Improved age estimation from semen using sperm-specific age-related CpG markers.

Accurate age estimation from semen has the potential to greatly narrow the pool of unidentified suspects in sexual assault investigations. However, previous efforts utilizing semen age-related CpG (AR-CpG) markers have shown lower accuracy compared to blood AR-CpG-based methods. This discrepancy may be attributed to DNA methylation (DNAm) interferences from "round cells" such as leukocytes and immature sperm cells in semen. This study aimed to develop age calculators based on sperm-specific AR-CpG markers and to achieve performance-improved age estimates from sperm DNA. Through an analysis of publicly available MethylationEPIC microarray data from 90 sperm samples of healthy males aged 22-51 years, we identified 31 sperm-specific AR-CpG markers with absolute Pearson's R values > 0.5 and Benjamini-Hochberg adjusted p values < 0.013. The top 19 AR-CpG markers with the largest absolute R values and beta ranges > 0.10, along with 3 reported semen AR-CpG markers (cg06304190, cg06979108, and cg12837463), were integrated into two methylation SNaPshot panels (Ⅰ and Ⅱ), each containing 11 markers. The 21 qualified AR-CpG markers showed absolute R values ≥ 0.427 in an independent validation cohort of 253 sperm DNA samples (22-67 years), with cg21843517 exhibiting the strongest age correlation (R = 0.853). The optimal models, constructed using sperm DNAm data of the training set (n = 214, 22-67 years) and markers from panel Ⅰ (n = 11), panel Ⅱ (n = 10), or both panels, achieved mean absolute errors (MAEs) of 2.526-4.746, 3.890-5.715, and > 9.800 years on the test sets of sperm (n = 39, 23-64 years), semen (same donors as the sperm test set), and whole blood (n = 40, 22-65 years), respectively. The simplified models incorporating 3, 5, 9, or 14 AR-CpG markers (MAE = 2.918-4.139 years for sperm) still outperformed the Lee et al. original model (MAE = 6.444 years for semen) and the reconstructed panel Lee model (MAE = 6.011 years for sperm). The final models, utilizing all sperm DNAm data (n = 253) and markers from panel Ⅰ, panel Ⅱ, or both panels, yielded mean MAEs of 2.587, 2.766, and 2.200 years, respectively, on the 50 test sets generated by 5 repeats of 10-fold cross-validations. Additionally, multiple markers in both panels demonstrated the ability to discern sperm or semen from blood with 100% accuracy. In summary, our study substantiates the potential of sperm-specific AR-CpG markers for precise age estimation from sperm DNA, providing an improved toolset for forensic investigations.

Identification of Mixtures of Two Types of Body Fluids Using the Multiplex Methylation System and Random Forest Models.

OBJECTIVE: Body fluid mixtures are complex biological samples that frequently occur in crime scenes, and can provide important clues for criminal case analysis. DNA methylation assay has been applied in the identification of human body fluids, and has exhibited excellent performance in predicting single-source body fluids. The present study aims to develop a methylation SNaPshot multiplex system for body fluid identification, and accurately predict the mixture samples. In addition, the value of DNA methylation in the prediction of body fluid mixtures was further explored. METHODS: In the present study, 420 samples of body fluid mixtures and 250 samples of single body fluids were tested using an optimized multiplex methylation system. Each kind of body fluid sample presented the specific methylation profiles of the 10 markers. RESULTS: Significant differences in methylation levels were observed between the mixtures and single body fluids. For all kinds of mixtures, the Spearman's correlation analysis revealed a significantly strong correlation between the methylation levels and component proportions (1:20, 1:10, 1:5, 1:1, 5:1, 10:1 and 20:1). Two random forest classification models were trained for the prediction of mixture types and the prediction of the mixture proportion of 2 components, based on the methylation levels of 10 markers. For the mixture prediction, Model-1 presented outstanding prediction accuracy, which reached up to 99.3% in 427 training samples, and had a remarkable accuracy of 100% in 243 independent test samples. For the mixture proportion prediction, Model-2 demonstrated an excellent accuracy of 98.8% in 252 training samples, and 98.2% in 168 independent test samples. The total prediction accuracy reached 99.3% for body fluid mixtures and 98.6% for the mixture proportions. CONCLUSION: These results indicate the excellent capability and powerful value of the multiplex methylation system in the identification of forensic body fluid mixtures.

