Validation of a single nucleotide polymorphism (SNP) typing assay with 49 SNPs for forensic genetic testing in a laboratory accredited according to the ISO 17025 standard

A multiplex assay with 49 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was validated for forensic genetic casework and accredited according to the ISO 17025 standard. The multiplex assay was based on the SNPforID 52plex SNP assay [J.J. Sanchez, C. Phillips, C. Børsting, K. Balogh, M. Bogus, M. Fondevila, C.D. Harrison, E. Musgrave-Brown, A. Salas, D. Syndercombe-Court, P.M. Schneider, A. Carracedo, N. Morling, A multiplex assay with 52 single nucleotide polymorphisms for human identification, Electrophoresis 27 (2006) 1713–1724], where 52 fragments were amplified in one PCR reaction. The SNPs were analysed by single base extension (SBE) and capillary electrophoresis. Twenty-three of the original SBE primers were altered to improve the overall robustness of the assay and to simplify the analysis of the SBE results. A total of 216 samples from 50 paternity cases and 33 twin cases were typed at least twice for the 49 SNPs. All electropherograms were analysed independently by two expert analysts prior to approval. Based on these results, detailed guidelines for analysis of the SBE products were developed. With these guidelines, the peak height ratio of a heterozygous allele call or the signal to noise ratio of a homozygous allele call is compared with previously obtained ratios. A laboratory protocol for analysis of SBE products was developed where allele calls with unusual ratios were highlighted to facilitate the analysis of difficult allele calls. The guidelines for allele calling proved to be highly efficient for the detection of DNA mixtures and contaminated DNA preparations. DNA from two individuals was mixed in seven different ratios ranging from 1:1 to 1:10; all mixtures were easily identified as mixtures.     

Performance of the SNPforID 52 SNP-plex assay in paternity testing

The performance of a multiplex assay with 52 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was tested on 124 mother–child–father trios. The typical paternity indices (PIs) were 10⁵–10⁶ for the trios and 10³–10⁴ for the child–father duos. Using the SNP profiles from the randomly selected trios and 700 previously typed individuals, a total of 83,096 comparisons between mother, child and an unrelated man were performed. On average, 9–10 mismatches per comparison were detected. Four mismatches were genetic inconsistencies and 5–6 mismatches were opposite homozygosities. In only two of the 83,096 comparisons did an unrelated man match perfectly to a mother–child duo, and in both cases the PI of the true father was much higher than the PI of the unrelated man. The trios were also typed for 15 short tandem repeats (STRs) and seven variable number of tandem repeats (VNTRs). The typical PIs based on 15 STRs or seven VNTRs were 5–50 times higher than the typical PIs based on 52 SNPs. Six mutations in tandem repeats were detected among the randomly selected trios. In contrast, there was not found any mutations in the SNP loci. The results showed that the 52 SNP-plex assay is a very useful alternative to currently used methods in relationship testing. The usefulness of SNP markers with low mutation rates in paternity and immigration casework is discussed.     

Population genetics of Y-chromosome STRs in a population of Northern Greeks

Seventeen Y STR loci were typed in a population sample of 191 unrelated male individuals from Northern Greece. Haplotypes are presented for the following loci: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448. The overall haplotype diversity was 0.9992. This database study provides significant additional information for the application of Y-chromosomal STRs to forensic identification efforts in Greece by nearly doubling both the number of individuals and the number of Y-loci typed from Greek populations. These samples have been previously typed for autosomal STRs [L. Kovatsi, T.J. Parsons, R.S. Just, J.A. Irwin, Genetic variation for 15 autosomal STR loci (PowerPlex 16) in a population sample from northern Greece, Forensic Sci. Int. 159 (2006) 61–63] and the mitochondrial DNA control region [J. Irwin, J. Saunier, K. Strouss, C. Paintner, T. Diegoli, K. Sturk, L. Kovatsi, A. Brandstatter, M.A. Cariolou, W. Parson, T.J. Parsons, Mitochondrial control region sequences from northern Greece and Greek Cypriots, Int. J. Legal Med. 122 (2008) 87–89].     

