Validation of the Utility of the Genetically Shared Regions of Chromosomes (GD-ICS) Measuring Method in Identifying Complicated Genetic Relatedness.

BACKGROUND: Relatives share more genomic regions than unrelated individuals, with closer relatives sharing more regions. This concept, paired with the increased availability of high-throughput single nucleotide polymorphism (SNP) genotyping technologies, has made it feasible to measure the shared chromosomal regions between individuals to assess their level of relation to each other. However, such techniques have remained in the conceptual rather than practical stages in terms of applying measures or indices. Recently, we developed an index called "genetic distance-based index of chromosomal sharing (GD-ICS)" utilizing large-scale SNP data from Korean family samples and demonstrated its potential for practical applications in kinship determination. In the current study, we present validation results from various real cases demonstrating the utility of this method in resolving complex familial relationships where information obtained from traditional short tandem repeats (STRs) or lineage markers is inconclusive. METHODS: We obtained large-scale SNP data through microarray analysis from Korean individuals involving 13 kinship cases and calculated GD-ICS values using the method described in our previous study. Based on the GD-ICS reference constructed for Korean families, each disputed kinship was evaluated and validated using a combination of traditional STRs and lineage markers. RESULTS: The cases comprised those A) that were found to be inconclusive using the traditional approach, B) for which it was difficult to apply traditional testing methods, and C) that were more conclusively resolved using the GD-ICS method. This method has overcome the limitations faced by traditional STRs in kinship testing, particularly in a paternity case with STR mutational events and in confirming distant kinship where the individual of interest is unavailable for testing. It has also been demonstrated to be effective in identifying various relationships without specific presumptions and in confirming a lack of genetic relatedness between individuals. CONCLUSION: This method has been proven effective in identifying familial relationships across diverse complex and practical scenarios. It is not only useful when traditional testing methods fail to provide conclusive results, but it also enhances the resolution of challenging kinship cases, which suggests its applicability in various types of practical casework.

A novel approach of kinship determination based on the physical length of genetically shared regions of chromosomes.

BACKGROUND: Determination of genetic relatedness between individuals plays a crucial role in resolving numerous civil cases involving familial relationships and in forensic investigation concerning missing persons. Short tandem repeats (STRs), known for their high degree of DNA polymorphism, have traditionally been the primary choice of DNA markers in genetic testing, but their application for kinships testing is limited to cases involving close kinship. SNPs have emerged as promising supplementary markers for kinship determination. Nevertheless, the challenging remains in discriminating between third-degree or more distant relatives, such as first cousins, using SNPs. OBJECTIVE: To investigate a kinship analysis method for distant degree of familial relationships using high-density SNP data. METHODS: A high-density SNP data from 337 individuals of Korean families using Affymetrix Axiom KORV1.0-96 Array was obtained for this study. SNPs were aligned by chromosomal positions, and identity-by-state (IBS) was determined, and then shared regions as consecutive SNPs with IBS of 1 or 2 were investigated. The physical lengths of these IBS segments were measured and summed them to create an Index, as a measure of kinship. RESULTS: The kinship was determined by the physical length of shared chromosomal regions that are distinguished by each kinship. Using this method, the relationship was able be distinguished up to the fourth degree of kinship, and non-relatives were clearly distinguished from true relatives. We also found a potential for this approach to be used universally, regardless of microarray platforms for SNP genotyping and populations. CONCLUSION: This method has a potential to determine the different degree of kinship between individuals and to distinguish non-relatives from true relatives, which can be of great help for practical applications in kinship determination.

Whole Mitochondrial Genome Analysis in Non-Small Cell Lung Carcinoma Reveals Unique Tumor-Specific Somatic Mutations.

