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.

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.

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.

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.

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.

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.

An 18 Multi-InDels panel for analysis of highly degraded forensic biological samples.

DNA genotyping from trace and highly degraded biological samples is one of the most significant challenges of forensic DNA identification. There is a lack of simple and effective methods for genotyping highly degraded samples. In this study, a multiple loci insertion/deletion polymorphisms (Multi-InDels) panel was designed for detecting 18 autosomal Multi-InDels through capillary electrophoresis (CE) with amplicon sizes no longer than 125 bp. Studies of sensitivity, degradation, and species specificity were performed and a population study was carried out using 192 samples from Han populations in Hunan province in the south of China. The combined random match probability (CMP) of these 18 Multi-InDels was 3.23 × 10-12 and the cumulative probability of exclusion (CPE) was 0.9989, suggesting this panel could be used independently for human identification and could provide efficient supporting information for parentage testing. Complete profiles were obtained from as low as 62.5 pg of total input DNA after increasing the number of PCR cycles. Moreover, all alleles were detected from artificially highly degraded DNA after 80 min of boiling water bath treatment. This 18 Multi-InDels panel is simple, fast, and effective for the forensic analysis of highly degraded DNA.

Considering the flanking region variants of nonbinary SNP and phenotype-informative SNP to constitute 30 microhaplotype loci for increasing the discriminative ability of forensic applications.

The flanking region variants of nonbinary SNPs and phenotype-informative SNPs (piSNPs) have been observed, which may greatly improve the discriminative ability after constituting microhaplotype. In this study, 30 microhaplotype loci based on the nonbinary SNPs and piSNPs (shown to be related to phenotypes such as hair and eye color) were selected. Genotyping were conducted on 100 unrelated northern Han Chinese, and the 26 populations from the 1000 Genome Project were also included for comparison of populations differentiation. The simulated study was conducted for evaluating the efficiency of kinship testing. These 30 microhaplotype loci we selected had good polymorphism, with a mean effective number of alleles (Ae) of 3.46. The average Ae increase was 1.27 compared with the target SNPs. The populations from the five regions worldwide could also be distinguished using these loci. The results of kinship testing showed that these microhaplotype loci had the similar ability as 15 STR loci of AmpFlSTRR IdentifilerR PCR Amplification Kit to identify the biological parent and a stronger ability to exclude the nonbiological parents. So, these 30 microhaplotype loci may be multifunctional for forensic application, including the ability of personal identification and kinship testing equivalent to 15 STR loci, and the power of ancestry inference for distinguishing the main intercontinental population. Moreover, our selected phenotypic microhaplotype loci may theoretically have phenotype prediction capabilities. But the phenotype prediction efficiency of these phenotypic microhaplotype loci may be worse than that of piSNPs and the detailed prediction accuracy of different populations needs to be further studied.

Forensic characteristics and genetic structure of 18 autosomal STR loci in the Sierra Leone population.

Population genetic analysis is of vital importance for personal identification and paternity testing in forensic science. However, the forensic characteristics of autosomal short tandem repeat (STR) loci in the Sierra Leone population have not been reported yet to the best of our knowledge. In this study, 528 unrelated individuals (256 males and 272 females) in Sierra Leone, West Africa, were genotyped using the DNA Typer19™ kit; forensic parameters and genetic relationships with 32 populations around the world were analyzed. A total of 239 alleles were detected, with corresponding allele frequencies ranged from 0.0009 to 0.4545. The cumulative power of discrimination (CPD) value of the 18 STR loci was 0.9999999999999999999999697; the cumulative probability of exclusion for duos (CPE duos) and exclusion for trios (CPE trios) were 0.99999343 and 0.9999999895, respectively. Genetic comparisons showed that the Sierra Leone population has a closer genetic relationship with the Bantu-speaking populations in Sub-Saharan Africa.

Population data and genetic structure analysis based on 29 Y-STR loci among the ethnolinguistic groups in Burkina Faso.

