Validation of 4 type-STR analysis for identification of 50 Korean

Short tandem repeat (STR) analysis provides genetic fingerprinting of individuals and is an indispensable technique for forensic human identification. Recently, this technique has been used in social areas, such as the identification of The Korean War, descendants of national merit, and missing children. STR analysis is performed by analyzing iteration number of repeating bases in the human genome, and currently FBI provides the Combined DNA Index System (CODIS) based on DNA databases. Among them, we used the autosomal short tandem repeats of loci D13S317, D16S539, D21S11, and amelogenin to validate this technique for identification. The samples were collected from unrelated 50 Korean individuals, and 4 STR loci of these samples were analyzed by ABI 3130 genetic analyzer. We demonstrated that 47 samples out of 50 were classified completely with only 4 STR markers, and perfect sex identification could be accomplished with amelogenin analysis.     

Forensically relevant SNP classes

Forensic samples that contain too little template DNA or are too degraded require alternate genetic marker analyses or approaches to what is currently used for routine casework. Single nucleotide polymorphisms (SNPs) offer promise to support forensic DNA analyses because of an abundance of potential markers, amenability to automation, and potential reduction in required fragment length to only 60-80 bp. The SNP markers will serve an important role in analyzing challenging forensic samples, such as those that are very degraded, for augmenting the power of kinship analyses and family reconstructions for missing persons and unidentified human remains, as well as for providing investigative lead value in some cases without a suspect (and no genetic profile match in CODIS). The SNPs for forensic analyses can be divided into four categories: identity-testing SNPs; lineage informative SNPs; ancestry informative SNPs; and phenotype informative SNPs. In addition to discussing the applications of these different types of SNPs, this article provides some discussion on privacy issues so that society and policymakers can be more informed.     

Effects of subpopulation structure on probability calculations of DNA profiles from forensic PCR analysis

DNA typing for forensic identification is a two-step process. The first step involves determining the profiles of samples collected at the crime scene and comparing them with the profiles obtained from suspects and the victims. In the case of a match that includes the suspect as the potential source of the material collected at the crime scene, the last step in the process is to answer the question, what is the likelihood that someone in addition to the suspect could match the profile of the sample studied? This likelihood is calculated by determining the frequency of the suspect's profile in the relevant population databases. The design of forensic databases and the criteria for comparison has been addressed by the NRC report of 1996 (National Research Council, 1996). However, the fact that geographical proximity, migrational patterns, and even cultural and social practices have effects on subpopulation structure establishes the grounds for further study into its effects on the calculation of probability of occurrence values. The issue becomes more relevant in the case of discrete polymorphic markers that show higher probability of occurrence in the reference populations, where several orders of magnitude difference between the databases may have an impact on the jury. In this study, we calculated G values for all possible pairwise comparisons of allelic frequencies in the different databases from the races or subpopulations examined. In addition, we analyzed a set of 24 unrelated Caucasian, 37 unrelated African-American, and 96 unrelated Sioux/Chippewa individuals for seven polymorphic loci (DQA1, LDLR, GYPA, HBGG, D7S8, GC, and D1S80). All three sets of individuals where sampled from Minnesota. The probability of occurrence for all seven loci were calculated with respect to nine different databases: Caucasian, Arabic, Korean, Sioux/Chippewa, Navajo, Pueblo, African American, Southeastern Hispanic, and Southwestern Hispanic. Analysis of the results demonstrated marked differences in the probabilities of occurrence when individuals were compared to the different populations and subpopulation databases. The possible genetic and forensic consequences of subpopulation structure on probability calculations are discussed.     