General Formulas for Calculating Commonly Used Kinship Index.

OBJECTIVES: To derive general formulas for calculating commonly used kinship index (KI). METHODS: By introducing the Kronecker symbol, the formulas used to calculate the same KI under different genotype combinations were summarized into a unified expression. RESULTS: The general formulas were successfully derived for KI in various case situations, including the paternity index, full sibling index, half sibling index, avuncular index, grandpaternity index, first-cousin index, and second-cousin index between two individuals without or with the mother being involved; grandpaternity index between grandparents and a grandchild without or with the mother being involved; half sibling index between two children with two mothers being involved; full sibling index among three children; and half sibling index among three children with no, one, or two mothers being involved. CONCLUSIONS: The general formulas given in this study simplify the calculation of KIs and facilitate fast and accurate calculation through programming.

Genetic polymorphisms of 19 X-STRs in populations of Hubei Han and Guangxi Zhuang and their comparisons with 13 other Chinese populations.

BACKGROUND: A prerequisite for applying short tandem repeat (STR) kits is obtaining population allele and/or haplotype frequencies and forensic parameters. AIM: Firstly, we aimed to investigate the population data of 19 X-chromosomal STRs (X-STRs) included in the AGCU X19 STR kit in the Han people residing in Hubei Province, Central China, and the Zhuang people residing in the Guangxi Zhuang Autonomous Region of South China. Furthermore, we compared these population data with those for other Chinese populations. SUBJECTS AND METHODS: In total, 509 unrelated Han males and 266 unrelated Zhuang males were genotyped using the AGCU X19 STR kit. Allele frequencies, haplotype frequencies, and forensic parameters were computed, and genetic differences among 15 Chinese populations were analysed. RESULTS: The 19 X-STRs showed a high power of discrimination and high mean chance of exclusion, whether calculated using allele or haplotype frequencies. Major differences were found between Han and Oroqen, Uyghur, Mongolian, Tibetan, Li, and Yi populations. Aberrant biallelic patterns at DXS10159, DXS10134, and DXS10079 and allelic dropouts at DXS10164 were observed. CONCLUSION: The 19 X-STRs were highly polymorphic in the Hubei Han and Guangxi Zhuang populations, and the AGCU X19 STR kit was shown to be suitable for forensic casework.

Accurate age estimation from blood samples of Han Chinese individuals using eight high-performance age-related CpG sites.

Age-related CpG sites (AR-CpGs) are currently the most promising biomarkers for forensic age estimation. In our previous studies, we first validated the age correlation of seven reported AR-CpGs in blood samples of Chinese Han population. Subsequently, we screened some good age predictors from blood samples of Chinese Han population, and built pyrosequencing-based age prediction models. However, it is still important to select a set of high-performance AR-CpGs in a specific racial group and establish a simple and efficient method for accurate age estimation for forensic purpose. In this study, eight AR-CpGs, namely chr6: 11,044,628 (ELOVL2), cg06639320 (FHL2), chr1: 207,823,723 (C1orf132), cg19283806 (CCDC102B), cg14361627 (KLF14), cg17740900 (SYNE2), cg07553761 (TRIM59), and cg26947034, were selected based on our previous studies, and a multiplex methylation SNaPshot assay was developed to investigate DNA methylation levels at these AR-CpGs in 529 blood samples (aged 2-82 years) from Han Chinese population. All selected CpG sites showed strong age correlation with the correlation coefficient (r) from 0.8363 to 0.9251. Multiple linear regression (MLR) and support vector regression (SVR) age prediction models were simultaneously established to fit change characteristics of DNA methylation levels of eight AR-CpGs with the age in 374 donors' blood samples. The MLR model enabled age prediction with R2 = 0.923, mean absolute error (MAE) = 3.52, while the SVR model enabled age prediction with R2 = 0.935, MAE = 2.88. One hundred fifty-five independent samples were used as a validation set to test the two models' performance, and the prediction MAE for the validation set was 3.71 and 3.34 for the MLR and SVR models, respectively. For the MLR and SVR models, the correct prediction rate at ± 5 years reached a high level of 79.35% and 83.23%, respectively. In general, these statistical parameters indicated that the SVR model outperformed the MLR model in age prediction of the Han Chinese population. In addition, our method provides sufficient sensitivity in forensic applications and allows for 100% efficiency when examining bloodstains kept in room conditions for up to 43 days. These results indicate that our multiplex methylation SNaPshot assay is a reliable, effective, and accurate method for age prediction in blood samples from the Chinese Han population.