Autosomal SNPs study of a population sample from North of Portugal and a sample of immigrants from the Eastern Europe living in Portugal

The use of autosomal single nucleotide polymorphisms (SNPs) for forensic research has been widely discussed in recent years, mainly because SNPs have important advantages compared to short tandem repeats (STRs).In this study a total of 131 non related individuals from the North of Portugal and 85 immigrant individuals from the Eastern Europe, mainly Ukrainians, equally non related and residing in Portugal, were typed for 52 loci included in the in the SNP for ID 52plex with the SNaPshot™ assay.     

A novel dimorphism in the human SRY gene: usefulness in human migration studies

A nucleotide polymorphism of C or T was detected at position 465 in the sex-determining region Y (SRY) gene. To evaluate the utility of this dimorphism in human population studies, the frequency and the frequency of the haplotype combined with the two polymorphic loci YAP and M9 were examined in a total of 130 unrelated Japanese and 130 unrelated German males. The T nucleotide was found in 24.6% (32/130) of the Japanese but not in any of the 130 German males. Accordingly, four of the eight possible combination haplotypes of SRY/YAP/M9 were identified in the Japanese population, but one of the four haplotypes comprising SRY(T) was absent in the German samples. This suggests that the C to T transition may be more recent than the YAP insertion or the M9 transversion and the change might have occurred in an ancestral Asian population. These results imply that the dimorphism at the SRY gene is one of the Y-linked markers useful for human population studies and also for ethnic identification of forensic samples. Received: 9 March 1999 / Accepted: 9 September 1999     

Autosomal SNPs study of a population sample from Southern Portugal and from a sample of immigrants from Guinea-Bissau residing in Portugal

In recent years, autosomal single nucleotide polymorphisms (SNPs) have been comprehensively investigated in forensic research due to their usefulness in certain circumstances in complementing short tandem repeats (STRs) analysis, or even for use on their own when analysis of STRs fails. However, as with STRs, in order to properly use SNP markers in forensic casuistic we need to understand the population and forensic parameters in question. As a result of Portugal’s colonial history during the time of empire, and the subsequent process of decolonization, some African individuals migrated to Portugal, giving rise to large African and African-descendent communities. One of these groups is the community originating from Guinea-Bissau, that in 2014, was enumerated to consist of more than 17,700 individuals with official residency status, more than the third major city of Guinea-Bissau.In order to study the population and forensic parameters mentioned above for the two populations important to our casuistic, a total of 142 unrelated individuals from the South of Portugal and 90 immigrants from Guinea-Bissau (equally non related and all residing in Portugal) were typed with SNaPshot™ assay for all 52 loci included in the SNPforID 52plex.     

Population data for 22 autosomal STR loci in the Uygur ethnic minority

Allele frequencies and forensically relevant population statistics parameter of 22 short tandem repeat (STR) loci were determined from 525 unrelated Uygur ethnic individuals. The samples were amplified with Microreader™ 23sp ID system. No significant deviation from Hardy–Weinberg equilibrium was detected, except for loci D7S3048, D21S1270, and D13S325. Investigated loci are very discriminating in Uygur ethnic population, with a combined discrimination power of 0.999999999999999999999999999920743. Furthermore, a comparison with previously published frequency data from Han population is presented.

     

Typing of Amerindian Kichwas and Mestizos from Ecuador with the SNPforID multiplex

A total of 119 unrelated individuals from two of the major ethnic groups in Ecuador were typed for 49 of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex using the SNapShot(?) assay. Of the above, 42 samples originated from Mestizos (an admixed population) and the remaining 77 were from Native Amerindian Kichwas. We obtained full SNP profiles in all individuals and concordance of duplicated analyses. No deviation from Hardy-Weinberg equilibrium (HWE) was observed for any SNP in the Mestizo and Kichwa populations and only one and four pairs of loci, respectively showed significant linkage disequilibrium. A relatively low genetic diversity and global positive F(IS) value was observed in Kichwas. A statistically significant global F(ST) value was obtained when the two Ecuadorian populations were compared with populations in Spain, Portugal, Argentina, Denmark, Greenland, China, Somalia and Mozambique. All pairwise F(ST) values were statistically significant. A multi-dimensional scaling based on pairwise F(ST) values showed that the Kichwa population differed from all other populations investigated and that the Mestizos had an intermediate position between Kichwas and Europeans. An admixture analysis indicated that the greater contributor to the Mestizo population was the Kichwas (71.2%) compared to the European contribution. The combined mean match probability and mean paternity exclusion probability were 3.3 × 10(-17) and 0.998, respectively, for the Mestizo population and 3.3 × 10(-14) and 0.993, respectively, for the Kichwa population.     