Burkina Faso (BF) is a landlocked Sahelian country located in the middle of West Africa. Sixty-three local languages are spoken in BF. Despite this high diversity, the BF population remains poorly investigated, and updated forensic parameters with a large number of Y chromosome short tandem repeats (Y-STRs) are still missing. Herein, 447 DNA samples were typed for a cocktail of 29 Y-STR loci. None of these 447 individuals in total shared a common haplotype. The overall Y-STR haplotypes were successfully uploaded online on the Y-STR Haplotype Reference Database (YHRD) with the accession numbers YA004690 and YA004691. The main haplotype diversity was 0.9999999965, which is much higher than that obtained with 12 Y-STRs in a previous study. Haploid Match Probability for the whole dataset was 0.002237. The phylogenetic analysis of 24 ethnic groups of BF shows that the ethnic group named BISSA is closer to Gur speakers than Mande speakers, where they belong. In addition, genetic structure analysis of 49 African subpopulations sheds light on the fact that geographic proximity turns out to be one of the best predictors of genetic affinity between populations.

Forensic parameters and genetic structure analysis of 30 autosomal InDels of the population in Freetown, Sierra Leone.

As the origin of modern humanity, African populations show high genetic diversity and are attracting increasing academic attention. However, populations living in West Africa have so far received less study and exploration. In this study, we analyze 30 insertion/deletion (InDel) loci of 516 samples from Freetown, Sierra Leone, to evaluate the forensic properties and reveal the genetic structure in Freetown, Sierra Leone, West Africa. No significant linkage disequilibrium (LD) between 30 InDels was observed after the Bonferroni correction. The random match probability (RMP), the combined power of exclusion for duos (CPE duos), and the combined power of exclusion for trios (CPE trios) were 6.823 × 10-11, 0.9168, and 0.9731, respectively. Null alleles and off-ladder alleles were observed, suggesting that we should be cautious when using this kit for forensic caseworks in African populations. In the population comparison study, we found that the Freetown population is genetically closer to geographically distinct West Africans and has a closer genetic relationship with the Bantu-speaking populations than other African populations.

Developmental validation of the STRscan-17LC kit: a 6 Dye STR kit enhanced stability and ability to detect degraded samples.

Genotyping of short tandem repeat (STR) markers is the basic method of forensic science. Enhanced technologies are needed to meet the requirements of databasing and casework samples. The STRscan-17LC kit is a 6 Dye STR kit which amplifies 16 STR loci: D3S1358, TPOX, D16S539, vWA, D2S1338, CSF1PO, D19S433, D7S820, FGA, D8S1179, D5S818, D13S317, D18S51, TH01, D12S391, and D21S11 and the sex-determinant locus amelogenin. This kit is designed for better tolerance to PCR inhibitors and analysis of mildly degraded samples with all fragments smaller than 330 bases. In this study, the STRscan-17LC kit is validated according to the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines, including PCR-based studies, sensitivity, precision and accuracy, inhibitors, species specificity, DNA mixture studies, population, and concordance studies. The validation results suggest that the STRscan-17LC kit is a useful tool for forensic application.

Feasibility of using probabilistic methods to analyse microRNA quantitative data in forensically relevant body fluids: a proof-of-principle study.

Several studies have confirmed that microRNAs (miRNAs) are promising markers for body fluid identification since they were introduced to this field. However, there is no consensus on the choice of reference genes and identification strategies. In this study, 13 potential candidate miRNAs were screened from three forensically relevant body fluid datasets, and the expression of 12 markers in five body fluids was determined using a real-time quantitative method. Two probabilistic approaches, Naive Bayes (NB) and partial least squares discriminant analysis (PLS-DA), were then applied to predict the origin of the samples to determine whether probabilistic methods are helpful in body fluid identification using miRNA quantitative data. Furthermore, 14 reference combinations were used to validate the influence of different reference choices on the predicted results simultaneously. Our results showed that in the NB model, leave-one-out cross-validation (LOOCV) achieved 100% accuracy and the prediction accuracy of the test set was 100% in most reference combinations. In the PLS-DA model, the first two components could interpret about 80% expression variance and LOOCV achieved 100% accuracy when miR-92a-3p was used as the reference. This study preliminarily proved that probabilistic approaches hold huge potential in miRNA-based body fluid identification, and the choice of references influences the prediction results to a certain extent.

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.

Genetic characterization of 19 X-STRs in Sierra Leone population from Freetown.