Assessment of Multiple Annealing and Looping-Based Amplification Cycle-Based Whole-Genome Amplification for Short Tandem Repeat Genotyping of Low Copy Number-DNA.

Aim: A common problem in forensic practice is the lack of sufficient amounts of good quality genomic DNA. A possible solution is the amplification of the available genomic DNA before locus-specific polymerase chain reaction (PCR) analysis. The aim of this study was to evaluate multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) for short tandem repeat (STR) genotyping of low copy number DNA (LCN-DNA). Materials and Methods: DNA isolated from five blood samples was quantified and diluted to 250, 150, 100, 50, 25, and 5 pg/µL. After preamplification with MALBAC, WGA products were quantified. PCR-STR genotyping was performed in triplicate using dilution or purification-treated WGA products for each level of DNA. STR profiles were analyzed and compared with that from non-WGA DNA. Results: The purification treatment performed better than dilution of the MALBAC-based WGA products. Compared with the non-WGA DNA, both the average number and peak heights of correct alleles were significantly improved after preamplification with the MALBAC-based WGA at DNA inputs of ≤50 pg. Like other WGA methods, allele dropout and allele drop-in were observed in the profiling results for many samples. Conclusions: MALBAC shows great potential in LCN-DNA analysis and could find broader application in the fields of forensics and genetics.

A novel multiplex assay system based on 10 methylation markers for forensic identification of body fluids.

Identifying the source of body fluids found at a crime scene is an essential forensic step. Some methods based on DNA methylation played significant role in body fluids identification. Since DNA methylation is related to multiple factors, such as race, age, and diseases, it is necessary to know the methylation profile of a given population. In this study, we tested 19 body fluid-specific methylation markers in a Chinese Han population. A novel multiplex assay system based on the selected markers with smaller variation in methylation and stronger tissue-specific methylation were developed for the identification of body fluids. The multiplex assay were tested in 265 body fluid samples. A random forest model was established to predict the tissue source based on the methylation data of the 10 markers. The multiplex assay was evaluated by testing the sensitivity, the mixtures, and old samples. For the result, the novel multiplex assay based on 10 selected methylation markers presented good methylation profiles in all tested samples. The random forest model worked extremely well in predicting the source of body fluids, with an accuracy of 100% and 97.5% in training data and test data, respectively. The multiplex assay could accurately predict the tissue source from 0.5 ng genomic DNA, six-months-old samples and distinguish the minor component from a mixture of two components. Our results indicated that the methylation multiplex assay and the random forest model could provide a convenient tool for forensic practitioners in body fluid identification.

Removal of Polymerase Chain Reaction Inhibitors by Electromembrane Extraction.

Polymerase chain reaction (PCR) technology has become the cornerstone of DNA analysis. However, special samples (e.g., forensic samples, soil, food, and mineral medicine) may contain powerful PCR inhibitors. High levels of inhibitors can hardly be sufficiently removed by conventional DNA extraction approaches and may result in the complete failure of PCR. In this work, the removal of PCR inhibitors by electromembrane extraction (EME) was investigated for the first time. To demonstrate the universality of the approach, EME formats with and without supported membranes (termed parallel-EME and µ-EME, respectively) were employed, and both anionic [humic acid (HA)] and cationic (Ca2+) PCR inhibitors were used as models. During EME, charged inhibitors in the sample migrate into the liquid membrane in the presence of an electric field and might further leech into the waste solution, while PCR analytes remain in the sample. After EME, the clearance values for HA at 0.2 and 2.5 mg mL-1 were 94 and 85%, respectively, and that for Ca2+ (275 mM) was 63%. Forensic PCR-short tandem repeat (PCR-STR) genotyping showed that EME significantly reduced the interference by HA in PCR-STR analysis and displayed a higher HA purge capability compared to existing methods. Furthermore, by combining EME with liquid-liquid extraction or solid-phase extraction, satisfactory STR profiles were obtained from HA-rich blood samples. In addition, false-negative reports of bacterial detection in mineral medicine and shrimps were avoided after the removal of Ca2+ by µ-EME. Our research demonstrates the great potential of EME for the purification of DNA samples containing high-level PCR inhibitors and opens up a new application direction for EME.

