Developmental validation of the AGCU YNFS Y Kit: A new 6-dye multiplex system with 44 Y-STRs and 5 Y-InDels for forensic application.

With the widespread use of the Y chromosome in genetics, a lot of commercially available Y chromosome kits were developed, validated, and applied to forensic science practice. The AGCU YNFS Y Kit is a new Y chromosome system containing forty-four preferred Y short tandem repeats (Y-STRs) and five common Y-InDels. In this study, the AGCU YNFS Y system was validated to verify its performance by following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM). A series of validation experiments included the following parameters: PCR-based studies, sensitivity studies, species specificity studies, stability studies, mixture studies, precision studies, stutter calculation, mutation and statistical analysis, population study, and case samples and degradation studies. The results suggested that appropriately changing PCR amplification conditions did not affect genotyping; the kit had good sensitivity for trace amounts of DNA (0.0625 ng), mixtures of multiple male individuals (minor: major = 1: 9), and three PCR inhibitors (more than 250 µM hematin, 250 ng/µL humic acid and 50 ng/µL tannic acid). The maximum standard deviation of allele size did not exceed 0.1552 reflecting the high accuracy of the system. By this, 87 DNA-confirmed pairs of father-son pairs were also analyzed for mutations. A total of 18 loci were mutated, with mutation rates ranging from 11.5×10-3 to 34.5×10-3 (95% CI 7.2×10-3-97.5×10-3, DYS627 and DYF404S1). In the population study, the haplotype diversity of 87 unrelated individuals was 0.9997, and discrimination capacity was 0.9885. Degradation studies have demonstrated that UV-C light exposure for up to 120 hours has no effect on male blood and semen-vaginal secretion mixtures. However, complete typing could no longer be obtained after 48 hours of UV exposure in single male saliva and in male saliva and female blood mixed samples. Collectively, the AGCU YNFS Y Kit is sensitive and accurate and can play its application value in forensic science practice.

Validation of the PowerPlex®35GY System: a novel eight-dye STR multiplex kit on the Spectrum Compact CE System.

The PowerPlex® 35GY System (Promega, USA) is an advanced eight-dye multiplex STR kit, incorporating twenty-three autosomal STR loci, eleven Y chromosome STR loci, one sex determining marker Amelogenin, and two quality indicators. This multiplex system includes 20 CODIS loci and up to 15 mini-STR loci with sizing values less than 250 bases. In this study, validation for PowerPlex® 35GY System was conducted following the guidelines of SWGDAM, encompassing sensitivity, precision, accuracy, concordance, species specificity, stutter, mixture, stability, and degraded DNA. The results from experiments demonstrated that the PowerPlex® 35GY System could effectively amplify DNA samples, with complete allele detection achieved at 125 pg. Moreover, over 90% of alleles from minor contributors were detected at a mixed ratio of 1:4. Additionally, the system was found to yield full profiles even in the presence of hematin, humic acid, and indigo. The PowerPlex® 35GY System demonstrated superior performance in the sensitivity and degraded DNA studies compared to a six-dye STR kit. Hence, it is evident that the PowerPlex® 35GY System is well-suited for forensic practice, whether in casework or for database samples. These findings provide strong support for the efficacy and reliability of the PowerPlex® 35GY System in forensic applications.

Discovery unbalanced DNA mixtures and evaluation mixing ratio via a droplet digital PCR method.

BACKGROUND: Small amounts of DNA from a perpetrator collected during crime-scene investigations can be masked by large amounts of DNA from the victim. These samples can provide important information for the perpetrator's conviction. Short tandem repeat (STR) detection system is not sensitive enough to detect trace amounts of minor components in unbalanced mixed DNA. We developed a system using droplet digital polymerase chain reaction (ddPCR) capable of discovering trace components and accurately determining the ratio of mixed DNA in extremely unbalanced mixtures. METHODS: The non-recombining regions of the X chromosome and Y chromosome were quantified in the DNA of male and female mixtures using duplex ddPCR. Absolute quantification of low-abundance portions of trace samples and unbalanced mixtures was done using different mixing ratios. RESULTS: The ddPCR system could be used to detect low-abundance samples with < 5 copies of DNA components in an extremely unbalanced mixture at a mixing ratio of 10000:1. The high sensitivity and specificity of the system could identify the mixing ratio of mixed DNA accurately. CONCLUSIONS: A ddPCR system was developed for evaluation of mixed samples of male DNA and female DNA. Our system could detect DNA quantities as low as 5 copies in extremely unbalanced mixed samples with good specificity and applicability. This method could assist forensic investigators in avoiding the omission of important physical evidence, and evaluating the ratio of mixed male/female trace samples.

