNA from eggshell recovered from the nest. For the second case, we utilized our provenance population database (nest sites), and identified the kinship and geographic origin of a white-tailed black-cockatoo, which was illegally harvested from the wild. The third case determined the number individual white-tailed black-cockatoos allegedly shot at a fruit grower's orchard from body part remains. These genetic investigations highlight the significance and statistical confidence of DNA profiling and associated databases for endangered taxa, such as exotic birds. Our cockatoo population databases are the first of their kind in Australia, and demonstrate the efficacy of such approaches to identify such illegal activity. With a robust set of genetic markers and methodologies in place, we aim to broaden our population databases to include other cockatoo species of conservation concern.

Genetic population studies on 15 NGM™ STR loci in central Poland population - Corrected Proof

Maciej Jedrzejczyk, Renata Jacewicz, Stefan Szram, Jaroslaw Berent — 2011-11-21

We investigated genetic polymorphism of 15 STR loci (D10S1248, D22S1045, D2S441, D1S1656, D12S391, D2S1338, D3S1358, D8S1179, D16S539, D18S51, D19S433, D21S11, FGA, TH01, vWA) included in the NGM™ amplification kit in a sample of 400 unrelated, adult individuals (189 females and 211 males) born in the Lodz region of central Poland.

Match probabilities for multiple siblings - Corrected Proof

K. Slooten — 2011-11-17

Abstract: A defendant whose dna profile matches that of a crime stain may argue that he has several, say n, brothers and that one of them may have been the origin of the crime stain. If the probability for any of the brothers considered separately to match the crime stain profile is p, we show that the probability that at least one of the n brothers match is strictly smaller than np. This latter quantity therefore is an easy to compute and conservative value to report.

Genome deletion and insertion polymorphisms (DIPs) in the Hungarian population - Corrected Proof

2011-10-19

Insertion and deletion (Indels or DIPs) loci are useful markers in tracing the history and diversity of the human population, as well as for forensic genetics. Their usefulness is demonstrated as they possess common features of both SNPs and STRs such as small amplicon sizes and the well established analysis methods including PCR and capillary electrophoresis. Insertion/deletions are length polymorphisms created by insertions or deletions of one or more nucleotides in the human genome . DIP loci with short amplicon sizes (maximum 158bp) and the absence of stutter peaks make them suitable for degraded samples. The 30 DIP loci included in the Investigator DIPplex PCR amplification kit are distributed over 19 autosomes that are at least 10Mbp away from any commercially used STR and SNP markers commonly used in forensic practice, as for example of the SNPforID 52 plex . The aim of this study was twofold; to test the Hungarian population on 30 DIP loci while calculating the statistical and forensic efficiency parameters. And secondly, to validate the Mentype DIPplex PCR amplification kit for forensic applications.

Y-chromosomal microsatellite diversity in three culturally defined regions of historical Tibet - Corrected Proof

2011-10-17

Abstract: In the present study, we analyzed 17 Y-STR loci in 350 Tibetan males from three culturally defined regions of historical Tibet: Amdo (88), Kham (109) and U-Tsang (153). A total of 299 haplotypes were observed, 272 (90.9%) of which were unique. Only one Y-STR profile is shared across the three Tibetan groups and, incidentally, is also the most frequent haplotype (4.0%), represented by two, five and seven individuals from U-Tsang, Kham and Amdo, respectively. The overall haplotype diversity for the three Tibetan populations at 17 Y-STR loci was 0.9978 and the corresponding values for the extended (11-loci) and minimal (9-loci) haplotypes were 0.9935 and 0.9909, respectively. Both neighbor-joining and Rst pairwise analyses suggest a close genetic relationship between the Amdo and Kham populations, while U-Tsang is genetically distinct from the aforementioned groups. The results demonstrate that the 17 Y-STR loci analyzed are highly polymorphic in all three Tibetan populations examined and hence useful for forensic cases, paternity testing and population genetic studies.