CONTEXT.­: Mitochondria and mitochondrial DNA have been suggested to play a role in cancer initiation and progression. Knowledge of mitochondrial DNA could provide a breakthrough to advance cancer management. OBJECTIVE.­: To identify the mitochondrial DNA landscape in non-small cell lung carcinoma. DESIGN.­: The adenocarcinoma set consisted of 365 pairs of adenocarcinomas and normal lung tissues, whereas the metastasis set included 12 primary non-small cell carcinomas, 15 metastatic tumors, and their normal counterparts. Tumor-specific somatic variants were identified, and if a variant showed heteroplasmy, the proportion of minor alleles was evaluated. Variants with greater than 10% change in allele frequency between tumor and normal pairs were identified as "heteroplasmic shifts." RESULTS.­: Tumor-specific variants appeared throughout the whole mitochondrial genome, without a common hot spot. Distinct variant profiles were seen in 289 (79.18%) of all individual adenocarcinomas. The presence of a unique profile and the number and loading of heteroplasmic shifts in tumors increased with higher stage or lymph node metastasis, and were related to shorter survival. In the metastasis set, the primary tumor variants were generally found in metastatic tumors. CONCLUSIONS.­: This study shows that somatic mitochondrial DNA mutations present with diverse locations and unique profiles in each individual tumor, implying their clinicopathologic utility.

Biogeographic origin and genetic characteristics of the peopling of Jeju Island based on lineage markers.

BACKGROUND: Jeju Island is the largest island of South Korea, located southwest far from the mainland of Korea, and has a unique history and its own cultures that are distinguished from those of the other regions of the Korean mainland. However, the Jeju population has not been deeply investigated to date to understand their genetic structure, which may reflect their historical and geographical background. OBJECTIVE: To identify the genetic characteristics and biogeographic origin of people of Jeju Island based on the statistical analysis of genetic data using lineage markers. METHODS: 17 Y-STRs data for 615 unrelated males and mitochondrial DNA haplogroup data for 799 unrelated individuals residing on Jeju Island were generated, and analyzed to investigate genetic diversity and genetic characteristics using statistical methods including pairwise Fst or Rst, Analysis of molecular variance (AMOVA) and Multidimensional scaling (MDS). RESULTS: For male individuals of Jeju Island, unique genetic characteristics were observed in the analysis of Y-STRs, including low haplotype diversity, strong association with surnames, genetic difference from other regions of Korea, and common genetic variation of the Y-STR loci known to be predominant in Northern populations, such as Mongolians. Statistical analysis of the mitochondrial DNA haplogroups also revealed similar results that showed low haplogroup diversity and high frequency of haplogroup Y prevalent mostly in ethnic populations around the Sea of Okhotsk in Northeastern Asia. All these results suggest that Jeju Island is genetically distinct from other regions of Korea, possibly being a subpopulation in Korea, and related closely to Northern Asian populations. CONCLUSION: The findings in the genetic approach could support understanding of the historical background of Jeju Island that is consistent with evidence from other multidisciplinary studies.

High-resolution melt analysis for the detection of skin/sweat via DNA methylation.

The determination of tissue type is important when reconstructing a crime scene as skin cells may indicate innocent contact, whereas other types of cells, such as blood and semen, may indicate foul play. Up to now, there has been no specific DNA methylation-based marker to distinguish skin cell DNA from other body fluids. The goal of this study is to develop a DNA methylation-based assay to detect and identify skin cells collected at forensic crime scenes for use in DNA typing. For this reason, we have utilized a DNA methylation chip array-based genome-wide association study to identify skin-specific DNA methylation markers. DNA obtained from skin along with other body fluids, such as semen, saliva, blood, and vaginal epithelia, were tested using five genes that were identified as sites for potential new epigenetic skin markers. Samples were collected, bisulfite converted, and subjected to real-time polymerase chain reaction (PCR) with high-resolution melt analysis. In our studies, when using WDR11, PON2, and NHSL1 assays with bisulfite-modified PCR, skin/sweat amplicons melted at lower temperatures compared to blood, saliva, semen, and vaginal epithelia. One-way analysis of variance demonstrates that these three skin/sweat markers are significantly different when compared with other body fluids (p < 0.05). These results demonstrate that high-resolution melt analysis is a promising technology to detect and identify skin/sweat DNA from other body fluids.