Interpreting anonymous DNA samples from mass disasters--probabilistic forensic inference using genetic markers

MOTIVATION: The problem of identifying victims in a mass disaster using DNA fingerprints involves a scale of computation that requires efficient and accurate algorithms. In a typical scenario there are hundreds of samples taken from remains that must be matched to the pedigrees of the alleged victim's surviving relatives. Moreover the samples are often degraded due to heat and exposure. To develop a competent method for this type of forensic inference problem, the complicated quality issues of DNA typing need to be handled appropriately, the matches between every sample and every family must be considered, and the confidence of matches need to be provided. RESULTS: We present a unified probabilistic framework that efficiently clusters samples, conservatively eliminates implausible sample-pedigree pairings, and handles both degraded samples (missing values) and experimental errors in producing and/or reading a genotype. We present a method that confidently exclude forensically unambiguous sample-family matches from the large hypothesis space of candidate matches, based on posterior probabilistic inference. Due to the high confidentiality of disaster DNA data, simulation experiments are commonly performed and used here for validation. Our framework is shown to be robust to these errors at levels typical in real applications. Furthermore, the flexibility in the probabilistic models makes it possible to extend this framework to include other biological factors such as interdependent markers, mitochondrial sequences, and blood type. AVAILABILITY: The software and data sets are available from the authors upon request.     

Ethnic-affiliation estimation by use of population-specific DNA markers.

During the past 10 years, DNA analysis has revolutionized the determination of identity in a forensic context. Statements about the biological identity of two human DNA samples now can be made with complete confidence. Although DNA markers are very powerful for distinguishing among individuals, most offer little power to distinguish ethnicity or to support any statement about the physical characteristics of an individual. Through a search of the literature and of unpublished data on allele frequencies we have identified a panel of population-specific genetic markers that enable robust ethnic-affiliation estimation for major U.S. resident populations. In this report, we identify these loci and present their levels of allele-frequency differential between ethnically defined samples, and we demonstrate, using log-likelihood analysis, that this panel of markers provides significant statistical power for ethnic-affiliation estimation. In addition to their use in forensic ethnic-affiliation estimation, population-specific genetic markers are very useful in both population- and individual-level admixture estimation and in mapping genes by use of the linkage disequilibrium created when populations hybridize.     

Human genome diversity

Human genome diversity studies analyse genetic variation among individuals and between populations in order to understand the origins and evolution of anatomically modern humans (Homo sapiens sapiens). The availability of thousands of DNA polymorphisms (genetic markers) brings analytic power to these studies. Human genome diversity studies have clearly shown that the large part of genetic variability is due to differences among individuals within populations rather than to differences between populations, effectively discrediting a genetic basis of the concept of ‘race’. Evidence from paleontology, archaeology and genetic diversity studies is quite consistent with an African origin of modern humans more than 100 000 years ago. The evidence favors migrations out of African as the source of the original peopling of Asia, Australia, Europe and Oceania. An international program for the scientific analysis of human genome diversity and of human evolution has been developed. The Human Genome Diversity Project (HGDP) aims to collect and preserve biologic samples from hundreds of populations throughout the world, make DNA from these samples available to scientists and distribute to the scientific community the results of DNA typing with hundreds of genetic markers.     

Familial identification: population structure and relationship distinguishability

With the expansion of offender/arrestee DNA profile databases, genetic forensic identification has become commonplace in the United States criminal justice system. Implementation of familial searching has been proposed to extend forensic identification to family members of individuals with profiles in offender/arrestee DNA databases. In familial searching, a partial genetic profile match between a database entrant and a crime scene sample is used to implicate genetic relatives of the database entrant as potential sources of the crime scene sample. In addition to concerns regarding civil liberties, familial searching poses unanswered statistical questions. In this study, we define confidence intervals on estimated likelihood ratios for familial identification. Using these confidence intervals, we consider familial searching in a structured population. We show that relatives and unrelated individuals from population samples with lower gene diversity over the loci considered are less distinguishable. We also consider cases where the most appropriate population sample for individuals considered is unknown. We find that as a less appropriate population sample, and thus allele frequency distribution, is assumed, relatives and unrelated individuals become more difficult to distinguish. In addition, we show that relationship distinguishability increases with the number of markers considered, but decreases for more distant genetic familial relationships. All of these results indicate that caution is warranted in the application of familial searching in structured populations, such as in the United States.     