Nucleotide sequence analysis of the hypervariable region III of mitochondrial DNA in Thais

This study analyzed the nucleotide sequences of the hypervariable region III (HVRIII) of mitochondrial DNA in Thai individuals. Buccal swab samples were randomly obtained from 100 healthy, unrelated, adult (18–60 years old), volunteer donors living in Thailand. Eighteen different haplotypes were found, of which 11 haplotypes were unique. The most frequent haplotypes observed were 522D-523D. Nucleotide transition from Thymine (T) to Cytosine (C) at position 489 (43%) was the most frequent substitution. Nucleotide transversions were also observed at position 433 (Adenine (A) to C, 1%) and position 499 (Guanine (G) to C, 1%). Fifty-three samples presented nucleotide insertion and deletion of C and A (CA) at position 514–523. Insertion of 1AC (3%) and 2AC (2%) were observed. Deletion of 1CA (53%) and 2CA (2%) at position 514–523 were revealed. The deletion of T at position 459 was observed. The haplotype diversity, random match probability, and discrimination power were calculated to be 0.7770, 0.2308, and 0.7692, respectively.     

Mitochondrial control region sequences from an African American population sample

Entire mitochondrial control region data were generated for 248 African American individuals, which had been previously typed for 15 autosomal STRs [J.M. Butler, R. Schoske, P.M. Vallone, J.W. Redman, M.C. Kline, Allele frequencies for 15 autosomal STR loci on U.S. Caucasian, African American, and Hispanic populations, J. Forensic Sci. 48 (2003) 908–911].     

Allele frequencies of six miniSTR loci in the population of Northern Portugal

A possible approach to try to recover information from degraded DNA is to reduce the size of the PCR products by designing primers that bind as close as possible to the STR repeat region, known as miniSTRs. Allele frequencies and forensic parameters for the six miniSTRs loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045 were investigated in a sample group consisting of 228 anonymous apparently healthy unrelated individuals living in North of Portugal. The results show that all loci were in Hardy–Weinberg equilibrium. The combined power of discrimination and power of exclusion for the six loci were 0.99999 and 0.9789, respectively. All but one (D4S2364) loci showed a moderate degree of polymorphism (observed heterozygosity >0.6). The allele sizes ranged between 66 and 118 bp in our population, which is beneficial for typing degraded samples than those of a commercial STR kit.     

Genetic polymorphism of 15 STR loci in central western Colombia

Fifteen autosomal short tandem repeat (STR) loci were analyzed using the AmpFISTR Identifiler PCR Amplification Kit in 1944 samples of healthy unrelated individuals, born in the regions of Caldas, Quindio and Risaralda, Colombia. Allele frequencies and statistical parameters of forensic interest are presented for all markers (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA). All loci of the pooled data were in agreement with Hardy–Weinberg equilibrium, after Bonferroni correction. Comparisons with neighboring Latin American populations and Andalucia (Spain) revealed significant differences in allele distributions. Genetic data presented in this study can be used for routine forensic applications in the region.     

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

Allele frequencies of five miniSTRs loci (D1S1656, D2S441, D12S391, D10S1248 and D22S1045) included in the new European Standard Set (ESS) were calculated from a sample of 150 unrelated individuals from Apulia, a Region of Southern Italy. Two different PCR Amplification Kits were used, in order to evaluate the concordance of the genotypes. The results obtained with the two kits showed no differences in all genotype profiles. No deviation from Hardy–Weinberg expectations was detected at either locus. Moreover genetic analysis using Fst estimation showed no evidence for differentiation at the five new loci between Apulia and Italian populations. The high levels of polymorphisms of the analyzed markers in the Apulian population allow to confirm that these markers are useful tools in paternity and forensic analysis from degraded DNA samples.     

Population genetics of six miniSTR loci (D1S1677, D2S441, D4S2364, D10S1248, D14S1434, D22S1045) in a Polish population

Allele frequency data and forensic efficiency parameters for six miniSTR loci: D1S1677, D2S441, D4S2364 (NC01), D10S1248, D14S1434, D22S1045 (NC02) were estimated from a sample of 116 unrelated individuals from Poland. No significant deviations from Hardy–Weinberg equilibrium expectations were detected. The combined power of discrimination for the six studied loci was 0.999995383.     