A total of 550 individuals (265 males and 285 females) from Sierra Leone, a west-African coastal country, were genotyped using the Microreader™ 19X ID System kit. No significant deviations from the Hardy-Weinberg equilibrium were observed. A total of 250 alleles were identified with corresponding allele frequencies spanning from 0.0012 to 0.6762. PIC of the loci ranged from 0.4615 to 0.9481. The CPE, CPDF, and CPDM were 0.9999997856, 0.999999999999999999995774, and 0.999999999998997, respectively. The highly combined MECKruger, MECKishida, MECDesmarais, and MECDesmarais Duo were achieved as 0.99999992508, 0.999999999990802, 0.999999999990836, and 0.99999998412, respectively. Genetic comparisons revealed that genetic homogeneity existed in similar ethno origin or geographic origin populations. This is a pioneering genetic investigation using the Microreader™ 19X ID System kit in the population of Sierra Leone.

Accuracy of the Demirjian and Willems methods of dental age estimation for children from central southern China.

The aim of this study was to compare the accuracy of the Demirjian method and the Demirjian method as revised by Willems for age estimation based on orthopantomograms from central southern Chinese Han population aged 8-16 years. Discrepancies between chronological and estimated ages were statistically evaluated by analyzing 1249 orthopantomograms from 603 girls and 646 boys. Using the Demirjian method, the mean age estimates underestimated chronological age by 0.03 years (p = 0.48) for girls and overestimated it by 0.03 years (p = 0.59) for boys; these differences with respect to chronological age were not statistically significant. In contrast, the Willems method underestimated chronological age by 0.54 years (p < 0.01) for girls and 0.44 years (p < 0.01) for boys; these differences with respect to chronological age were statistically significant. Compared to the Demirjian method, the overall mean absolute error generated using the Willems method was slightly higher (0.85 and 0.86 years, respectively). Since the Demirjian method was more accurate, we highly recommend that it should be applied when estimating dental age in the Chinese Han population. Further modifications of these two methods for populations from other regions and additional studies of other age groups are warranted.

Genetic polymorphisms of new 22 autosomal STR loci in the Mongolian ethnic group.

The Microreader™ 23SP ID System is a novel STR kit, but there are no Mongolian data related to this kit. In this study, allelic frequencies and forensic parameters were obtained from 505 unrelated healthy Mongolians. These samples were amplified using the kit. The dataset successfully passed quality control after being submitted to STRidER (STRidER dataset reference STR000198). A total of 264 alleles were observed, with corresponding allelic frequencies ranged from 0.001 to 0.378. The combined power of discrimination (CPD) and combined probability of exclusion (CPE) of the 22 autosomal STR loci were 0.999999999999999999999999999217318 and 0.999999999776042, respectively. Furthermore, population differentiation comparisons involving previously reported groups were conducted.

The forensic value of X-linked markers in mixed-male DNA analysis.

Autosomal genetic markers and Y chromosome markers have been widely applied in analysis of mixed stains at crime scenes by forensic scientists. However, true genotype combinations are often difficult to distinguish using autosomal markers when similar amounts of DNA are contributed by multiple donors. In addition, specific individuals cannot be determined by Y chromosomal markers because male relatives share the same Y chromosome. X-linked markers, possessing characteristics somewhere intermediate between autosomes and the Y chromosome, are less universally applied in criminal casework. In this paper, X markers are proposed to apply to male mixtures because their true genes can be more easily and accurately recognized than the decision of the genotypes of AS markers. In this study, an actual two-man mixed stain from a forensic case file and simulated male-mixed DNA were examined simultaneously with the X markers and autosomal markers. Finally, the actual mixture was separated successfully by the X markers, although it was unresolved by AS-STRs, and the separation ratio of the simulated mixture was much higher using Chr X tools than with AS methods. We believe X-linked markers provide significant advantages in individual discrimination of male mixtures that should be further applied to forensic work.

Third molar mineralization in relation to chronologic age estimation of the Han in central southern China.

The purpose of this study is to provide a forensic reference data about estimating chronologic age by evaluating the third molar mineralization of Han in central southern China. The mineralization degree of third molars was assessed by Demirjian's classification with modification for 2519 digital orthopantomograms (1190 males, 1329 females; age 8-23 years). The mean ages of the initial mineralization and the crown completion of third molars were around 9.66 and 13.88 years old in males and 9.52 and 14.09 years old in females. The minimum ages of apical closure were around 16 years in both sexes. Twenty-eight at stage C and stage G and 38 and 48 at stage F occurred earlier in males than in females. There was no significant difference between maxillary and mandibular teeth in males and females except that stage C in males. Two formulas were devised to estimate age based on mineralization stages and sexes. In Hunan Province, the person will probably be over age 14, when a third molar reaches the stage G. The results of the study could provide reference for age estimation in forensic cases and clinical dentistry.