An optimized protocol for forensic application of the PreCR™ Repair Mix to multiplex STR amplification of UV-damaged DNA - Corrected Proof

Toni M. Diegoli, Matthew Farr, Carter Cromartie, Michael D. Coble, Todd W. Bille — 2011-10-17

Abstract: Damage to the DNA molecule can occur through exposure to environmental conditions such as ultraviolet light, heat and humidity. Forensic samples are particularly prone to such damage due to their prolonged exposure after deposition at crime scenes or mass disasters. Current methods for typing such samples rely heavily on the intact DNA template, and can be adversely affected by damage that is present. Proposed solutions center around increased access to the smaller remaining fragments and/or increased sensitivity. However, all rely on the polymerase chain reaction to copy the starting material; the required polymerase can be impeded by certain types of damage such as dimer-formation after ultraviolet light exposure, resulting in stochastic effects that can complicate interpretation. In vitro repair of such damage offers the ability to generate high quality profiles using traditional methods without changes to the current amplification reagents or conditions. Typically, repair reactions required large quantities of starting material and a separate repair reaction. Forensic samples, however, usually consist of small quantities, and quality control measures necessitate laboratory procedures that minimize sample manipulation. Here, an optimized protocol for forensic application of the PreCR™ Repair Mix to current typing methods is demonstrated for samples damaged by ultraviolet light exposure.

Discrimination power of Investigator DIPplex loci in Finnish and Somali populations - Corrected Proof

Anu M. Neuvonen, Jukka U. Palo, Minttu Hedman, Antti Sajantila — 2011-10-17

Abstract: In this study, the suitability of the Investigator DIPplex insertion/deletion polymorphism (indel) kit for forensic casework was assessed through the genotyping of 151 Finns and 175 Somalis. Allele frequency and heterozygosity (H) of this 30-indel marker set were determined, and forensic efficacy was evaluated through estimation of discrimination power (DP), match probability (MP), typical paternity index (TPI), power of paternity exclusion (PE), and polymorphic information content (PIC). A high level of discrimination power was observed for the marker set in both sample groups (CDP>0.9999). East-west population substructure found previously in uniparental markers within Finland was not evident for this autosomal set (E-W FST=0.003). High exclusion probability and low subdivision together demonstrate that these markers are well-suited for identification of individuals in Finland. However, values for typical paternity index and power of paternity exclusion were low (TPI range Finns=0.750–1.190, PE=0.996; TPI Somalis=0.680–1.090, PE=0.986) in comparison to standard STR sets, and thus indels are not recommended for use in paternity or kinship investigations, except as a supplement to other more powerful tools.

Selective quantification of human DNA by real-time PCR of FOXP2 - Corrected Proof

Mikiko Soejima, Kenichi Hiroshige, Joji Yoshimoto, Yoshiro Koda — 2011-10-17

Abstract: We established a simple quantitative PCR procedure with high specificity and sensitivity using TaqMan probes targeting the FOXP2 sequence. This assay distinguished human and nonhuman, including primates, samples with the exception of mouse, turtle, lizard, and fishes. However, the specific amplification of mouse, lizard, and turtle fragments of FOXP2 could be confirmed by electrophoresis after real-time PCR. Because the CT values obtained for human DNA were not affected by contaminating animal DNA at concentrations up to 30 times that of human DNA, we were able to estimate the concentration of human DNA in mixed specimens. This assay provides a reliable and useful method for routine quantification of human-specific DNA in forensic practice.

Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis - Corrected Proof

2011-10-17

Abstract: The identification of forensically relevant human body fluids through messenger RNA (mRNA) profiling is of interest to the forensic community. Previous studies have proposed several tissue-specific mRNA markers to achieve this goal. Seven markers for the following genes were selected for evaluation in this study: histatin 3 (HTN3) and statherin (STATH) for saliva, mucin 4 (MUC4) for vaginal secretions, matrix metalloproteinase 7 (MMP7) for menstrual blood, delta-aminolevulinate synthase 2 (ALAS2) for peripheral blood, and protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen. The expression of these markers was examined in each body fluid. All mRNA markers were present in their target body fluids. Peripheral blood and saliva showed little cross-reactivity with the selected markers. However, a high level of cross-reactivity was observed between the vaginal secretion marker MUC4 and saliva stains. Semen showed a high level of cross-reactivity with the selected markers. Co-expression of the predicted body fluid markers was detected in menstrual blood and vaginal secretion stains. The expression pattern of these mRNA markers varied through the menstrual cycle time points tested. Differences in gene expression levels and marker cross-reactivity were observed in the donors tested. Despite the presence of cross-reactivity and co-expression, each of the body fluids examined have distinct gene expression profiles, allowing for body fluid identification based on mRNA profiling.