Validation of BMI genetic risk score and DNA methylation in a Korean population.

When DNA profiles obtained from biological evidence at a crime scene fail to match suspects or anyone in the database, forensic DNA phenotyping, which is the prediction of externally visible characteristics, can facilitate a traced search for an unknown suspect by limiting the search range. Therefore, age, trait, or lifestyle predictors, as well as the predictor for colorations, have been researched in the forensic field. In the present study, for the development of a prediction model for BMI or obesity, we investigated several previously reported BMI- or obesity-associated genetic and epigenetic markers that included four CpGs (cg06500161, cg00574958, cg12593793, and cg10505902 of the ABCG1, CPT1A, LMNA, and PDE4DIP genes, respectively), and eight SNPs (rs12463617, rs1558902, rs591166, rs11030104, rs11671664, rs6545814, rs16858082, and rs574367 near the TMEM18, FTO, MC4R, BDNF, GIPR/QPCTL, ADCY3/RBJ, GNPDA2, and SEC16B genes, respectively) in 700 Koreans within the BMI ranging from 16.1 to 40.6 (27.6 ± 4.5) kg/m2. Linear regression analysis showed that DNA methylation of the four CpG sites explained 10.9% total variance in BMI, and the model constructed using age information, genetic score from eight SNPs, and DNA methylation at four CpG sites could account for 17.4% of BMI variance. Using data mining techniques, i.e., decision tree (Entropy and Gini), random forest, and bagging, a total of eight models with BMI 31 or 32 as a cutoff value were also constructed based on the data obtained from 490 training samples with age and sex as a covariate. Among them, a random forest model with a cutoff value of 31 showed the best performance with 63.3% accuracy and the AUC value of 0.682 in 210 test set samples. In the present study, we could replicate the previous finding that DNA methylation contributes more to BMI than do genetic factors. In addition, although the accuracy for the prediction of BMI was not high, our study is meaningful in respect of the ability to use a small number of markers to achieve similar prediction accuracy to that obtained from a model composed of more than a thousand markers, which adds support to continued research to identify a small set of predictive markers for practical application in the forensic field.

Improved STR analysis of degraded DNA from human skeletal remains through in-house MPS-STR panel.

DNA analysis of degraded samples and low-copy number DNA derived from skeletal remains, one of the most challenging forensic tasks, is common in disaster victim identification and genetic analysis of historical materials. Massively parallel sequencing (MPS) is a useful technique for STR analysis that enables the sequencing of smaller amplicons compared with conventional capillary electrophoresis (CE), which is valuable for the analysis of degraded DNA. In this study, 92 samples of human skeletal remains (70+ years postmortem) were tested using an in-house MPS-STR system designed for the analysis of degraded DNA. Multiple intrinsic factors of DNA from skeletal remains that affect STR typing were assessed. The recovery of STR alleles was influenced more by DNA input amount for amplification rather than DNA degradation, which may be attributed from the high quantity and quality of libraries prepared for MPS run. In addition, the higher success rate of STR typing was achieved using the MPS-STR system compared with a commercial CE-STR system by providing smaller sized fragments for amplification. The results can provide constructive information for the analysis of degraded sample, and this MPS-STR system will contribute in forensic application with regard to skeletal remain sample investigation.

A case of 46,XX/46,XX chimerism in a phenotypically normal woman.

Chimerism is the presence of two genetically different cell lines within a single organism, which is rarely observed in humans. Usually, chimerism in the human body is revealed by the finding of an abnormal phenotype during a medical examination or is unexpectedly detected in routine genetic analysis. However, the incidence or underlying mechanism of chimerism remains unclear due to the lack of information on this infrequent biological event. A phenotypically normal woman with a 46,XX karyotype and atypical short tandem repeat (STR) allelic patterns observed in DNA analysis was investigated with various genetic testing methods, including STR typing based on capillary electrophoresis and massively parallel sequencing, genome-wide SNP array, and a differentially methylated parental allele assay (DMPA). The proband's parents were not available for testing to discriminate the parental allelic contribution, but the parents' alleles were recovered from testing the proband's siblings. Based on the results consistently found in multiple analyses using STR and single nucleotide polymorphism (SNP) polymorphism markers, dispermic fertilization was suggested as the underlying mechanism. The application of various molecular genetic testing methods was used to elucidate the chimerism observed in the proband in this study. In the future, the development of novel genetic markers or techniques, such as DMPA, may have potential use in the investigation of chimerism.