A preliminary study on identification of the blood donor in a body fluid mixture using a novel compound genetic marker blood-specific methylation-microhaplotype.

Blood-containing mixtures are frequently encountered at crime scenes involving violence and murder. However, the presence of blood, and the association of blood with a specific donor within these mixtures present significant challenges in forensic analysis. In light of these challenges, this study sought to address these issues by leveraging blood-specific methylation sites and closely linked microhaplotype sites, proposing a novel composite genetic marker known as "blood-specific methylation-microhaplotype". This marker was designed to the detection of blood and the determination of blood donor within blood-containing mixtures. According to the selection criteria mentioned in the Materials and Methods section, we selected 10 blood-specific methylation-microhaplotype loci for inclusion in this study. Among these loci, eight exhibited blood-specific hypomethylation, while the remaining two displayed blood-specific hypermethylation. Based on data obtained from 124 individual samples in our study, the combined discrimination power (CPD) of these 10 successfully sequenced loci was 0.999999298. The sample allele methylation rate (Ram) was obtained from massive parallel sequencing (MPS), which was defined as the proportion of methylated reads to the total clustered reads that were genotyped to a specific allele. To develop an allele type classification model capable of identifying the presence of blood and the blood donor, we used the Random Forest algorithm. This model was trained and evaluated using the Ram distribution of individual samples and the Ram distribution of simulated shared alleles. Subsequently, we applied the developed allele type classification model to predict alleles within actual mixtures, trying to exclude non-blood-specific alleles, ultimately allowing us to identify the presence of blood and the blood donor in the blood-containing mixtures. Our findings demonstrate that these blood-specific methylation-microhaplotype loci have the capability to not only detect the presence of blood but also accurately associate blood with the true donor in blood-containing mixtures with the mixing ratios of 1:29, 1:19, 1:9, 1:4, 1:2, 2:1, 7:1, 8:1, 31:1 and 36:1 (blood:non-blood) by DNA mixture interpretation methods. In addition, the presence of blood and the true blood donor could be identified in a mixture containing four body fluids (blood:vaginal fluid:semen:saliva = 1:1:1:1). It is important to note that while these loci exhibit great potential, the impact of allele dropouts and alleles misidentification must be considered when interpreting the results. This is a preliminary study utilising blood-specific methylation-microhaplotype as a complementary tool to other well-established genetic markers (STR, SNP, microhaplotype, etc.) for the analysis in blood-containing mixtures.

Internal validation of the GA118-24B Genetic Analyzer, a stable capillary electrophoresis system for forensic DNA identification.

The GA118-24B Genetic Analyzer (hereafter, "GA118-24B") is an independently developed capillary electrophoresis instrument. In the present research, we designed a series of validation experiments to test its performance at detecting DNA fragments compared to the Applied Biosystems 3500 Genetic Analyzer (hereafter, "3500"). Three commercially available autosomal short tandem repeat multiplex kits were used in this validation. The results showed that GA118-24B had acceptable spectral calibration for three kits. The results of accuracy and concordance studies were also satisfactory. GA118-24B showed excellent precision, with a standard deviation of less than 0.1 bp. Sensitivity and mixture studies indicated that GA118-24B could detect low-template DNA and complex mixtures as well as the results generated by 3500 in parallel experiments. Based on the experimental results, we set specific analytical and stochastic thresholds. Besides, GA118-24B showed superiority than 3500 within certain size ranges in the resolution study. Instead of conventional commercial multiplex kits, GA118-24B performed stably on a self-developed eight-dye multiplex system, which were not performed on 3500 Genetic Analyzer. We compared our validation results with those of previous research and found our results to be convincing. Overall, we conclude that GA118-24B is a stable and reliable genetic analyzer for forensic DNA identification.

