Genetics and genomics of core short tandem repeat loci used in human identity testing

Over the past decade, the human identity testing community has settled on a set of core short tandem repeat (STR) loci that are widely used for DNA typing applications. A variety of commercial kits enable robust amplification of these core STR loci. A brief history is presented regarding the selection of core autosomal and Y-chromosomal STR markers. The physical location of each STR locus in the human genome is delineated and allele ranges and variants observed in human populations are summarized as are mutation rates observed from parentage testing. Internet resources for additional information on core STR loci are reviewed. Additional topics are also discussed, including potential linkage of STR loci to genetic disease-causing genes, probabilistic predictions of sample ethnicity, and desirable characteristics for additional STR loci that may be added in the future to the current core loci. These core STR loci, which form the basis for DNA databases worldwide, will continue to play an important role in forensic science for many years to come.     

武汉汉族人群D20S85和D6S477基因座遗传多态性调查

目的对武汉地区280名汉族无关个体的D20S85和D6S477位点遗传多态性进行调查,研究其在法医学检验中的应用价值。方法应用PCR和PAGE技术进行分型检验。结果分别检出10个、9个等位基因,获得各等位基因在该地区汉族人群分布频率,基因型频率分布符合Hardy-Weinberg平衡。两位点的DP值分别为0.9085、0.9127。结论D20S85和D6S477是法医学中重要的遗传标记。     

Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA

DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.     

Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible-often directly next to the identified repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.     

Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits

The AmpFlSTR MiniFiler polymerase chain reaction amplification kit developed by Applied Biosystems enables size reduction on eight of the larger STR loci amplified in the Identifiler kit, which will aid recovery of information from highly degraded DNA samples. The MiniFiler Kit amplifies CSF1PO, FGA, D2S1338, D7S820, D13S317, D16S539, D18S51, and D21S11 as well as the sex-typing locus amelogenin. A total of 1308 samples were evaluated with both the MiniFiler and Identifiler STR kits: 449 African American, 445 Caucasian, 207 Hispanic, and 207 Asian individuals. Full concordance between Identifiler and MiniFiler Kits was observed in 99.7% (10,437 out of 10,464) STR allele calls compared. The 27 differences seen are listed in Table 1 and encompass the loci D13S317 (n = 14) and D16S539 (n = 10) as well as D18S51 (n = 1), D7S820 (n = 1), and CSF1PO (n = 1). Genotyping discrepancies between the Identifiler and MiniFiler kits were confirmed by reamplification of the samples and further testing using the PowerPlex 16 kit in many cases. DNA sequence analysis was also performed in order to understand the nature of the genetic variations causing the allele dropout or apparent repeat unit shift.     

Bioinformatics and human identification in mass fatality incidents: the world trade center disaster

Victim identification initiatives undertaken in the wake of Mass Fatality Incidents (MFIs) where high-body fragmentation has been sustained are often dependent on DNA typing technologies to complete their mandate. The success of these endeavors is linked to the choice of DNA typing methods and the bioinformatic tools required to make the necessary associations. Several bioinformatic tools were developed to assist with the identification of the victims of the World Trade Center attacks, one of the most complex incidents to date. This report describes one of these tools, the Mass Disaster Kinship Analysis Program (MDKAP), a pair-wise comparison software designed to handle large numbers of complete or partial Short Tandem Repeats (STR) genotypes, and infer identity of, or biological relationships between tested samples. The software performs all functions required to take full advantage of the information content of processed genotypic data sets from large-scale MFIs, including the collapse of victims data sets, remains re-association, virtual genotype generation through gap-filling, parentage trio searching, and a consistency check of reported/inferred biological relationships within families. Although very few WTC victims were genetically related, the software can detect parentage trios from within a victim's genotype data set through a nontriangulated approach that screens all possible parentage trios. All software-inferred relationships from WTC data were confirmed by independent statistical analysis. With a 13 STR loci complement, a fortuitous parentage trio (FPT) involving nonrelated individuals was detected. Additional STR loci would be required to reduce the risk of an FPT going undetected in large-scale MFIs involving related individuals among the victims. Kinship analysis has proven successful in this incident but its continued success in larger scale MFIs is contingent on the use of a sufficient number of STR loci to reduce the risk of undetected FPTs, the use of mtDNA and Y-STRs to confirm parentage and of bioinformatics that can support large-scale comparative genotyping schemes capable of detecting parentage trios from within a group of related victims.     

