Forensic Science International: Genetics

Editorial Board

2010-07-01

A universal strategy to interpret DNA profiles that does not require a definition of low-copy-number

Peter Gill, John Buckleton — 2009-10-12

Abstract: In this paper we critically examine the causes of the underlying confusion that relates to the issue of low-template (LT) DNA profile interpretation. Firstly, there is much difficulty in attempting to distinguish between LT-DNA vs. conventional DNA because there is no discrete ‘cut-off’ point that can be reasonably defined or evaluated. LT-DNA is loosely characterised by drop-out (where alleles may be missing) and drop-in (where additional alleles may be present). We have previously described probabilistic methods that can be used to incorporate these phenomena using likelihood ratio (LR) principles. This is preferred to the random man not excluded (RMNE) method, because we cannot identify a coherent way forward within the restrictions provided by this framework. Most LT-DNA profiles are interpreted using a ‘consensus’ profile method, we called this the ‘biological model’, where only those alleles that are duplicated in consecutive tests are reported. We recognise that there is an increased need for probabilistic models to take precedence over the biological model. These models are required for all kinds of DNA profiles, not just those that are believed to be low-template. We also recognise that there is a need for education and training if the methods we recommend are to be widely introduced.

Shotgun metagenomics of biological stains using ultra-deep DNA sequencing

B. Brenig, J. Beck, E. Schütz — 2009-11-02

Abstract: A detailed molecular analysis of blood or other biological stains at a crime scene is often hampered by the low quantity and quality of the extractable DNA. However, the determination of the origin and composition of a stain is in most cases a prerequisite for the final elucidation of a criminal case. Standard methodologies, e.g. amplification of DNA followed by microsatellite typing or mitochondrial DNA sequencing, are often not sensitive enough to result in sufficient and conclusive data. We have applied ultra-deep DNA sequencing using the 454 pyrosequencing technology on a whole genome amplified (WGA) environmental biological stain, which was analysed unsuccessfully with standard methodologies following WGA. With the combination of WGA and 454 pyrosequencing, however, we were able to generate 7242 single sequences with an average length of 195bp. A total of 1,441,971bp DNA sequences were generated and compared with public DNA sequence databases. Using RepeatMasker and basic logical alignment search tool (BLAST) searches against known microbial and mammalian genomes it was possible to determine the metagenomic composition of the stain, i.e. 4.2% bacterial DNA, 0.3% viral DNA, 2.7% fungal DNA, 10.3% mammalian repetitive DNA, 0.9% porcine DNA, 0.13% human DNA and 81.5% DNA of unknown origin. Our data demonstrate that 454 pyrosequencing has the potential to become a powerful tool not only in basic research but also in the metagenomic analysis of biological trace materials for forensic genetics.

Use of DNA profiles for investigation using a simulated national DNA database: Part I. Partial SGM Plus® profiles

T. Hicks, F. Taroni, J. Curran, J. Buckleton, O. Ribaux, V. Castella — 2009-11-04

Abstract: In traditional criminal investigation, uncertainties are often dealt with using a combination of common sense, practical considerations and experience, but rarely with tailored statistical models. For example, in some countries, in order to search for a given profile in the national DNA database, it must have allelic information for six or more of the ten SGM Plus® loci for a simple trace. If the profile does not have this amount of information then it cannot be searched in the national DNA database (NDNAD). This requirement (of a result at six or more loci) is not based on a statistical approach, but rather on the feeling that six or more would be sufficient. A statistical approach, however, could be more rigorous and objective and would take into consideration factors such as the probability of adventitious matches relative to the actual database size and/or investigator's requirements in a sensible way. Therefore, this research was undertaken to establish scientific foundations pertaining to the use of partial SGM Plus® loci profiles (or similar) for investigation.

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

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

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

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

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

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

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

2009-11-09

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

Nilotes from Karamoja, Uganda: Haplotype data defined by 17 Y-chromosome STRs

2009-08-03

Abstract: In this work 118 Nilote male samples were genotyped from Karamoja region, in Northeast Uganda, through 17 Y-chromosomal short tandem repeats (STRs)—DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and GATA H4.1. A total of 94 different haplotypes were found, where 19 were shared by at least two individuals, and haplotype diversity amounted to 0.9958±0.0017. When considering only the nine Y-STRs included in the minimal haplotype (YHRD) the haplotype diversity decreased to 0.9807±0.0048, a similar value to those found in other African populations such as Mozambique, Angola and Guinea-Bissau. Population comparisons were performed between our sample and nine other African populations. Significant Rst genetic distances were obtained between the Nilote population from Karamoja and all African populations used for comparison, except Xhosa sample from South Africa. In the multidimensional scaling (MDS) plot, the Karamoja sample is well separated from all other populations, standing between the Ethiopia and the Bantu samples, although closer to this last group.