Population data of the AmpFISTR® NGM™ STR loci in a North of Portugal sample - Corrected Proof

M.L. Pontes, M.F. Pinheiro — 2011-10-17

The AmpFℓSTR® NGM™ PCR Amplification Kit includes 15 STRs loci and 12 of them (D3S1358, VWA, D8S1179, D21S11, D18S51, TH01, FGA, D1S1656, D2S441, D10S1248, D12S391 and D22S1045) are part of the European Standard Set (ESS). Five of these loci, D10S1248, D22S1045, D2S441, D1S1656, D12S391, were recommended by the ENFSI and EDNAP groups to be adopted for the analysis of degraded DNA samples, in order to improve the power of national databases and to encourage standardization within Europe . Allele frequencies and other forensically relevant statistic data for the STR loci included in the AmpF2STR® NGM™ PCR Amplification Kit, were generated. No significant deviations from Hardy–Weinberg expectations were found.

FTIR spectroscopy: A new diagnostic tool to aid DNA analysis from heated bone - Corrected Proof

Jamie Daniel Fredericks, Phil Bennett, Anna Williams, Keith Derek Rogers — 2011-10-03

Abstract: Deoxyribonucleic acid (DNA) extracted from skeletal tissue can be invaluable in genetic profiling applications, as it is often the only source available. Like all forensic samples, skeletal tissue may have been exposed to a variety of environmental insults, including heat. This study has focussed upon characterising changes in the material properties of bone that has been compromised by controlled heat treatments. These changes were then examined in relation to the subsequent success or failure of nuclear DNA (nDNA) amplification, using a range of differently sized amplicons, relevant to alternate profiling strategies. The results presented demonstrate that the ability to amplify nDNA correlates well with particular changes in mineral and organic content of bone. As such, we propose the application of a ‘diagnostic triage tool’ that can be performed quickly and at low cost on individual bone samples, in order to determine whether nDNA analysis is likely to be a viable option.

Quadriplex real-time PCR (qPCR) assay for human–canine–feline species identification and nuclear DNA quantification - Corrected Proof

S. Kanthaswamy, A. Premasuthan — 2011-10-03

This letter is an update to a recent publication in the Forensic Science International Genetics journal, “Quantitative real-time PCR (qPCR) assay for human–dog–cat species identification and nuclear DNA quantification”: S. Kanthaswamy, A. Premasuthan, et al. This triplex qPCR assay quantifies and identifies the presence of as little as 0.4pg of human, feline and canine DNA in a mixed sample . The assay did not include an Internal Positive Control (IPC) due to the three-dye limit in the Applied Biosystems 7300 machine. The assay is now modified to include an IPC using the 7500 Fast Real Time PCR system ().

UK population data generated with the PowerPlex® ESI 16 system - Corrected Proof

Valerie C. Tucker, Coralie Baumgartner, Gareth R. Stead, Andrew J. Hopwood — 2011-09-26

In order to prepare our laboratory for the implementation of STR data exchange with Europe , we have undertaken to genotype DNA samples from three ethnic populations common in the UK. Allele frequencies generated using the PowerPlex® ESI 16 System (ESI 16) are presented, along with observations of discordance with alternative multiplexes.

MtDNA SNP multiplexes for efficient inference of matrilineal genetic ancestry within Oceania - Corrected Proof

2011-09-23

Abstract: Human mitochondrial DNA (mtDNA) is a convenient marker for tracing matrilineal bio-geographic ancestry and is widely applied in forensic, genealogical and anthropological studies. In forensic applications, DNA-based ancestry inference can be useful for finding unknown suspects by concentrating police investigations in cases where autosomal STR profiling was unable to provide a match, or can help provide clues in missing person identification. Although multiplexed mtDNA single nucleotide polymorphism (SNP) assays to infer matrilineal ancestry at a (near) continental level are already available, such tools are lacking for the Oceania region. Here, we have developed a hierarchical system of three SNaPshot multiplexes for genotyping 26 SNPs defining all major mtDNA haplogroups for Oceania (including Australia, Near Oceania and Remote Oceania). With this system, it was possible to conclusively assign 74% of Oceanian individuals to their Oceanian matrilineal ancestry in an established literature database (after correcting for obvious external admixture). Furthermore, in a set of 161 genotyped individuals collected in Australia, Papua New Guinea and Fiji, 87.6% were conclusively assigned an Oceanian matrilineal origin. For the remaining 12.4% of the genotyped samples either a Eurasian origin was detected indicating likely European admixture (1.9%), the identified haplogroups are shared between Oceania and S/SE-Asia (5%), or the SNPs applied did not allow a geographic inference to be assigned (5.6%). Sub-regional assignment within Oceania was possible for 32.9% of the individuals genotyped: 49.5% of Australians were assigned an Australian origin and 13.7% of the Papua New Guineans were assigned a Near Oceanian origin, although none of the Fijians could be assigned a specific Remote Oceanian origin. The low assignment rates of Near and Remote Oceania are explained by recent migrations from Asia via Near Oceania into Remote Oceania. Combining the mtDNA multiplexes for Oceania introduced here with those we developed earlier for all other continental regions, global matrilineal bio-geographic ancestry assignment from DNA is now achievable in a highly efficient way that is also suitable for applications with limited material such as forensic case work.