HumanMSD and MouseMSD: generating genetic maps for human and murine microsatellite markers

We present two Web interfaces that generate genetic maps for given sets of human or mouse microsatellite markers. The genetic maps are generated from available databases using linear interpolation of physical map distances to infer genetic map positions for missing markers in these databases.     

Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci

In this study, we have evaluated the efficacy and the validity of the AmpFISTR SGM plus multiplex PCR typing system when Low Copy Number (LCN) amounts of DNA are processed. The characteristics of SGM plus profiles produced under LCN conditions were studied on the basis of heterozygote balance, between loci balance and stutter proportion based on profiles that were obtained from a variety of mock casework samples. These experiments clearly showed that LCN DNA profiles carry their own characteristic features, which must be taken into account during interpretation. Herewith, we confirmed the data of recent other studies that a comprehensive interpretation strategy is dependent upon multiple replication of the PCR using the same extract together with the proper use of extraction and amplification controls. The limitations of LCN DNA analysis were further studied in a series of single cell PCR experiments using an amplification regime of 34 PCR cycles. The allele dropout phenomenon was demonstrated to its full extent when single cells were analysed. However, the "consensus profile" which was obtained from separate single cell PCR experiments matched the actual profile of the cell donor. Single cell PCR experiments also showed that a further increase of the number of PCR cycles did not result in enhanced sensitivity and had a highly negative effect on the balance of this multiplex PCR system which hampered correct interpretation of the profile. Also, the potential of LCN typing in analysing mixtures of DNA was investigated. It was clearly shown that LCN typing had no advantages over 28 cycles amplification in the detection of the minor component of DNA-mixtures. In addition to the 34 cycles PCR amplification regime, the utility of a new approach that involved reamplification of the 28 cycle SGM plus PCR products with an extra 6 PCR cycles after the addition of fresh AmpliTaq Gold DNA Polymerase was investigated. This approach provides the scientist with an extra typing result that enhances the reliability of the consensus profile, which is commonly retrieved from two separate 34 cycle PCR results. Furthermore, the 28 + 6 cycles approach may be used to screen LCN samples for their potential to produce a 34 PCR cycle profile. Finally and as a last resort the 28 + 6 cycles approach can be used in those cases where no further extract from the crime sample is available. Finally, the potential of LCN typing was demonstrated in typing samples from non-probative and actual casework examples. From a high proportion of samples that failed to demonstrate SGM plus typing results using the standard protocol of 28 cycles, at least partial profiles could be obtained after LCN methods were used. For example, LCN typing was applied in a case where 10-year old samples from bones and teeth that were retrieved from a mass grave had to be identified. This study resulted in the positive identification of a number of victims by comparing the LCN DNA profiles with the profiles from putative relatives. The value of LCN DNA typing was further demonstrated in a strangulation case. The throat of the victim was sampled and only after 34 PCR cycles were we able to reveal that the evidential sample contained a distinct mixture of the victim's own DNA and the DNA of the defendant.     

Ancient DNA: Using molecular biology to explore the past

Ancient DNA has been discovered in many types of preserved biological material, including bones, mummies, museum skins, insects in amber and plant fossils, and has become an important research tool in disciplines as diverse as archaeology, conservation biology and forensic science. In archaeology, ancient DNA can contribute both to the interpretation of individual sites and to the development of hypotheses about past populations. Site interpretation is aided by DNA-based sex typing of fragmentary human bones, and by the use of genetic techniques to assess the degree of kinship between the remains of different individuals. On a broader scale, population migrations can be traced by studying genetic markers in ancient DNA, as in recent studies of the colonisation of the Pacific islands, while ancient DNA in preserved plant remains can provide information on the development of agriculture.     