Allele frequency data for 17 short tandem repeats in a Czech population sample

Allele frequencies for 17 short tandem repeats (STRs) autosomal loci (D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, PentaD, PentaE, TH01, TPOX, vWA) were studied in an extensive sample (max. N = 1411) of unrelated individuals originating from the Czech Republic. Population and forensic parameters were estimated. Except for FGA and Penta E loci, no deviations from the Hardy–Weinberg equilibrium were detected. A comparative analysis with published data revealed significant differences in allele frequencies for some loci from the Polish population and three Hungarian populations (Ashkenazim population and Romany populations from Debrecen and Baranya County, respectively). A combination of these 17 STR loci provides a powerful tool for forensic identification in the native Czech population.     

Linkage disequilibrium analysis of D12S391 and vWA in U.S. population and paternity samples

Recently, the European Network of Forensic Science Institutes voted to adopt five additional STR loci (D12S391, D1S1656, D2S441, D10S1248, and D22S1045) to their existing European Standard Set of seven STRs (TH01, vWA, FGA, D8S1179, D18S51, D21S11, and D3S1358). The D12S391 and vWA loci are located 6.3megabases (Mb) apart on chromosome 12. Ideally for use in forensic analyses, genetic markers on the same chromosome should be more than 50Mb in physical distance in order to ensure full recombination and thus independent inheritance. The purpose of this study was to evaluate if the closely located D12S391 and vWA loci are independent and, consequently, if these loci can be included in the product rule calculation for forensic and kinship analyses. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium between the D12S391 and vWA loci were tested using n=654 unrelated U.S. African American, Caucasian, and Hispanic samples, and n=764 father/son paternity samples. In the unrelated U.S. population samples, no significant departures from HWE were detected for D12S391 or vWA. No significant evidence of linkage disequilibrium was observed between the loci in the population samples. However, significant linkage disequilibrium was detected in U.S. African American, Caucasian, and Asian father/son samples with phased genotypes. No significant linkage disequilibrium was detected for U.S. Hispanic paternity samples. The use of phased father/son pairs allowed for robust detection of linkage disequilibrium between D12S391 and vWA. In unrelated population samples, linkage disequilibrium is present but more difficult to detect due to the large number of possible haplotype combinations and unknown allelic phase. For casework analyses that involve unrelated or related individuals, the single-locus genotype probabilities for D12S391 and vWA should not be multiplied to determine the match probability of an autosomal STR profile. Since the D12S391 and vWA loci are not independent, it is recommended that the observed combination of alleles at D12S391 and vWA should be treated as a non-independent diplotype for profile probability calculations. The observed haplotype frequencies for U.S. African American, Caucasian, Hispanic, and Asian populations are provided for match probability calculations.     

Forensic validation of the SNPforID 52-plex assay

The advantages of single nucleotide polymorphism (SNP) typing in forensic genetics are well known and include a wider choice of high-throughput typing platforms, lower mutation rates, and improved analysis of degraded samples. However, if SNPs are to become a realistic supplement to current short tandem repeat (STR) typing methods, they must be shown to successfully and reliably analyse the challenging samples commonly encountered in casework situations.The European SNPforID consortium, supported by the EU GROWTH programme, has developed a multiplex of 52 SNPs for forensic analysis, with the amplification of all 52 loci in a single reaction followed by two single base extension (SBE) reactions which are detected with capillary electrophoresis.In order to validate this assay, a variety of DNA extracts were chosen to represent problems such as low copy number and degradation that are commonly seen in forensic casework. A total of 40 extracts were used in the study, each of which was sent to two of the five participating laboratories for typing in duplicate or triplicate. Laboratories were instructed to carry out their analyses as if they were dealing with normal casework samples. Results were reported back to the coordinating laboratory and compared with those obtained from traditional STR typing of the same extracts using Powerplex^® 16 (Promega). These results indicate that, although the ability to successfully type good quality, low copy number extracts is lower, the 52-plex SNP assay performed better than STR typing on degraded samples, and also on samples that were both degraded and of limited quantity, suggesting that SNP analysis can provide advantages over STR analysis in forensically relevant circumstances. However, there were also additional problems arising from contamination and primer quality issues and these are discussed.     