Large-scale identification of human bone remains via SNP microarray analysis with reference SNP database.

Single nucleotide polymorphisms (SNPs) are valuable markers complementary to conventional forensic short tandem repeat (STR) markers in genetic typing, with potential advantages in challenging forensic casework. With the advent of high-throughput technologies, such as microarrays and massively parallel sequencing, the use of SNP typing has now expanded to large-scale forensic applications. Herein, a forensic case is presented to demonstrate the usefulness of SNP typing in identifying large-scale human bone remains with reference database construction. A total of 402 bone remains were recovered from an island in the Jeju Province of Korea where a massive disaster occurred in 1948. The first phase of the identification process was accomplished via conventional DNA typing methods including autosomal and Y-chromosomal STR typing, and mitochondrial DNA sequencing, which resulted in the identification of 74 of 402 remains. The second phase of the identification involved the remaining 327 unidentified remains using SNP typing as a supplementary tool based on Affymetrix resequencing array. The SNP markers of 782 family members were also analyzed and a reference database was constructed for comparison. An additional 51 bone remains were identified in the second phase. SNP data obtained from the supplementary genotyping yielded additional genetic information as well as contributed to kinship testing to determine the second degrees of relationship. In addition SNPs are useful in discriminating ambiguous relationship when only STR data are available. A software program developed for SNP typing system enabled efficient kinship analysis for large-scale forensic identification. The results and the casework are described and discussed.

Development of a body fluid identification multiplex via DNA methylation analysis.

The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers - BCAS4, CG06379435, VE_8, and ZC3H12D - were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.

Detection of Innate and Artificial Mitochondrial DNA Heteroplasmy by Massively Parallel Sequencing: Considerations for Analysis.

BACKGROUND: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS. METHODS: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared. RESULTS: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates. CONCLUSION: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.

Genetic resolution of applied biosystems™ precision ID Ancestry panel for seven Asian populations.

Massively parallel sequencing (MPS) offers additional information in cases that lack reference samples for comparison. The HID-Ion AmpliSeq Ancestry Panel is a forensic multiplex platform consisting of 165 autosomal markers designed to provide biogeographic ancestry information. We analyzed seven different population groups from Asia to assess the accuracy and reliability of analysis, using this panel. In this study, we have designated 750 unrelated Asians, from southern China (n = 99), Beijing (n = 100), Japan (n = 101), Korea (n = 100), Vietnam (n = 100), Nepal (n = 100), India (n = 51), and Pakistan (n = 99). The likelihood ratios of 750 Asians were calculated, using the Torrent Server and the HID SNP Genotyper Plugin Version 4.3.2. The results reveal that all Northeast Asians (China, Japan, and Korea), and Vietnamese, were predicted as East Asians with the highest ethnicity likelihood values from reference data. Although the samples from Nepal, India and Pakistan (Southwest Asians), were predominantly predicted as South Asians, there were seven cases of which results revealed as Europeans, with similar likelihood patterns. Population structure analysis indicated that Southwest Asians have a genetic profile that is distinguishable from those of other Asian populations. This panel was validated for potential usefulness in forensic analysis and the findings could be used as a basis for building additional Asian specific reference databases. Nevertheless, additional analyses comprising larger sample sizes will be necessary, especially Southeast Asians, to fully understand the Asian population structure, and to discriminate them with further details.

Assessment of mitochondrial DNA heteroplasmy detected on commercial panel using MPS system with artificial mixture samples.