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

Yuvaneswari Chandramoulee Swaran, Lindsey Welch — 2011-09-19

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

Forensic implications of PCR inhibition—A review - Corrected Proof

Reza Alaeddini — 2011-09-14

Abstract: Polymerase chain reaction (PCR) is currently the method of choice for the identification of human remains in forensic coursework. DNA samples from crime scenes often contain co-purified impurities which inhibit PCR. PCR inhibition is the most common cause of PCR failure when adequate copies of DNA are present. Inhibitors have been routinely reported in forensic investigations of DNA extracted from a variety of templates. Humic compounds, a series of substances produced during decay process have been considered as the materials contaminating DNA in soil, natural waters and recent sediments. Those compounds have been frequently assigned as PCR inhibitors. The current report reviews the characteristics of PCR inhibition, including the proposed mechanisms of inhibition, detection methods and the available technologies to remove or overcome the inhibitory activities.

X chromosomal recombination study in three-generation families in Hungary - Corrected Proof

2011-09-14

X chromosome STR studies have a curious role in forensic and population genetics. These markers are present as a single copy in males and as double copies in females. Analysis of X-chromosomal loci can be beneficial in deficient paternity cases, when the offspring is female and the alleged father is missing, or in maternity cases . So far, a very limited number of X recombination and linkage disequilibrium studies (LD) has been published . Hering et al. have analyzed three-generation family studies and Inturri et al. have studied two-generation families in the past. Inturri et al. stated that two-generation family studies are less accurate than three-generation studies, as the unavailability of the maternal grandfather does not allow a direct count of recombinant chromosomes. Furthermore, only Tillmar et al. performed the LD test using data of male haplotypes for 8 X-STR loci, but not for 12 X-STR loci.

A new SNP assay for identification of highly degraded human DNA - Corrected Proof

2011-09-12

Abstract: There is growing evidence that the histone–DNA complexes found in nucleosomes offer protection from DNA degradation processes, including apoptotic events in addition to bacterial and environmental degradation. We sought to locate human nucleosome regions and build a catalogue of SNPs sited near the middle of these genomic segments that could be combined into a single PCR multiplex specifically for use with extremely degraded human genomic DNA samples. Using recently optimized bio-informatics tools for the reliable identification of nucleosome sites based on sequence motifs and their positions relative to known promoters, 1395 candidate loci were collected to construct an 18-plex single base extension assay. Genotyping performance of the nucleosome SNPs was tested using artificially degraded DNA and 24 casework samples where the likely state of degradation of DNA was established by comparison to profile completeness in four other forensic assays: a standard 15-plex STR identification test, a miniaturized STR multiplex and two autosomal SNP multiplexes. The nucleosome SNP assay gave genotyping success rates 6% higher than the best existing forensic SNP assay: the SNPforID Auto-2 29-plex and significantly higher than the mini-STR assay. The nucleosome SNPs we located and combined therefore provide a new type of marker set that can be used to supplement existing approaches when the analysed DNA is likely to be extremely degraded and may fail to give sufficient STR genotypes for a reliable identification.

Analysis of matches and partial-matches in a Danish STR data set - Corrected Proof

2011-09-07

Abstract: Over the recent years, the national databases of STR profiles have grown in size due to the success of forensic DNA analysis in solving crimes. The accumulation of DNA profiles implies that the probability of a random match or near match of two randomly selected DNA profiles in the database increases.We analysed 53,295 STR profiles from individuals investigated in relation to crime case investigations at the Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark. Incomplete STR profiles (437 circa 0.8% of the total), 48 redundant STR profiles from monozygotic twins (0.09%), 6 redundant STR profiles of unknown cause and 1283 STR profiles from repeated testing of individuals were removed leaving 51,517 complete 10 locus STR profiles for analysis. The number corresponds to approximately 1% of the Danish population. We compared all STR profiles to each other, i.e. 1.3×109 comparisons.With these large number of comparisons, it is likely to observe DNA profiles that coincide on many loci, which has concerned some commentators and raised questions about “overstating” the power of DNA evidence. We used the method of Weir and Curran et al. to compare the observed and expected number of matches and near matches in the data set. We extended the methods by computing the covariance matrix of the summary statistic and used it for the estimation of the identical-by-descent parameter, θ. The analysis demonstrated a number of close relatives in the Danish data set and substructure. The main contribution to the substructure comes from close relatives. An overall θ-value of 1% compensated for the observed substructure, when close familial relationships were accounted for.