Mitochondrial DNA HV1 and HV2 variation in Danes

Martin Mikkelsen, Erik Sørensen, Erik Michael Rasmussen, Niels Morling — 2009-08-27

Abstract: Sequences from the two hypervariable regions (HV1 and HV2) of the control region of the mitochondrial DNA were obtained from a total of 201 Danes and five individuals who later were recognized to be of non-West European origin. Two fractions of each region were amplified separately and sequenced at least twice. The samples were sequenced using flanking sequencing primes and both terminator and primer chemistry. Sequence evaluation was performed by two independent scientists. The haplogroup distribution of the samples resembles that found in other European population. All the sequences have been made available in the EMPOP database.

Genetic population data of 12 Y-chromosome STRs loci in Mendoza population (Argentina)

Miguel Marino, Sandra Furfuro — 2009-09-11

Abstract: The 12 Y-chromosome STRs included in the “minimal haplotype” plus “SWGDAM core set” (DYS438 and DYS439) and DYS437, were typed in 224 unrelated males from Mendoza province, Argentina. The amplifications were performed using PowerPlex®Y Systems (Promega Corp.) and AmpFISTR YFiler Amplification Kit (AB Applied Biosystems). A total of 203 different haplotypes were identified, of which 186 were unique and 17 were found in two or more individuals. Allele frequency and haplotype diversity (HD) were calculated.

An X-chromosome pentaplex in two linkage groups: Haplotype data in Alagoas and Rio de Janeiro populations from Brazil

2009-09-02

Abstract: Genotype data were obtained for five X-Chr STRs (DXS10079, DXS10074, DXS10075, DXS7424 and DXS101) in two different populations from Brazil, namely Alagoas and Rio de Janeiro. Observed genotype distributions in female samples for each locus do not show deviations from Hardy–Weinberg equilibrium expectations. Gametic association was tested for all pairs of loci in male samples. Significant association values were found between all pairs including DXS10079, DXS10074 and DXS10075, as well as between DXS7424 and DXS101, proving that these two groups of markers must be treated as haplotypes. No significant association could be found between markers from the two groups (DXS10079, DXS10074 or DXS10075 vs. DXS7424 or DXS101), although distances between them varied from 24 to 25cM. When comparing haplotype frequencies in Alagoas, Rio de Janeiro, Germany and Ghana, significant differences were found between the Brazilian and the Germany sample and that from Ghana. Nevertheless, no significant differences were found between Alagoas and Rio de Janeiro, as well as between these two populations and Germany. The combined power of discrimination in males and females were high in both Brazilian populations (≥0.9996 and ≥0.9999998, respectively), showing the utility of these markers for human identification and paternity testing.

Icelandic population data for the STR loci in the AMPFlSTR®SGM Plus™ system and the PowerPlex® Y-system

Rune Andreassen, Luísa Pereira, Berit M. Dupuy, Bente Mevaag — 2009-09-22

Abstract: We present allele frequencies and statistical parameters of forensic interest for 10 autosomal STR loci and 12 Y-STR loci obtained from an Icelandic population sample. The testing of the STR loci in the AmpFlSTR®SGM Plus™ kit in 151 unrelated individuals showed heterozygosity frequencies ranging from 0.775 (vWA) to 0.874 (D2S1338). A significant deviation from Hardy–Weinberg equilibrium was observed in vWA, but it was not statistically significant after application of Bonferroni correction. The exact test of differentiation analysis revealed one significant departure from differentiation out of 45 pairwise comparisons, but the departure was not significant after Bonferroni's correction. Seventy-five different haplotypes were observed in the 100 male samples analysed for the twelve Y-STRs included in the PowerPlex® Y-system. No haplotype was observed more than four times. Pairwise comparisons for genetic distances based on the minimal haplotype diversity showed Iceland to be closer to Norway and Denmark than to Sweden, UK, Ireland and Greenland. As expected, the higher percentage of variation was observed within than among populations (90.40% versus 9.60%, respectively, for RST).

Genetic polymorphisms of eight X-chromosomal STR loci in the population of Japanese

Jian Tie, Seisaku Uchigasaki, Shigemi Oshida — 2009-09-27

Abstract: The genetic polymorphisms of eight X-chromosomal short tandem repeats (STR) DXS10135, DXS8378, DXS7132, DXS10074, HPRTB, DXS10101, DXS10134 and DXS7423 were analyzed in a sample of 492 unrelated males (283) and females (209) from the Japanese population. Multiplex PCR amplification was performed using the Mentype® Argus X-8 PCR amplification kit. The haplotype frequencies within the four linkage groups were studied for the 283 examined Japanese males. Allele frequencies of eight X-STR loci were calculated separately for males and females, and exact tests demonstrated no significant deviations from Hardy–Weinberg equilibrium. Several microvariant and rare alleles were observed, and forensic efficiency parameters were calculated. The combined powers of discrimination of the loci in men and women were 0.999995 and 0.9999999999988, respectively.