A preliminary exploration for co-detecting RNA virus and STR type on capillary electrophoresis in forensic practice.

RNA virus infection such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection shows severe respiratory symptoms on human and could be an obvious individual characteristic for investigations in forensic science. As for biological samples suspected to contain RNA virus in forensic casework, it requires respective detection of viral RNA and human DNA: reverse transcriptase polymerase chain reaction and DNA type (short tandem repeat [STR] analysis). Capillary electrophoresis (CE) has been shown to be a versatile technique and used for a variety of applications, so we preliminarily explored the co-detection of RNA virus and STR type on CE by developing a system of co-detecting SARS-CoV-2 and STR type under ensuring both the efficiency of forensic DNA analysis and safety of the laboratory. This study investigated the development and validation of the system, including N and ORF1ab primer designs, polymerase chain reaction amplification, allelic ladder, CE detection, thermal cycling parameters, concordance, sensitivity, species specificity, precision, and contrived and real SARS-CoV-2 sample studies. Final results showed the system could simultaneously detect SARS-CoV-2 and STR type, further indicating that CE has possibilities in the multi-detection of RNA viruses/STR type to help to prompt individual characteristics (viral infection) and narrow the scope of investigation in forensic science.

Combining the third molar mineralization to further improve the accuracy of the Kvaal's method in dental age estimation of subadults in northern China.

The morphological changes based on deposition of secondary dentin and mineralization of the third molar have been proven to be related to chronological age. However, Kvaal's method on the theory of deposition of secondary dentin was controversial with respect to dental age estimation in the recent research. The aim of this study was to combine the parameters of Kvaal's method with relatively high correlation coefficients and mineralization stages of the third molar to improve the accuracy of predicting the dental age of subadults in northern China. A total of 340 digital orthopantomograms of subadults aged from 15 to 21 years were analysed. A training group was used to test the accuracy of the original Kvaal's method and to establish novel methods for subadults in northern China. A testing group was used to compare the accuracy of the newly established methods with the Kvaal's original method and with published method specifically used in northern China. To increase the feasibility of our estimation model, we combined the mineralization of the third molar to build a combined specific formula. The results showed that the combined specific model increased the coefficient of determination to 0.513, and the standard error of the estimate was reduced to 1.482 years. We concluded that the combined specific model based on the deposition of secondary dentin and mineralization of the third molar could improve the accuracy of dental age assessment of subadults in northern China. Key Points: The decrease in the dental pulp cavity based on deposition of secondary dentin is a useful variable for assessing age.A total of 340 orthopantomographs were used in this research, including 278 in training groups and 62 in testing groups.Original Kvaal's method underestimated the dental age for subadults in northern China.The equation of combined specific method constructed in our study was proved more suitable to calculate dental age for subadults in northern China.

Preliminary study on genetic factors related to Demirjian's tooth age estimation method based on genome-wide association analysis.

The age determination of individuals, especially minors, is critical in forensic research. In forensic practice, dental age estimation is one of the most commonly used methods for determining age as teeth are easy to preserve and relatively resistant to environmental factors. Tooth development is affected and regulated by genetic factors; however, these are not incorporated into current commonly used tooth age inference methods, leading to unreliable results. Here, we established a Demirjian and a Cameriere tooth age estimation-based methods suitable for use in children in southern China. By using the difference between the inferred age and the actual age (MD) as the phenotype, we identified 65 and 49 SNPs related to tooth age estimation from 743,722 loci among 171 children in southern China through a genome-wide association analysis (p<0.0001). We also conducted a genome-wide association study on dental development stage (DD) using the Demirjian tooth age estimation method and screened two sets of SNP sites (52 and 26) based on whether age difference was considered. The gene function enrichment analysis of these SNPs found that they were related to bone development and mineralization. Although SNP sites screened based on MD seem to improve the accuracy of tooth age estimation, there is little correlation between these SNPs and an individual's Demirjian morphological stage. In conclusion, we found that individual genotypes can affect tooth age estimation, and based on different phenotypic analysis models, we have identified some novel SNP sites related to tooth age inference and Demirjian's tooth development stage. These studies provide a reference for subsequent phenotypic selection based on tooth age inference analysis, and the results could possibly be used in the future to make forensic age estimation more accurate.