武汉汉族人群短串联重复序列D5S818， D7S820位点多态性研究

目的研究Ｄ５Ｓ８１８,Ｄ７Ｓ８２０的多态性及法医学应用价值。方法应用聚合酶链反应（ＰＣＲ）、聚丙烯酰胺凝胶电泳分离及银染显带技术对武汉地区汉族２３２例无关个体作Ｄ５Ｓ８１８,Ｄ７Ｓ８２０位点分型调查。结果Ｄ５Ｓ８１８和Ｄ７Ｓ８２０位点分别检出８个和６个等位基因,获汉族人群基因频率分布。二位点基因型频率分布符合ＨａｒｄｙＷｅｉｎｂｅｒｇ平衡。位点杂合度分别为０８１２１和０７９３４,个人识别能力分别为０９４１６和０９２５５,非父排除率分别为０５８４２和０５８１６。结论Ｄ５Ｓ８１８和Ｄ７Ｓ８２０ＳＴＲ位点均是高杂合度、高鉴别能力的遗传标记系统,在法医学个人识别和亲子鉴定中有较高实用价值     

STR位点D19S253和D8S1179的法医学意义及应用研究

为评估STR位点D19S253和D8S1179的法医学应用价值，应用PCR和PAG垂直电泳技术对两位点的种属特异性，检测灵敏度，以及同一个体不同组织分型的同一性及不同基质和不同保存时间的斑痕分型等与法医应用有关的问题进行了研究，D19S253和D8S1179位点的检测灵敏度分别为0．25ng及0．5ng，同时两位点具有较高的种属特异性，同一性及较好重复性，且能够复合扩增，表明D19S253和D8S1179是法医学检案中较实用的两个STR标记。     

武汉汉族人群STR基因座D3S1358、D13S317、D12S391的多态性研究

目的　调查中国武汉地区汉族人群STR基因座—D3S1 358、D1 3S31 7、D1 2S391基因频率分布和群体遗传数据。方法　从 2 0 8个汉族无关个体收集血液标本 ,应用PCR技术及聚丙烯酰胺凝胶垂直板电泳对D3S1 358、D1 3S31 7和D1 2S391基因座分型。结果　D3S1 358检出 7个等位基因和 4 1个基因型。三基因座基因型分布符合Hardy-Weinberg平衡。观察 2 31次减数分裂均未发现突变基因。另外 ,调查结果计算显示D3S1 358、D1 3S31 7和D1 2S391基因座的杂合度 (H)分别为 0 70 98、 0 80 56和 0 84 0 0 ;三个人识别能力 (DP)分别为 0 851 6、 0 9332和 0 952 3;非父排除率 ( pE)分别为 0 4 463、 0 60 1 6和 0 681 8。结论　D3S1 358、D1 3S31 7和D1 2S391基因座在群体遗传学研究和法医学亲子鉴定及个人识别中具有较高实用价值     

Comparison of two methods for isolating DNA from human skeletal remains for STR analysis

Abstract: The quality and efficiency of a standard organic DNA isolation method and a silica‐based method using the QIAGEN Blood Maxi Kit were compared to obtain human DNA and short tandem repeats (STRs) profiles from 39 exhumed bone samples for paternity testing. DNA samples were quantified by real‐time PCR, and STR profiles were obtained using the AmpFlSTR^® Identifiler^® PCR amplification kit. Overall, the silica‐based method recovered less DNA ranging from 0 to 147.7 ng/g (average 7.57 ng/g, median = 1.3 ng/g) than did the organic method ranging from 0 to 605 ng/g (average 44.27 ng/g, median = 5.8 ng/g). Complete profiles (16/16 loci tested) were obtained from 37/39 samples (95%) using the organic method and from 9/39 samples (23%) with the silica‐based method. Compared with a standard organic DNA isolation method, our results indicate that the published silica‐based method does not improve neither the quality nor the quantity of DNA for STR profiling.     

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

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

Allele frequency distribution based on 17 STR markers in three major Dravidian linguistic populations of Andhra Pradesh, India

Allele frequencies for 15 tetranucleotides and 2 pentanucleotides repeat loci were determined in 317 unrelated, healthy individuals of Andhra Pradesh, India, belonging to three pre-dominant endogamous populations namely, Kappu Naidu, Kamma Chaudhary and Kapu Reddy. Adherence to the expectations of the Hardy-Weinberg equilibrium (HWE) was confirmed for all loci with few exceptions, which were not significant after applying Bonferroni's correction. Statistical parameters of forensic interest; observed heterozygosity, probability of homozygosity, probability of extact test, power of discrimination, match probability, polymorphism information content, power of exclusion and mean paternity index were determined for all loci. The present study reveals that Penta E and D2S1338 are the most informative loci in all the studied populations. The combined power of discrimination was greater than 0.976, whereas the cumulative power of exclusion gave an expected value of 0.9999 for all the tested microsatellite loci. No difference was observed in the discriminatory power of 15 loci in studied populations on comparison with other populations of India. Population differentiation tests revealed significant differences between the studied and neighboring populations at several loci. Analyzed parameters indicate the utility and efficacy of the studied 17 STR systems as a powerful tool in forensic human identification, paternity testing and human population genetic studies.     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.     

Genetic Data Analysis of Nine STRs in Two Caribbean Colombian Populations: César and Guajira

POPULATION:  We have analyzed the distribution of the allele frequencies and parameters of forensic interest at nine short tandem repeat (STR) loci (CSF1PO, D3S1358, D5S818, D7S820, D13S317, FGA, TH01, TPOX, and vWA) in a sample population of 165 unrelated individuals from César and Guajira, Caribbean populations of Colombia.     