Application of the new insertion–deletion polymorphism kit for forensic identification and parentage testing on the Czech population

Insertion-deletion polymorphisms (INDELs) are diallelic markers derived from a single mutation event. Their low mutation frequency makes them suitable for forensic and parentage testing. The examination of INDELs thus combines advantages of both short tandem repeats (STR) and single nucleotide polymorphisms (SNP). This type of polymorphisms may be examined using as small amplicon size as SNP (about 100 bp) but could be analyzed by techniques used for routine STR analysis. For our population study, we genotyped 55 unrelated Czech individuals. We also genotyped 11 trios to analyze DIPplex Kit (QIAGEN, Germany) suitability for parentage testing. DIPplex Kit contains 30 diallelic autosomal markers. INDELs in DIPplex Kit were tested with linkage disequilibrium test, which showed that they could be treated as independent markers. All 30 loci fulfill Hardy–Weinberg equilibrium. There were several significant differences between Czech and African populations, but no significant ones within European population. Probability of a match in the Czech population was 1 in 6.8?×?10¹²; combined power of discrimination was 99.9999999999%. Average paternity index was 1.13–1.77 for each locus; combined paternity index reached about 27,000 for a set of 30 loci. We can conclude that DIPplex kit is useful as an additional panel of markers in paternity cases when mutations in STR polymorphisms are present. For application on degraded or inhibited samples, further optimization of buffer and primer concentrations is needed.     

Corrigendum to “Linkage disequilibrium analysis of D12S391 and vWA in U.S. population and paternity samples” [Forensic Sci. Int.: Genet. (in press), doi:10.1016/j.fsigen.2010.09.003]

Recently, the European Network of Forensic Science Institutes voted to adopt five additional STR loci (D12S391, D1S1656, D2S441, D10S1248, and D22S1045) to their existing European Standard Set of seven STRs (TH01, vWA, FGA, D8S1179, D18S51, D21S11, and D3S1358). The D12S391 and vWA loci are located 6.3 megabases (Mb) apart on chromosome 12. Ideally for use in forensic analyses, genetic markers on the same chromosome should be more than 50 Mb in physical distance in order to ensure full recombination and thus independent inheritance. The purpose of this study was to evaluate if the closely located D12S391 and vWA loci are independent and, consequently, if these loci can be included in the product rule calculation for forensic and kinship analyses. Departures from Hardy–Weinberg equilibrium and linkage disequilibrium between the D12S391 and vWA loci were tested using n = 654 unrelated U.S. African American, Caucasian, and Hispanic samples, and n = 764 father/son paternity samples. In the unrelated U.S. population samples, no significant departures from HWE were detected for D12S391 or vWA. No significant evidence of linkage disequilibrium was observed between the loci in the population samples. However, significant linkage disequilibrium was detected in U.S. African American, Caucasian, and Asian father/son samples with phased genotypes. No significant linkage disequilibrium was detected for U.S. Hispanic paternity samples. The use of phased father/son pairs allowed for robust detection of linkage disequilibrium between D12S391 and vWA. In unrelated population samples, linkage disequilibrium is present but more difficult to detect due to the large number of possible haplotype combinations and unknown allelic phase. For casework analyses that involve unrelated or related individuals, the single-locus genotype probabilities for D12S391 and vWA should not be multiplied to determine the match probability of an autosomal STR profile. Since the D12S391 and vWA loci are not independent, it is recommended that the observed combination of alleles at D12S391 and vWA should be treated as a non-independent diplotype for profile probability calculations. The observed haplotype frequencies for U.S. African American, Caucasian, Hispanic, and Asian populations are provided for match probability calculations.     

15 STR loci frequencies in the population from Paraná, Southern Brazil

Allele frequencies for 15 short tandem repeats (STR) loci were obtained from a sample of 4076 unrelated individuals undergoing paternity testing. The population is from Paraná, Southern Brazil. The loci are the most commonly used in forensic and paternity testing, being analyzed by the AmpFlSTR^® Identifiler™ (Applied Biosystems) commercial kit. The most polymorphic loci were D2S1338 and D18S51. Excepting the D13S317, all loci were in Hardy–Weinberg equilibrium. Comparative analyses between our population data and other populations are presented.