Preliminary DNA identification for the tsunami victims in Thailand

The 2004 Southeast Asia Tsunami killed nearly 5,400 people in Southern Thailand, including foreign tourists and local residents. To recover DNA evidence as much as possible from the seriously decomposed bodies, we explored procedures of sample preparation from both bone and tooth samples as well as both mitochondrial and nuclear markers. Despite having failed to recover enough DNA for nuclear marker typing, we succeeded in obtaining fully informative results for mitochondrial markers （HV1 and HV2） from 258 tooth samples with a success rate of 51% （258/507）. Using an organic DNA extraction method coupled with an ultrafiltration step, we obtained 16 STR （including 13 CODIS loci, one sex discrimination locus, and two Identifiler loci） profiles for 834 samples with a success rate of 79% （834/1,062）. In addition, by comparing the allelic frequencies between the typed samples as a group and other index populations, we conclude that the Thai tsunami victims are a combined group of several populations. Our results provide valuable evidence and protocols for the future forensic practice.     

Authenticated DNA from ancient wood remains

BACKGROUND: The reconstruction of biological processes and human activities during the last glacial cycle relies mainly on data from biological remains. Highly abundant tissues, such as wood, are candidates for a genetic analysis of past populations. While well-authenticated DNA has now been recovered from various fossil remains, the final 'proof' is still missing for wood, despite some promising studies. SCOPE: The goal of this study was to determine if ancient wood can be analysed routinely in studies of archaeology and palaeogenetics. An experiment was designed which included blind testing, independent replicates, extensive contamination controls and rigorous statistical tests. Ten samples of ancient wood from major European forest tree genera were analysed with plastid DNA markers. CONCLUSIONS: Authentic DNA was retrieved from wood samples up to 1,000 years of age. A new tool for real-time vegetation history and archaeology is ready to use.     

Improving human forensics through advances in genetics, genomics and molecular biology

Forensic DNA profiling currently allows the identification of persons already known to investigating authorities. Recent advances have produced new types of genetic markers with the potential to overcome some important limitations of current DNA profiling methods. Moreover, other developments are enabling completely new kinds of forensically relevant information to be extracted from biological samples. These include new molecular approaches for finding individuals previously unknown to investigators, and new molecular methods to support links between forensic sample donors and criminal acts. Such advances in genetics, genomics and molecular biology are likely to improve human forensic case work in the near future.     

Correlation of DNA fragment sizes within loci in the presence of non-detectable alleles

At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size resulting from one allele remains undetected. In this work, we evaluate the effect of such allelic non-detectability on correlation of fragment sizes within individuals at a locus, and its impact on the inference of independence of fragment sizes within loci. We show that when non-detectable alleles are present in a population at a locus, positive correlations of fragment sizes are expected, which increase with the proportion of non-detectable alleles at the locus. Therefore, a non-zero positive correlation is not a proof of allelic dependence within individuals. Applications of this theory to the current forensic RFLP databases within the US show that there is virtually no evidence of significant allelic dependence within any of the loci. Therefore, the assumption that DNA fragment sizes within loci are independent is valid, and hence, the population genetic principles of computing DNA profile frequencies by multiplying binned frequencies of fragment sizes are most likely to be appropriate for forensic applications of DNA typing data.Editor's commentsThe presence of non-detectable alleles for VNTR loci has plagued the use of these highly-discriminating systems in human identification. The authors take explicit account of these alleles, and are able to show independence of the frequencies of detectable alleles. They raise the troubling issue of how to account for occasional significant results when multiple tests are performed. By invoking Bonferroni corrections, they regard all tests, even those performed on different loci, as addressing the same hypothesis—the absence of dependence at any VNTR locus.     

Recent advances and considerations for the future in forensic analysis of degraded DNA by autosomic miniSTR multiplex genotyping

STR genotyping from degraded DNA samples requires genetic profiles to be obtained from DNA fragments no bigger than 200-300 bp. It requires the use of miniSTRs, which are smaller than the STRs used in standard typing. This paper reviews recent advances in miniSTR genotyping, beginning with a brief introduction to the processes involved in DNA fragmentation and how it hinders standard STR genotyping before proceeding further to the loci included in the main DNA databases and finishing with the International Workgroups' recommended design strategies for developing miniSTR reactions. The results of the efforts of many laboratories achieving different STR multiplexes and patents are also described and compared. Finally, a consideration of the perspectives for the future in this area is presented.     