Typing of 30 insertion/deletions in Danes using the first commercial indel kit—Mentype® DIPplex - Corrected Proof

2011-09-07

Abstract: In this study, we tested the first commercial kit with insertion/deletion (indel) polymorphisms, the Mentype® DIPplex PCR Amplification Kit (DIPplex kit). A total of 30 biallelic autosomal indels and Amelogenin were amplified with the DIPplex kit. All loci were amplified in one PCR multiplex and all amplicon lengths were shorter than 160bp. Full indel profiles were generated from as little as 100pg of DNA. A total of 117 individuals from Danish paternity cases were successfully typed. No deviation from Hardy–Weinberg equilibrium was observed for any of the indels. The combined mean match probability was 3.3×10−13, the mean paternity exclusion probability was 99.7% and the typical paternity indices for trios and duos were 2350 and 165, respectively. Furthermore, we typed five highly degraded DNA samples with the DIPplex kit, the AmpFlSTR® SGM Plus kit and the AmpFlSTR® SEfiler Plus kit. Full indel profiles were obtained with the DIPplex kit, whereas only partial profiles were obtained with the STR kits. In general, the DIPplex kit performed well and it would be a valuable assay for forensic genetic testing, especially in crime cases with partially degraded DNA or low amounts of template DNA. However, some difficulties with pull-ups were observed at DNA concentrations of 1000pg. Rearrangement of the allele windows by changing the lengths of some of the PCR primers would greatly improve the assay, and more robustness towards higher amounts of DNA would allow the use of the DIPplex kit without prior quantification of the samples.

A model for data analysis of microRNA expression in forensic body fluid identification - Corrected Proof

Zheng Wang, Haibo Luo, Xiongfei Pan, Miao Liao, Yiping Hou — 2011-09-07

Abstract: MicroRNAs (miRNAs, 18–25 bases in length) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. MiRNA expression patterns, including presence and relative abundance of particular miRNA species, provide cell- and tissue-specific information that can be used for body fluid identification. Recently, two published studies reported that a number of body fluid-specific miRNAs had been identified. However, the results were inconsistent when different technology platforms and statistical methods were applied. To further study the role of miRNAs in identification of body fluids, this study sets out to develop an accurate and reliable model for data analysis of miRNA expression. To that end, the relative expression levels of three miRNAs were studied using the mirVana™ miRNA Isolation Kit, high-specificity stem-loop reverse transcription (RT) and high-sensitivity hydrolysis probes (TaqMan) quantitative real-time polymerase chain reaction (qPCR) in forensically relevant biological fluids, including venous blood, vaginal secretions, menstrual blood, semen and saliva. Accurate quantification of miRNAs requires not only a highly sensitive and specific detection platform for experiment operation, but also a reproducible methodology with an adequate model for data analysis. In our study, the efficiency-calibrated model that incorporated the impact of the quantification cycle (Cq) values and PCR efficiencies of target and reference genes was developed to calculate the relative expression ratio of miRNAs in forensically relevant body fluids. Our results showed that venous blood was distinguished from other body fluids according to the relative expression ratio of miR16 using as little as 50pg of total RNA, while the expression level of miR658 was unstable and that of miR205 was nonspecific among different body fluids. Collectively, the findings may constitute a basis for future miRNA-based research on body fluid identification and show miRNAs as a promising biomarker in forensic identification of body fluids.

Interpreting lineage markers in view of subpopulation effects - Corrected Proof

Sarah Cockerton, Kurt McManus, John Buckleton — 2011-09-05

Abstract: The interpretation of lineage markers is usually carried out as a count in a database. The count is a factual statement and does not take into account subpopulation effects that may be acting on the data. Subpopulation effects are usually taken into consideration for autosomal DNA genotype interpretation by the incorporation of a correction, θ. The question has arisen as to whether lineage markers should also have such a correction. This paper discusses if and how subpopulation effects could be considered.