Mutation analysis of 28 autosomal short tandem repeats in the Chinese Han population.

Short tandem repeats (STRs) have been extensively used in forensic genetics. However, according to previous studies, the mutation rates of STRs are relatively high and are affected by many factors. Therefore, it is important to analyze STR mutations and determine the influence of underlying factors on STR mutation rates. Mutation rates of 28 autosomal STRs were determined from 8708 paternity testing cases in the Chinese Han population, and the relationships between STR mutation rates and population, sex, age, allele length and heterozygosity were investigated. A total of 279 mutations were observed at 27 loci in a total of 233,530 meiosis cases, including 273 (97.8%) one-step, 5 (1.8%) two-step and 1 (0.4%) three-step mutations. The overall average mutation rate was 1.19 × 10-3 (95% CI 1.06 × 10-3 - 1.34 × 10-3) ranging from 0 (TPOX) to 2.79 × 10-3 (D13S325). Mutation rate comparisons revealed statistically significant differences at several STRs among populations. Paternal mutations occurred more frequently than maternal mutations, at a ratio of 6.04:1, and the mutation rate tended to increase with paternal age. Moreover, our study revealed a bias towards contraction mutations for long alleles and expansion mutations for short alleles. No obvious bias was observed in the overall mutation direction. In addition, STR loci with higher expected heterozygosity (Hexp) tended to have higher mutation rates. This work revealed the relationships between STR mutation rates and several influencing factors, providing useful data and information for further research on STR mutations in forensic genetics.

Genome-wide identification of age-related CpG sites for age estimation from blood DNA of Han Chinese individuals.

Age-related CpG (AR-CpG) sites are currently the most promising molecular markers for forensic age estimation. However, the AR-CpG sites of Han Chinese population remains to be systematically characterized. In this study, we performed genome-wide methylation analyses on 42 whole blood DNA from healthy Han Chinese volunteers (aged from 18 to 62 years) using the Illumina MethylationEPIC BeadChip microarray. As expected, both known and novel AR-CpG sites were identified. Considering the sex difference in aging rate, we then separately selected AR-CpG candidates and built pyrosequencing-based multiple linear regression models for age estimation of males and females. The model constructed from the male sample group (n = 167, aged from 1.50 to 85.71 years) explained 95.22% of variation in age using five AR-CpG sites (chr6:11044864 ELOVL2, chr1:207997068 C1orf132, cg19283806 CCDC102B, cg17740900, and chr10:73740306 CHST3) and yielded a mean absolute error (MAE) of 2.79 years. The model constructed from the female sample group (n = 141, aged from 3.33 to 80.38 years) explained 94.90% of variation in age with six AR-CpG sites (chr6:11044867 ELOVL2, chr1:207997060 C1orf132, chr2:106015757 FHL2, cg26947034, chr16: 67184108 B3GNT9, and chr20:44658203 SLC12A5) and yielded an MAE of 2.53 years. Besides, the estimated age was highly correlated with the actual age (R > 0.97). The robustness of these AR-CpG markers was demonstrated by 10-fold cross-validations. In conclusion, we updated the AR-CpG sites of Han Chinese population and provided two sets of AR-CpG sites for accurate age estimation.

Genetic analysis of 32 InDels in four ethnic minorities from Chinese Xinjiang.

The present study used the previously constructed 32-plex InDels panel to investigated the genetic diversity of four ethnic minorities (Hui, Mongol, Uygur and Kazakh) from Xinjiang, and analyzed the genetic relationships between the four populations and 27 reference populations. No significant deviations were observed from the Hardy-Weinberg equilibrium (HWE) at the 32 InDels for each population. The average observed heterozygosity (Hexp), average polymorphic information content (PIC), combined power of discrimination (CPD) and cumulative probability of exclusion (CPE) for the 32 InDels were all higher than the Qiagen Investigator DIPplex kit in the four populations from Xinjiang. The CPD ranged from 0.999999999999903 (Kazakh) to 0.999999999999952 (Hui) and CPE ranged from 0.9971 (Uygur) to 0.9985 (Hui), which indicated that the 32 InDels were capable for individual identification and could be a supplementary tool in paternity test for these populations. Population genetic analysis by the method of analysis of molecular variance (AMOVA), FST, phylogenetic tree, TreeMix-based topology, multi-dimensional scale analysis (MDS), principal components analysis (PCA) and STRUCTURE analysis showed that Xinjiang Hui population has a close relationship with East Asians (EAS), especially Chinese Han, and the populations of Xinjiang Mongol, Uygur and Kazakh showed mixed ancestral components related to EAS and Europeans (EUR).