Developing a male-specific age predictive model based on Y-CpGs for forensic analysis.

In forensic work, predicting the age of the criminal suspect or victim could provide beneficial clues for investigation. Epigenetic age estimation based on age-correlated DNA methylation has been one of the most widely studied methods of age estimation. However, almost all available epigenetic age prediction models are based on autosomal CpGs, which are only applicable to single-source DNA samples. In this study, we screened the available methylation data sets to identify loci with potential to meet the objectives of this study and then established a male-specific age prediction model based on 2 SNaPshot systems that contain 13 Y-CpGs and the mean absolute deviation (MAD) values were 4-6 years. The multiplex methylation SNaPshot systems and age-predictive model have been validated for sensitivity (the DNA input could be as low as 0.5 ng) and male specificity. They are supposed to have feasibility in forensic practice. In addition, it demonstrated that the method was also applicable to bloodstains, which were commonly found at crime scenes. The results showed good performance (the training set: R2 = 0.9341, MAD = 4.65 years; the test set: R2 = 0.8952, MAD = 5.73 years) in case investigation for predicting male age. For mixtures, when the male to female DNA ratio is 1:1, 1:10, the deviation between the actual age and the predicted age obtained by the model was less than 8 years, which offers great hope for future prediction of the age of males in mixtures and will be a powerful tool for special cases, such as sexual assault. Furthermore, the work provides a basis for the application of Y-CpGs in forensic science.

Large fragment Sanger sequencing identifies the newly encountered variant that caused null alleles in parentage testing.

Short tandem repeat (STR) is regarded as a crucial tool for personal identification as well as parentage testing. Thus, genotyping errors of STRs could have negative effects on the reliability of forensic identification. A null allele at the combined DNA index system (CODIS) core loci D2S1338 was found in a father-daughter pair with the AGCU Expressmarker 22 kit which was a commonly used commercial kit during our daily laboratory work. This null allele caused the father and daughter to not conform to the laws of inheritance, thus potentially generating erroneous conclusions that excluded parentage. To figure out the reason for this phenomenon, re-amplification with new primers and then large fragment Sanger sequencing was conducted. We found a G to G/T variation at the position which is fifty-nine bases away from the 3' end of the core repeat in both samples. This probably could be considered a novel variant at the primer binding region which had not been reported that resulted in the emergence of the null allele. We also found that there was more than one single-nucleotide polymorphism (SNP) with minor allele frequency (MAF) greater than 0.1 in the upstream and downstream sequences of D2S1338. When designing primers for amplification of D2S1338, the possible adverse results of these SNPs should be taken into account and avoided.

Corrigendum: A novel SNP-STR system based on a capillary electrophoresis platform.

[This corrects the article DOI: 10.3389/fgene.2021.636821.].

The application of short and highly polymorphic microhaplotype loci in paternity testing and sibling testing of temperature-dependent degraded samples.