Comparative allele distribution at 16 STR loci between the Andean and coastal population from Peru

2009-10-05

Abstract: In the present study, we analysed the allelic distribution of 16 autosomal short tandem repeats (STRs) performed on unrelated individuals from seven different Peruvian cities, three highland Andean cities and four coastal ones. The loci investigated were F13A01, FESFPS, vWA, CSF1PO, TPOX, TH01, D16S539, D7S820, D13S317, D5S818, D19S253, F13B, D21S11, LPL and D8S1179 y D3S1358. The allele frequency, statistical parameters, Hardy–Weinberg equilibrium and population pair comparison across all loci were determinate. The combined matching probability for the 16 loci was 5.41136×10−15 and the combined probability of exclusion (PE) was 0.999998307. The results showed new local databases for the evaluation of Andean and coastal Peruvian populations in human identity testing.

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

2009-11-09

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

Genetic polymorphisms of 15 autosomal STR loci in three isolated tribal populations of Bangladesh

2009-09-04

We determined the allele frequencies for the 15 autosomal STR loci included in AmpFlSTR® Identifiler™ kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) in a total of 279 unrelated individuals belonging to 3 major tribal groups: Chakma (n=113), Marma (n=83) and Tripura (n=83) residing in Chittagong Hill Tracts (CHT) of Bangladesh.

Genetic data for D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045 miniSTR loci from Libya

2009-09-07

We determined the allelic frequencies for six miniSTR loci D1S1677, D2S441, D4S2364 (miniplex NC02) and D10S1248, D14S1434, D22S1045 (miniplex NC01) in a sample of 124 unrelated Libyans. Libya, a Northern African country, was first inhabited by Berbers, followed by Phoenicians, Greeks, Romans, Arabs and Ottomans. Libya became independent in 1951 after a brief period as Italian colony.

Manufacturer contamination of disposable plastic-ware and other reagents—An agreed position statement by ENFSI, SWGDAM and BSAG

Peter Gill, Diane Rowlands, Gillian Tully, Ingo Bastisch, Ted Staples, Pam Scott — 2009-09-07

Recently, a number of laboratories in Europe, the United States and New Zealand have obtained results with STR analysis that indicated apparent links from unconnected cases that had been processed in geographically different areas in different laboratories. Further investigation suggested that the DNA had been introduced during the process of manufacturing consumables and products used in the DNA analysis process. This was confirmed after comparing DNA profiles obtained from staff working in the plastic-ware factory and DNA profiles derived during the course of casework in the United Kingdom . Contamination of disposable plastic-ware at a manufacturing source was originally suggested in relation to the analysis of mitochondrial DNA. SWGDAM recommends that this position statement be limited to nuclear DNA.

Admixture estimates and statistical parameters of forensic importance based on PowerPlex® 16 system in Mexican-Mestizos from the States of Guanajuato (Center) and Veracruz (East)

2009-10-05

We determined the allele frequencies for 15 STR loci included in PowerPlex® 16 System PCR amplification kit (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S539, D18S51, D21S11, Penta D, and Penta E) in 534 unrelated Mexican individuals, including 334 and 200 individuals from the states of Guanajuato (Center), and Veracruz (East), respectively. Sampled individuals were self-denominated Mestizos because anyone belonged to some specific Mexican ethnic group. Mexican-Mestizos are the result of admixture, principally between Native Americans and Spaniards, during and after the Conquest of the New World; using Spanish language as a selection criterion, they constitute ca 93% of the present-day Mexican population . DNA was extracted from dried blood spotted on FTA paper or buccal swabs by Chelex® 100. Amplifications were carried out in 8μL volume containing 1–2ng of DNA template, following the manufacturer's recommendations. Amplified products were analyzed by capillary electrophoresis using the ABI Prism™ 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Results were analyzed using the Genescan 3.1 and Genotyper software's. Genotypes were designed by comparison with allelic ladders provided with the kit. Allele frequencies and statistical parameters of forensic importance were computed with the software PowerStats . Hardy–Weinberg expectations (HWE) for each and combined loci were calculated by exact tests, and pairwise comparisons (FST p-values) were performed with the software Arlequin 3.0 . Bonferroni correction, according to the loci number, was implemented to evaluate p-values of HWE test (p<0.0033). In order to characterize the ancestral genetic pool within Mexican-Mestizos (admixture proportions), we implemented a supervised clustering method with the program STRUCTURE driving these populations to fit into one cluster . We utilized a 10,000-iteration burn-in period followed by 10,000 iterations; with the 25 replicates obtained, we used the Cluster matching and permutation program (CLUMPP) to identify the best correspondence among runs . For this purpose, CODIS-STR population data from 114 Andalusians (Sothern Spain) , 130 Fangs (Guinea Ecuatorial) , and 161 Purépechas (Western Mexico) were employed to represent the African, European, and Amerindian ancestral components, respectively.

Graydon et al. provide no new evidence that forensic STR loci are functional

Kirk E. Lohmueller — 2009-10-05

Abstract: Graydon et al. recently reported “there is a correspondence between STR polymorphism and physical traits, suggesting that STRs may not be just genetic ‘junk’, but may play a role in influencing phenotypic differences between people.”. We argue that this conclusion is unwarranted in light of past and present work in human population genetics. Instead, Graydon et al.’s results can be explained solely by population history.