D6S1043和D12S391基因座在亲权鉴定中的应用

目的研究D6S1043和D12S391基因座在亲权关系鉴定案件中的应用价值。方法应用荧光标记复合扩增系统对日常检案中所收集的192名汉族无关个体血样DNA进行PCR扩增,用ABI3100-Avant遗传分析仪对扩增产物进行毛细管电泳,用GeneMapperv3.2软件进行基因分型,统计分析D6S1043和D12S391基因座的多态信息。结果在D6S1043和D12S391基因座分别发现12个等位基因,它们在中国汉族人群中的个体识别能力分别为0.9656和0.9510,二联体非父排除率分别为0.573和0.510,三联体非父排除率分别为0.731和0.679。结论D6S1043和D12S391基因座具有高度多态性,在亲权鉴定中具有重要应用价值。     

壮族人群3个STR基因座基因频率分布及其法医学应用

研究3个STR基因座（D21S11、HumFGA、D19S253）在广西壮族人群中的基因频率分布及其在实际检案中的应用价值。以自制等位基因Ladder样品作为标准对照,用PCR结合PAGE技术对3个STR基因座的扩增产物进行分型。结果显示：D21S11基因座有14个等位基因,有44个基因型;HumFGA基因座有15个等位基因,40个基因型;D195253基因座有9个等位基因,23个基因型。经检验,3个STR基因座基因型分布均符合Hardy－Weinberg平衡,累计个体识别力（DP）为0．9995。3个STR基因座在壮族人群属高识别力遗传标记系统,在法医学个体识别及亲权鉴定方面有重要价值。     

D3S1754、D18S535基因座在中国北方汉族的多态性分析

目的 了解D3S1754、D18S535基因座多态性在中国北方人群中的分布特点及其应用价值。方法 使用PCR、聚丙烯酰胺垂直板电泳及银染的方法。结果D3S1754基因座检出9个等位基因（n=184）,D185535基因座检出8个等位基因（n=201）,两个位点的等位基因频率在群体中的分布符合Hardy-Weinberg平衡（P>0.05）,它们的杂合率（He）分别为0.706和0.807,个人识别机率（DP）分别是0.859和0.934,非父排除率（EPP）分别为0.464和0.629。结论 D3S1754、D18S535两个遗传标记的个人识别率高、非父排除能力较强且能稳定遗传,具有较高的应用价值。     

Developmental validation of the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit: an established multiplex assay with improved performance

Abstract: Analysis of length polymorphism at short tandem repeat (STR) loci utilizing multiplex polymerase chain reaction (PCR) remains the primary method for genotyping forensic samples. The AmpF?STR^® Identifiler^® Plus PCR Amplification Kit is an improved version of the AmpF?STR^® Identifiler^® PCR Amplification Kit and amplifies 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, amelogenin, and two internationally accepted loci, D2S1338 and D19S433. While the primer sequences and dye configurations were unchanged, the AmpF?STR^® Identifiler^® Plus PCR Amplification Kit features an enhanced buffer formulation and an optimized PCR cycling protocol that increases sensitivity, provides better tolerance to PCR inhibitors, and improves performance on mixture samples. The AmpF?STR^® Identifiler^® Plus PCR Amplification Kit has been validated according to the FBI/National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The validation results support the use of the AmpF?STR^® Identifiler^® Plus PCR Amplification Kit for human identity and parentage testing.     

Detection of genotype recycling fraud in U.S. immigrants

Abstract: Relationship testing laboratories provide genetic evidence to support or refute claims of kinship between U.S. citizen petitioners and potential immigrant beneficiaries. One female beneficiary presented a male amelogenin type and alleles at 15 autosomal loci that were identical to an alleged brother’s. Laboratory records showed that her alleged father had petitioned to have 15 children emigrate from Ghana. The petitioner’s 15 paternity indices exceeded 10⁵, but the children shared only four short tandem repeat (STR) profiles, suggesting fraudulent reuse of genotypes in this alleged pedigree (AP). To determine the extent of this “genotype recycling,” I examined the laboratory’s 555 APs from Ghana and 532 control APs from Nigeria. Seventeen Ghanaian APs (3.1%) but no Nigerian APs showed genotype recycling. Of 90 tested people in the 17 APs, 56 shared identical STR profiles with others in their AP. Of these 56 people, 10 were petitioners with unexpectedly high parentage indices. Seven of 56 had amelogenin types that disagreed with their declared genders. Database searches for identical multilocus genotypes in allegedly different people would best detect this fraud.     

荧光标记3个miniSTR基因座复合扩增系统的建立

目的建立扩增片段<135bp,包括D5S818,D8S1179,D16S539 3个miniSTR基因座复合扩增系统。方法采用不同荧光染料标记引物,通过PCR扩增,利用ABI 3100遗传分析仪进行片段长度分析,对100份无关个体血样,10个家系样本以及30份高度降解检材进行检测。结果本系统DNA分型结果与AmpFLSTR Identifiler试剂盒完全一致,且灵敏度高于AmpFLSTR Identifiler试剂盒。结论本系统可以应用于个人识别和亲权鉴定,为降解DNA样本分型提供了新的方法。