Paternity testing and sibling testing become more complex and difficult when samples degrade. But the commonly used genetic markers (STR and SNP) cannot completely solve this problem due to some disadvantages. The novel genetic marker microhaplotype proposed by Kidd's research group combines the advantages of STR and SNP and is expected to become a promising genetic marker for kinship testing in degraded samples. Therefore, in this study, we intended to select an appropriate number of highly polymorphic SNP-based microhaplotype loci, detect them by the next-generation sequencing technology, analyze their ability to detect degraded samples, calculate their forensic parameters based on the collected 96 unrelated individuals, and evaluate their effectiveness in paternity testing and sibling testing by simulating kinship relationship pairs, which were also compared to 15 STR loci. Finally, a short and highly polymorphic microhaplotype panel was developed, containing 36 highly polymorphic SNP-based microhaplotype loci with lengths smaller than 100 bp and A e greater than 3.00, of which 29 microhaplotype loci could not reject the Hardy-Weinberg equilibrium and linkage equilibrium after the Bonferroni correction. The CPD and CPE of these 29 microhaplotype loci were 1-2.96E-26 and 1-5.45E-09, respectively. No allele dropout was observed in degraded samples incubated with 100°C hot water for 40min and 60min. According to the simulated kinship analysis, the effectiveness at the threshold of 4/-4 reached 98.39% for relationship parent-child vs. unrelated individuals, and the effectiveness at the threshold of 2/-2 for relationship full-sibling vs. unrelated individuals was 93.01%, which was greater than that of 15 STR loci (86.75% for relationship parent-child vs. unrelated individuals and 81.73% for relationship full-sibling vs. unrelated individuals). After combining our 29 microhaplotype loci with other 50 short and highly polymorphic microhaplotype loci, the effectiveness values at the threshold of 2/-2 were 82.42% and 90.89% for relationship half-sibling vs. unrelated individuals and full-sibling vs. half-sibling. The short and highly polymorphic microhaplotype panel we developed may be very useful for paternity testing and full sibling testing in degraded samples, and in combination with short and highly polymorphic microhaplotype loci reported by other researchers, may be helpful to analyze more distant kinship relationships.

Bacterial Succession in Microbial Biofilm as a Potential Indicator for Postmortem Submersion Interval Estimation.

Bacteria acts as the main decomposer during the process of biodegradation by microbial communities in the ecosystem. Numerous studies have revealed the bacterial succession patterns during carcass decomposition in the terrestrial setting. The machine learning algorithm-generated models based on such temporal succession patterns have been developed for the postmortem interval (PMI) estimation. However, the bacterial succession that occurs on decomposing carcasses in the aquatic environment is poorly understood. In the forensic practice, the postmortem submersion interval (PMSI), which approximately equals to the PMI in most of the common drowning cases, has long been problematic to determine. In the present study, bacterial successions in the epinecrotic biofilm samples collected from the decomposing swine cadavers submerged in water were analyzed by sequencing the variable region 4 (V4) of 16S rDNA. The succession patterns between the repeated experimental settings were repeatable. Using the machine learning algorithm for establishing random forest (RF) models, the microbial community succession patterns in the epinecrotic biofilm samples taken during the 56-day winter trial and 21-day summer trial were determined to be used as the PMSI predictors with the mean absolute error (MAE) of 17.87 ± 2.48 ADD (≈1.3 day) and 20.59 ± 4.89 ADD (≈0.7 day), respectively. Significant differences were observed between the seasons and between the substrates. The data presented in this research suggested that the influences of the environmental factors and the aquatic bacterioplankton on succession patterns of the biofilm bacteria were of great significance. The related mechanisms of such influence need to be further studied and clarified in depth to consider epinecrotic biofilm as a reliable predictor in the forensic investigations.

DNA methylation analysis for smoking status prediction in the Chinese population based on the methylation-sensitive single-nucleotide primer extension method.