Automatable full demineralization DNA extraction procedure from degraded skeletal remains - Corrected Proof

Sylvain Amory, René Huel, Ana Bilić, Odile Loreille, Thomas J. Parsons — 2011-09-02

Abstract: During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGEN's Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop a more efficient extraction protocol for bone samples that uses significantly less starting material while increasing the success in obtaining DNA results from smaller, more challenging samples. In this study we compared the performance of ICMP's original protocol against an automatable full demineralization approach. In order to provide reliable results and to simulate a wide variety of cases, we analyzed 40 bone samples in a comparative study based on DNA concentrations and quality of resulting STR profiles. The new protocol results in the dissolution of the entire bone powder sample, thus eliminating the possibility that DNA is left behind, locked in remaining solid bone matrix. For the majority of samples tested, the DNA concentrations obtained from half a gram of fully digested bone material were equivalent to or greater than the ones obtained from 2g of partially demineralized bone powder. Furthermore, the full demineralization process significantly increases the proportion of full profiles reflecting the correlation with better DNA quality. This method has been adapted for the QIAcube robotic platform. The performance of this automated full demineralization protocol is similar to the manual version and increases overall lab throughput. It also simplifies the process by eliminating quality control procedures that are advisable in manual procedures, and overall reduces the chance of human error. Finally we described a simple and efficient post-extraction clean-up method that can be applied to DNA extracts obtained from different protocols. This protocol has also been adjusted for the QIAcube platform.

Botanical DNA evidence in criminal cases: Knotgrass (Polygonum aviculare L.) as a model species - Corrected Proof

2011-08-31

Abstract: The possibilities and strategies for using DNA characteristics to link a botanical sample to a specific source plant or location vary with its breeding system. For inbreeding species, which often form small patches of identical genotypes, knotgrass (Polygonum aviculare L.) is a suitable model species because of its (1) occurrence in a wide range of natural environments, (2) abundant presence in pieces of evidence, and (3) ease in molecular processing. The value of knotgrass for forensic casework was demonstrated using data from a homicide case. Using the DNA fingerprinting technique AFLP® we were able to identify the knotgrass population at the crime site as the most likely origin of the botanical evidence. We expect that the development of tailored marker systems for knotgrass and other frequently occurring (model) species will considerably accelerate the use of botanical DNA evidence in criminal cases.

The recombination landscape around forensic STRs: Accurate measurement of genetic distances between syntenic STR pairs using HapMap high density SNP data - Corrected Proof

C. Phillips, D. Ballard, P. Gill, D. Syndercombe Court, Á. Carracedo, M.V. Lareu — 2011-08-29

Abstract: Family studies can be used to measure the genetic distance between same-chromosome (syntenic) STRs in order to detect physical linkage or linkage disequilibrium. However, family studies are expensive and time consuming, in many cases uninformative, and lack a reliable means to infer the phase of the diplotypes obtained. HapMap provides a more comprehensive and fine-scale estimation of recombination rates using high density multi-point SNP data (average inter-SNP distance: 900 nucleotides). Data at this fine scale detects sub-kilobase genetic distances across the whole recombining human genome. We have used the most recent HapMap SNP data release 22 to measure and compare genetic distances, and by inference fine-scale recombination rates, between 29 syntenic STR pairs identified from 39 validated STRs currently available for forensic use. The 39 STRs comprise 23 core loci: SE33, Penta D & E, 13 CODIS and 7 non-CODIS European Standard Set STRs, plus supplementary STRs in the recently released Promega CS-7™ and Qiagen Investigator HDplex™ kits. Also included were D9S1120, a marker we developed for forensic use unique to chromosome 9, and the novel D6S1043 component STR of SinoFiler™ (Applied Biosystems). The data collated provides reliable estimates of recombination rates between each STR pair, that can then be placed into haplotype frequency calculators for short pedigrees with multiple meiotic inputs and which just requires the addition of allele frequencies. This allows all current STR sets or their combinations to be used in supplemented paternity analyses without the need for further adjustment for physical linkage. The detailed analysis of recombination rates made for autosomal forensic STRs was extended to the more than 50 X chromosome STRs established or in development for complex kinship analyses.

Germline mutations of STR-alleles include multi-step mutations as defined by sequencing of repeat and flanking regions - Corrected Proof