Mitochondrial DNA (mtDNA) heteroplasmy is a potential genetic marker for forensic mtDNA analysis as well as phylogenic studies. Frequency of mtDNA heteroplasmy has been investigated in different populations through massively parallel sequencing (MPS) analysis, revealing various levels of frequency based on different MPS systems. For accurate heteroplasmy identification, it is essential to explore reliable detection threshold on various MPS systems. In addition, software solutions and pipelines need to be evaluated to analyze data effectively. In this study, heteroplasmy analysis was conducted on a commercially available mtDNA analysis system developed for forensic caseworks with artificially mixed DNA samples known for ratios and variant positions for assessment. mtDNA heteroplasmy > 10% was detectable with Torrent Variant Caller (TVC) while lower levels were identified using GeneMarker® HTS specialized software for minor variant detection. This study implies that analytical parameters and tools need to be optimized and evaluated for low-level heteroplasmy identification. Automated system with simple and efficient workflow is needed for forensic caseworks.

Y chromosomal deletion pattern in Koreans inhabiting Jeju Island.

ABSTRACT: Mutations occur in Y chromosome genes similar to autosomal genes. However, unlike autosomal genes, Y chromosome genes do not undergo recombination, which produce distinctive characteristics and distribution patterns in different geographic regions. Therefore, detailed analysis of mutations of Y chromosome genes might provide information for personal identification or analysis of phylogenetic history. In Y-STR (short tandem repeat) analysis tests on 668 habitants of Jeju Island, the largest island in the Korean peninsula located apart from the mainland, a deletion at DYS448 was found in 10 samples. The length of deletion was estimated by confirming specific Sequence Tagged Site (STS) markers ranging from G66018 to sY1201. Patterns found were similar to those of the Kalmyks, a tribe that has had strong social and genetic influences in Jeju Island in the past. Historically from 1273 on, Jeju Island was governed by Mongolian for about one hundred years. The results of this study suggest such historical aspects affected the genetic composition of people living in Jeju Island. Furthermore, previous reports showed that Y chromosomal deletions and region specific Y chromosomal mutations depended on regional differences. This study may be useful for a better understanding of the genetic structure of Jeju habitants as well as Korean population for the purpose of forensic practice and population genetics.

Independent validation of DNA-based approaches for age prediction in blood.

Numerous molecular biomarkers have been proposed as predictors of chronological age. Among them, T-cell specific DNA rearrangement and DNA methylation markers have been introduced as forensic age predictors in blood because of their high prediction accuracy. These markers appear highly promising, but for better application to forensic casework sample analysis the proposed markers and genotyping methods must be tested further. In the current study, signal-joint T-cell receptor excision circles (sjTRECs) and DNA methylation markers located in the ELOVL2, C1orf132, TRIM59, KLF14, and FHL2 genes were reanalyzed in 100 Korean blood samples to test their associations with chronological age, using the same analysis platform used in previous reports. Our study replicated the age association test for sjTREC and DNA methylation markers in the 5 genes in an independent validation set of 100 Koreans, and proved that the age predictive performance of the previous models is relatively consistent across different population groups. However, the extent of age association at certain CpG loci was not identical in the Korean and Polish populations; therefore, several age predictive models were retrained with the data obtained here. All of the 3 models retrained with DNA methylation and/or sjTREC data have a CpG site each from the ELOVL2 and FHL2 genes in common, and produced better prediction accuracy than previously reported models. This is attributable to the fact that the retrained model better fits the existing data and that the calculated prediction accuracy could be higher when the training data and the test data are the same. However, it is notable that the combination of different types of markers, i.e., sjTREC and DNA methylation, improved prediction accuracy in the eldest group. Our study demonstrates the usefulness of the proposed markers and the genotyping method in an independent dataset, and suggests the possibility of combining different types of DNA markers to improve prediction accuracy.

Set up of cutoff thresholds for kinship determination using SNP loci.