In some criminal cases, the identity of suspect is unknown and there is no matching DNA profile in the DNA database. Forensic DNA Phenotyping can provide useful investigative information for these cases. Most forensic studies focus on visible characteristics rather than behavioral characteristics. However, smoking is prevalent in the Chinese population, and DNA methylation is the most promising biomarker for smoking. We collected 204 whole blood samples from the Chinese population and measured methylation levels of 9 smoking-related CpG loci using the methylation-sensitive single-nucleotide primer extension method (Ms-SnuPE). But the single-base extension primers of loci cg12803068 and cg21566642 contained other CpG sites, which may introduce bias, and only the other 7 CpG loci were included in subsequent statistical analysis. The methylation level of locus cg05575921 near the aromatic hydrocarbon receptor repressor (AHRR) gene was much lower in the current smoker group than in the never smoker group. To evaluate the ability of each of 7 CpG loci to predict smoking status, the logistic regression (LR) models were established separately, and locus cg05575921 had the best ability to predict smoking status compared with the other 6 loci. Then, combined (including loci cg19572487, cg05575921, cg23480021, cg23576855, cg21161138, cg01940273, and cg09935388) and stepwise (including loci cg05575921 and cg01940273) multinomial logistic regression (MLR) models were also established. Both combined and stepwise MLR models had good efficiencies in predicting smoking status, and outperformed the above 7 LR models. However, the accuracy, specificity and area under the curve (AUC) of stepwise MLR model in the testing dataset were slightly higher than those of combined MLR model, and the stepwise MLR model required less loci information. Therefore, the stepwise MLR model based on 2 significant CpG loci was more recommended model for predicting smoking status in the Chinese population, and the formula was as follow: P = 1/(1 +e-(10.621-10.005*cg05575921-8.770*cg01940273)). Mainly 2 CpG loci (cg05575921 and cg01940273) played a major role in the prediction of smoking status, and the other 5 CpG loci contributed less. Moreover, for evaluating the ability of each of 7 CpG loci to predict cigarette consumption, the polynomial regression formulas were established separately. As the adjusted R2 was between 0.00 and 0.20, the methylation levels of these 7 loci were not closely associated with the cigarette consumption. Our methylation assay is simple, economical, and available in conventional forensic laboratories, and may be useful in assessing the smoking status of unknown suspects.

Application of Cameriere's method for dental age estimation in children in South China.

The aim of this study was to evaluate the applicability of Cameriere's European formula for age estimation in children in South China and to adapt the formula to establish a more suitable formula for these children. Moreover, the performance of dental age estimation based on Cameriere's method combining the developmental information of permanent teeth (PT) and third molar (TM) was also analysed. Orthopantomographs of 720 healthy children in Group A, and orthopantomographs of 320 children and 280 subadults in Group B were assessed. The samples of Group A were divided into training dataset 1 and test dataset 1, and the samples of Group B were also divided into training dataset 2 and test dataset 2. A South China-specific formula was established based on the training dataset 1, and the comparison of accuracy between the Cameriere's European formula and the South China-specific formula was conducted with the test dataset 1. Additionally, a PT regression model, a TM regression model, and a combined regression model (PT + TM) were established based on the training dataset 2, and the performance of these three models were validated on the test dataset 2. The Cameriere's European formula underestimated chronological age with a mean difference (ME) of -0.47 ± 1.11 years in males and -0.69 ± 1.19 years in females. However, the South China-specific formula underestimated chronological age, with a mean difference (ME) of -0.02 ± 0.71 years in males and -0.14 ± 0.73 years in females. Compared with PT model and TM model, the PT and TM combined model obtained the smallest root mean square error (RMSE) of 1.29 years in males and 0.93 years in females. In conclusion, the South China-specific formula was more suitable for assessing the dental age of children in South China, and the PT and TM combined model can improve the accuracy of dental age estimation in children.Key pointsOrthopantomographs of 720 healthy children in Group A, and orthopantomographs of 320 children and 280 subadults in Group B were assessed.A South China-specific formula was established based on the training dataset 1, and the comparison of accuracy between the Cameriere's European formula and the South China-specific formula was conducted with the test dataset 1.A PT regression model, a TM regression model, and a combined regression model (PT + TM) were established based on the training dataset 2, and the performance of these three models were validated on the test dataset 2.The South China-specific formula was more suitable for assessing the dental age of children in South China, and the PT and TM combined model can improve the accuracy of dental age estimation in children.

Validation of the AGCU Expressmarker 20 + 20Y kit: A 6-dye multiplex assay for forensic application.