2011-08-29

Abstract: Well defined estimates of mutation rates are a prerequisite for the use of short tandem repeat (STR-) loci in relationship testing. We investigated 65 isolated genetic inconsistencies, which were observed within 50,796 allelic transfers at 23 STR-loci (ACTBP2 (SE33), CD4, CSF1PO, F13A1, F13B, FES, FGA, vWA, TH01, TPOX, D2S1338, D3S1358, D5S818, D7S820, D8S1132, D8S1179, D12S391, D13S317, D16S539, D17S976, D18S51, D19S433, D21S11) in Caucasoid families residing in Austria and Switzerland. Sequencing data of repeat and flanking regions and the median of all theoretically possible mutational steps showed valuable information to characterise the mutational events with regard to parental origin, change of repeat number (mutational step size) and direction of mutation (losses and gains of repeats). Apart from predominant single-step mutations including one case with a double genetic inconsistency, two double-step and two apparent four-step mutations could be identified. More losses than gains of repeats and more mutations originating from the paternal than the maternal lineage were observed (31 losses, 22 gains, 12 losses or gains and 47 paternal, 11 maternal mutations and 7 unclear of parental origin). The mutation in the paternal germline was 3.3 times higher than in the maternal germline. The results of our study show, that apart from the vast majority of single-step mutations rare multi-step mutations can be observed. Therefore, the interpretation of mutational events should not rigidly be restricted to the shortest possible mutational step, because rare but true multi-step mutations can easily be overlooked, if haplotype analysis is not possible.

Fourteen short tandem repeat loci Y chromosome haplotypes: Genetic analysis in populations from northern Brazil - Corrected Proof

2011-08-29

Abstract: Fourteen Y-STR loci (DYS458, DYS439, Y-GATA H4, DYS576, DYS447, DYS460, DYS456, YGATA A10, DYS437, DYS449, DYS570, DYS635 or Y-GATA C4, DYS448 and DYS438) were analysed in 873 males from eight northern Brazil populations: Belém (N=400), Santarém (N=69), Manaus (N=75), Macapá (N=65), Palmas (N=30), Rio Branco (N=32), Porto Velho (N=135) and Boa Vista (N=67). A total of 871 different haplotypes were identified, of which 869 were unique. The panel's estimated total haplotype diversity (HD) is 0.9988, and its discrimination capacity (DC) is 0.9980. The lowest estimates of genetic diversity correspond to markers Y-GATA H4 (0.550) and DYS460 (0.581), and the greatest (above 0.700) to markers DYS458, DYS576, DYS447, YS449, DYS570 and DYS635. The genetic parameters obtained were higher for the 14-Y-STR panel than that for the minimum haplotype set (HD=0.9969; DC=0.76) and the parameters were similar to those obtained with the panel of 17 YSTR of YHRD (HD=0.9987; DC=0. 9870). The analysis of molecular variance (AMOVA) indicated that most of the genetic variance is found within populations and a smaller, but significant part, is found among populations (RST=0.027, p value=0.009). The data when compared with those from African, Amerindian and European populations have shown no significant genetic distance between northern Brazil populations and Europeans, but there is a significant genetic distance when compared to Africans and Amerindians. The discrimination capacity of the markers shows a high potential for forensic analysis.

Analysis of a claimed distant relationship in a deficient pedigree using high density SNP data - Corrected Proof

2011-08-25

Abstract: DNA markers are routinely used to reveal both simple and complex family relationships. Likelihood based approaches have been traditionally used to estimate relationships using relatively few unlinked markers. However it is widely recognized that when using such limited numbers of loci distant relationships between two individuals cannot be distinguished from the average level of allele sharing found in random pairwise comparisons in the same population. As a real example, we demonstrate the usefulness of genome-wide SNP genotyping to analyze a claimed second cousin relationship that could not be resolved using standard forensic markers, confirming theoretical expectations for very distant relationships. Genome profiles derived from Affymetrix 6.0 SNP arrays obtained from the claimed second cousins were compared to profiles obtained from unrelated individuals and simulated data. Significance of the high estimated probabilities in favor of the second cousin relationship hypothesis was proved from the results obtained with both real and simulated unrelated pairs. As a final cautionary note, it is important to consider that successful identification of the claimed distant relationship reported here is largely due to a well-founded hypothesis being compared to the alternative hypothesis of the claimants being unrelated, but where there are several possible alternative hypotheses, the approach we outline here can yield false indications of unfounded alternative relationships.

Composite profiles in DNA analysis - Corrected Proof

Jo-Anne Bright, Peter Gill, John Buckleton — 2011-08-16

Abstract: Composite profiles are created by combining DNA profiling information from replicate profiles derived from the same DNA extract. In this paper we have shown that, provided the probability of drop-in is low or nil, the creation of composite profiles is an acceptable approximation to a Bayesian approach, as long as simple samples are analysed.

Autosomal STR allele frequencies for the CODIS system from a large random population sample in Chile - Corrected Proof

Ismael A. Vergara, Pamela Villouta, Sandra Herrera, Francisco Melo — 2011-08-03

Abstract: The thirteen autosomal STR loci of the CODIS system were typed from DNA of 732 unrelated male individuals sampled from different locations in Chile. This is the first report of allele frequencies for the thirteen STRs loci defined in the CODIS system from the Chilean population.