Straightforward inference of ancestry and admixture proportions through ancestry-informative insertion deletion multiplexing

Ancestry-informative markers (AIMs) show high allele frequency divergence between different ancestral or geographically distant populations. These genetic markers are especially useful in inferring the likely ancestral origin of an individual or estimating the apportionment of ancestry components in admixed individuals or populations. The study of AIMs is of great interest in clinical genetics research, particularly to detect and correct for population substructure effects in case-control association studies, but also in population and forensic genetics studies. This work presents a set of 46 ancestry-informative insertion deletion polymorphisms selected to efficiently measure population admixture proportions of four different origins (African, European, East Asian and Native American). All markers are analyzed in short fragments (under 230 basepairs) through a single PCR followed by capillary electrophoresis (CE) allowing a very simple one tube PCR-to-CE approach. HGDP-CEPH diversity panel samples from the four groups, together with Oceanians, were genotyped to evaluate the efficiency of the assay in clustering populations from different continental origins and to establish reference databases. In addition, other populations from diverse geographic origins were tested using the HGDP-CEPH samples as reference data. The results revealed that the AIM-INDEL set developed is highly efficient at inferring the ancestry of individuals and provides good estimates of ancestry proportions at the population level. In conclusion, we have optimized the multiplexed genotyping of 46 AIM-INDELs in a simple and informative assay, enabling a more straightforward alternative to the commonly available AIM-SNP typing methods dependent on complex, multi-step protocols or implementation of large-scale genotyping technologies.     

Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.

We have developed a new method for forensic identification of individuals, in which a panel of biallelic DNA markers are amplified by the PCR, and the variable nucleotides are detected in the amplified DNA fragments by the solid-phase minisequencing method. A panel of 12 common polymorphic nucleotides located on different chromosomes with reported allele frequencies close to .5 were chosen for the test. The allele frequencies for most of the markers were found to be similar in the Finnish and other Caucasian populations. We also introduce a novel approach for rapid determination of the population frequencies of biallelic markers. By this approach we were able to determine the allele frequencies of the markers in the Finnish population, by quantitative analysis of three pooled DNA samples representing 3,000 individuals. The power of discrimination and exclusion of the solid-phase minisequencing typing test with 12 markers was similar to that of three VNTR markers that are routinely used in forensic analyses at our institute. The solid-phase minisequencing method was successfully applied to type paternity and forensic case samples. We also show that the quantitative nature of our method allows typing of mixed samples.     

Inferring coancestry in population samples in the presence of linkage disequilibrium

In both pedigree linkage studies and in population-based association studies there has been much interest in the use of modern dense genetic marker data to infer segments of gene identity by descent (ibd) among individuals not known to be related, to increase power and resolution in localizing genes affecting complex traits. In this article, we present a hidden Markov model (HMM) for ibd among a set of chromosomes and describe methods and software for inference of ibd among the four chromosomes of pairs of individuals, using either phased (haplotypic) or unphased (genotypic) data. The model allows for missing data and typing error, but does not model linkage disequilibrium (LD), because fitting an accurate LD model requires large samples from well-studied populations. However, LD remains a major confounding factor, since LD is itself a reflection of coancestry at the population level. To study the impact of LD, we have developed a novel simulation approach to generate realistic dense marker data for the same set of markers but at varying levels of LD. Using this approach, we present results of a study of the impact of LD on the sensitivity and specificity of our HMM model in estimating segments of ibd among sets of four chromosomes and between genotype pairs. We show that, despite not incorporating LD, our model has been quite successful in detecting segments as small as 10(6) bp (1 Mpb); we present also comparisons with fastIBD which uses an LD model in estimating ibd.     

Short tandem repeat typing technologies used in human identity testing

Short tandem repeat (STR) typing methods are widely used today for human identity testing applications including forensic DNA analysis. Following multiplex PCR amplification, DNA samples containing the length-variant STR alleles are typically separated by capillary electrophoresis and genotyped by comparison to an allelic ladder supplied with a commercial kit. This article offers a brief perspective on the technologies and issues involved in STR typing.