The AGCU Expressmarker 20 + 20Y Kit is a newly devised short tandem repeat (STR) multiplex system that simultaneously analyses a set of 19 autosomal STR loci, 20 Y chromosomal STR loci and the amelogenin locus with six-dye fluorescent labelling. Here, the AGCU Expressmarker 20 + 20Y system was validated following the guidelines published by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including PCR-condition, sensitivity, mixtures, species specificity, models of inhibition, precision, stutter percentage, concordance, population genetic studies, performance on three kinds of degraded DNA and a type of casework samples. The results indicated that the kit had high sensitivity when there was a small amount of DNA (0.0625 ng), more than one male (minor: major=1:19), or a mixture of males and females (male: female=1:32), models of inhibition (250 µM hematin, 1500 ng/µL humic acid and more than 100 ng/µL tannic acid) and degraded samples. The kit showed high precision level with standard deviation of allele size no more than 0.0930. Furthermore, this system was also tested in 444 random male samples of Chinese Han and Hui population, showing its high discrimination capability in Han and Hui population. Meanwhile, the system was applicable to the case of the AMELY abnormality. In short, the kit was verified and proved to be a robust, reliable and suitable tool for human identification and casework samples.

Forensic examination and application of areca nuts as material evidence.

In this study, we aimed to explore the possibility of DNA analysis of areca nut as material evidence and the value of short tandem repeat (STR) typing of areca nut as material evidence under the condition of simulating external environment. In this study, water soaking, soil burial, sun exposure, and wet environment were used to treat areca nut residues. Chelex 100 was used to extract DNA, the PowerPlex21 kit to amplify, and the ABI PRISM® 310 Genetic Analyzer to analyze the DNA of areca nut residues. DNA and STR typing were performed to analyze the residue after chewing. The results showed that the number of residual sites decreased with time under the conditions of water soaking, soil burial, sun exposure, and wet environment. Thus, areca nut can be used as forensic material evidence for DNA analysis and individual identification.

Comparing the accuracy of Demirjian and Nolla methods and establishing a new method for dental age estimation in northeastern Chinese children.

Dental age estimation plays an important role in the field of clinic medicine and forensic medicine. The Demirjian and Nolla methods are common scoring methods for dental age estimation but there was no research about the comparison of accuracy of these two methods in northeastern Chinese children. Hence, in this study, we compared the accuracy of these two methods to explore more suitable method for our studied population. We collected 535 orthopantomograms from northern Chinese children aged from 6 to 15 years and divided them into training dataset and testing dataset according to the ratio of 7:3. The dental age of training dataset were estimated using Demirjian and Nolla methods, respectively. The results suggested that the mean differences of these two methods were 0.24 and -0.40 years, and mean absolute difference were 0.65 and 0.59 years. Then to further improve the accuracy of dental age assessment, the new improved formulas and dental age conversion tables were established after analyzing the relationship between the sum scores based on Nolla method and chronology age in training dataset. According to the new method used in testing dataset, the minimum value of mean difference (0.00) and mean absolute difference (0.49) were obtained, which are largely smaller than that of Demirjian and Nolla methods. The new developed method and dental age conversion scales may be more suitable dental age estimation method for northeastern Chinese children.

Development and validation of novel 8-dye short tandem repeat multiplex system for forensic applications.

DNA profiling of short tandem repeats (STRs) is the primary method for genotyping forensic samples. However, degraded DNA and trace samples are still major problems for commercial 5- or 6-dye STR kits. In order to improve the performance of this method, we developed a novel 8-dye STR multiplex system containing 18 autosomal loci (D3S1358, D1S1656, TPOX, D16S539, vWA, D6S1043, D2S1338, CSF1PO, D19S433, D7S820, FGA, D8S1179, D5S818, D13S317, TH01, D21S11, D12S391, and PentaD) and the sex-determining locus Amelogenin, with all fragments smaller than 330 bases. Validation was carried out as recommended by the Scientific Working Group on DNA Analysis Methods. The results showed that complete profiles were obtainable when the input DNA was as low as 0.0625 ng. Full profiles were obtained even in the presence of inhibitors such as humic acid (< 300 ng/µl), hematin (< 100 µM), and indigo (0.01%). The 8-dye STR multiplex system also showed good performance in the detection degraded DNA samples. These results indicate that the 8-dye STR multiplex system is suitable for human DNA genotyping, including for difficult forensic materials.