Pentaplex typing of new European Standard Set (ESS) STR loci in Indian population - Corrected Proof

D. Kalpana, Tania Ghosh, Sanjukta Mukerjee, Meeta Mukherjee, Anil Kumar Sharma — 2011-08-03

Autosomal short tandem repeats (STRs) are effective tools for human identification. The amplicon size of the conventional STR markers used for the forensic casework ranges from 75 to 450bp. Due to fragmented DNA template or PCR inhibitors present in the samples, often loss of signal or partial profiles are observed for the conventional STR markers. Previous studies have demonstrated utility of size-reduced amplicons (miniSTRs) for the analysis of highly degraded DNA by choosing primers close to the repeat regions . European DNA profiling group (EDNAP) recommended five new European Standard Set (ESS) markers D10S1248, D2S441, D22S1045, D1S1656 and D12S391 for the forensic casework and database . Evaluating polymorphisms of these miniSTR markers in the Indian population would enable their efficient use for the forensic casework. Hence, a pentaplex for the simultaneous analysis of these five miniSTR loci was optimized and their polymorphisms was studied in 11 endogamous populations of India belonging to four linguistic (Indo-European, Dravidian, Tibeto-Burman and Austro-Asiatic), six geographic (north, east, central, west, south and north-east) and two socio-cultural (caste and tribe) groups.

Updated Brazilian STR allele frequency data using over 100,000 individuals: An analysis of CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, Penta D, Penta E, TH01, TPOX and vWA loci - Corrected Proof

2011-08-03

Abstract: The Brazilian population is one of the most heterogeneous populations of the world, formed mainly by an admixture of European, African and Native American populations. Brazil is the fifth largest country in the world (8,511,960km2), being divided into five geographical regions. This study provides population genetic data of up to 137,161 unrelated individuals representing the entire Brazilian territory. Allelic frequencies and other population data analysis are reported for the 15 autosomal STR loci included in the PowerPlex®16 kit (Promega Corporation, Madison, WI, USA). In order to guarantee that individuals were not related, we have considered only F1 data from couples undergoing paternity testing. The number of individuals genotyped for each locus was: CSF1PO (113,526); D3S1358 (135,133); D5S818 (135,181); D7S820 (137,136); D8S1179 (134,211); D13S317 (137,161); D16S539 (136,942); D18S51 (136,739); D21S11 (130,014); FGA (135,839); Penta D (110,333); Penta E (128,055); TH01 (112,695); TPOX (123,102); vWA (127,415). Allele sizes ranged from 1 to 48.2. Statistic parameters (PD, PIC and Ho; considering values ≥0.75) suggest that markers D13S317, D16S539, D18S51, D21S11, D7S820, D8S1179, Penta D, Penta E, TH01, FGA and vWA were more informative for genetic identification purposes in the Brazilian population.

DNA microarray as a tool in establishing genetic relatedness—Current status and future prospects - Corrected Proof

2011-08-03

Abstract: In the past decades, microarray technology has definitely put an edge to the field of genetic research. Our aim was to determine whether single nucleotide polymorphism (SNP) microarrays could be used as a tool in establishing genetic relationships where current molecular genetic methods are not sufficient. We used the Genechip, Affymetrix GenomeWide SNP Array 6.0, which detects more than 900,000 SNP markers dispersed throughout the human genome. The intention was to find a good selection of SNP markers that could be used for statistical evaluation of relatedness in a forensic setting. We conducted pairwise comparisons in the R-package FEST as well as pedigree comparisons in Merlin. Our methods were applied on two separate families, where relationships as distant as 3rd cousins were known. In addition, a question about a possible common ancestry between the two families was tested. Relationships as distant as 2nd cousins could be readily distinguished both from unrelated and other, genetically, closer relationships. This was achieved with a selection of 5774 markers, where each pair of markers was separated by a genetic distance of at least 0.5cM (centiMorgan). When considering 3rd cousins, and more distant relationships, the number of markers needs to be extended, consequently decreasing the genetic distance between the markers. However, inclusion of a too large number of markers presents new challenges and our results imply that the use of too dense sets of markers always yields the highest probability for the genetically closest relationship hypothesis. Simulations confirm that this is most probably caused by the fact that the computational model assumes linkage equilibrium between markers, a problem that will be further evaluated. Our results do however suggest that SNP-data derived from microarrays are well suited for kinship determination provided linkage disequilibrium is properly accounted for.