The usefulness of single nucleotide polymorphism (SNP) loci for kinship testing has been demonstrated in many case works, and suggested as a promising marker for relationship identification. For interpreting results based on the calculation of the likelihood ratio (LR) in kinship testing, it is important to prepare cutoffs for respective relatives which are dependent on genetic relatedness. For this, analysis using true pedigree data is significant and reliable as it reflects the actual frequencies of markers in the population. In this study, the kinship index was explored through 1209 parent-child pairs, 1373 full sibling pairs, and 247 uncle-nephew pairs using 136 SNP loci. The cutoffs for LR were set up using different numbers of SNP loci with accuracy, sensitivity, and specificity. It is expected that this study can support the application of SNP loci-based kinship testing for various relationships.

Entire Mitochondrial DNA Sequencing on Massively Parallel Sequencing for the Korean Population.

Mitochondrial DNA (mtDNA) genome analysis has been a potent tool in forensic practice as well as in the understanding of human phylogeny in the maternal lineage. The traditional mtDNA analysis is focused on the control region, but the introduction of massive parallel sequencing (MPS) has made the typing of the entire mtDNA genome (mtGenome) more accessible for routine analysis. The complete mtDNA information can provide large amounts of novel genetic data for diverse populations as well as improved discrimination power for identification. The genetic diversity of the mtDNA sequence in different ethnic populations has been revealed through MPS analysis, but the Korean population not only has limited MPS data for the entire mtGenome, the existing data is mainly focused on the control region. In this study, the complete mtGenome data for 186 Koreans, obtained using Ion Torrent Personal Genome Machine (PGM) technology and retrieved from rather common mtDNA haplogroups based on the control region sequence, are described. The results showed that 24 haplogroups, determined with hypervariable regions only, branched into 47 subhaplogroups, and point heteroplasmy was more frequent in the coding regions. In addition, sequence variations in the coding regions observed in this study were compared with those presented in other reports on different populations, and there were similar features observed in the sequence variants for the predominant haplogroups among East Asian populations, such as Haplogroup D and macrohaplogroups M9, G, and D. This study is expected to be the trigger for the development of Korean specific mtGenome data followed by numerous future studies.

Influence of immunologic status on age prediction using signal joint T cell receptor excision circles.

Age estimation based on quantifying signal joint T cell receptor excision circle (sjTREC) in T cells has been established to be a promising approach in forensic practice and demonstrated in different ethnic groups. Considering that the homeostasis of T cells carrying sjTRECs is closely related to the immunologic status of a person, it is important to investigate the influence of various immunologic statuses on the age estimation model. In this study, quantification of sjTREC contents was performed for groups of people with various immune system statuses, and the result showed less correlation with chronological age (r 2 = 0.424) than in the healthy group (r 2 = 0.648). The simulation model indicated that this influence could increase the range of prediction in the age estimation model, and the mean absolute deviation (MAD) between chronological age and predicted age. Through this study, it was demonstrated that immunologic status is a factor that affects the accuracy of age prediction using sjTREC quantification.

Kinship Testing Based on SNPs Using Microarray System.

BACKGROUND: Kinship testing using biallelic SNP markers has been demonstrated to be a promising approach as a supplement to standard STR typing, and several systems, such as pyrosequencing and microarray, have been introduced and utilized in real forensic cases. The Affymetrix microarray containing 169 autosomal SNPs developed for forensic application was applied to our practical case for kinship analysis that had remained inconclusive due to partial STR profiles of degraded DNA and possibility of inbreeding within the population. CASE REPORT: 169 autosomal SNPs were typed on array with severely degraded DNA of two bone samples, and the kinship compared to genotypes in a reference database of their putative family members. RESULTS: Two bone samples remained unidentified through traditional STR typing with partial profiles of 10 or 14 of 16 alleles. Because these samples originated from a geographically isolated population, a cautious approach was required when analyzing and declaring true paternity only based on PI values. In a supplementary SNP typing, 106 and 78 SNPs were obtained, and the match candidates were found in each case with improved PI values than using only STRs and with no discrepant SNPs in comparison. CONCLUSION: Our case showed that the utility of multiple SNPs on array is expected in practical forensic caseworks with an establishment of reference database.