The evaluation of seven age-related CpGs for forensic purpose in blood from Chinese Han population.

Age prediction of biological samples is one of the important tasks in forensic DNA phenotyping, and DNA methylation is regarded as the most promising biomarker for forensic age prediction. To date, numerous CpG sites have been reported to be age-related based on the changes in methylation. In this study, seven age-related CpG (AR-CpG) sites, cg02228185 (ASPA), cg09809672 (EDARADD), cg19283806 (CCDC102B), cg04208403 (ZNF423), chr17: 44,390,358 of GRCh38/hg38 (ITGA2B), cg14361627 (KLF14), and cg06639320 (FHL2), were selected and analyzed in 310 blood samples using a multiplex methylation SNaPshot assay to evaluate the value of selected AR-CpGs in age prediction in blood from Chinese Han population. The study confirmed the correlation of all the investigated markers with human age, and the correlation of cg19283806 with age is the highest while cg04208403 is the lowest in the Chinese Han population. Two different age prediction models, stepwise regression and support vector regression (SVR), were established based on the methylation SNaPshot data using 230 blood samples from donors aged 2-86 years old. The stepwise regression model included six AR-CpGs (except cg09809672) and enabled age prediction with R2 = 0.85, mean absolute deviation (MAD) = 4.22, while the SVR model enabled age prediction with R2 = 0.86, MAD = 4.01. An independent set of 80 samples was used to test the two models' performance and the prediction MAD for the validation set was 4.71 and 4.56 for the stepwise regression and SVR models, respectively. The number of correct predictions for ±5 years achieved a high level of 67.50 % and 73.75 %, respectively for the stepwise regression and SVR models. In general, the SVR model was superior to the linear regression model in age prediction. These results suggest that these seven CpG sites would be useful for age prediction in blood samples from the Chinese Han population.

Characterisation, verification and genetic basis of anomalous STR patterns: a report of four cases of X-chromosome STR biallelic patterns in human males.

Analysis of the characteristics and genetic basis of the anomalous short tandem repeat (STR) pattern encountered in forensic cases has been shown to be useful for analysing STR profiles in routine forensic casework. Here, we report biallelic patterns at several X-chromosome STR (X-STR) loci in human males revealed by forensic parameters investigation using the commercial AGCU X19 Kit. The presence of these patterns was verified by reanalysis using new samples and bidirectional Sanger sequencing of the singleplex polymerase chain reaction (PCR) products. And the genetic basis for their production was inferred based on the relative peak heights at the amelogenin locus and the affected locus (DXS10159, DXS10134 and DXS10079) and the normalised peak height ratios between the affected locus and adjacent loci relative to the control sample 9947A. The inference results suggested that two cases of biallelic pattern at the DXS10159 locus would be caused by local duplications, while in the other two cases, both the biallelic patterns at loci DXS10134 and DXS10079 would be due to somatic mutations. One case where the male showed a biallelic pattern at the DXS10159 locus (Xp11.21) was further analysed. Quantitative PCR (qPCR) revealed a microduplication (< 0.2 Mb) spanning at least 13.9 kb in Xp11.21 encompassing the DXS10159 locus. Finally, a workflow for analysing anomalous STR patterns was summarised. In conclusion, this study is a detailed report of X-STR biallelic patterns in human males, which serves as an effective complement to the database and provides an example for the analysis of anomalous STR patterns.

Development of a new 32-plex InDels panel for forensic purpose.

Insertion/deletion polymorphisms (InDels) have attracted more and more attention of forensic researchers because of their low mutation rate and small amplicons. In the face of challenging forensic cases and degraded DNA, InDels break through the limitations of traditional STRs and provided a new direction for forensic identification. In this study, a multiplex panel consisting of 32 InDels and amelogenin was established and the InDels were selected with minimum allele frequencies (MAF) ≥ 0.4 in Chinese Han population based on the 1000 Genomes Project and published articles. The 32-plex InDels assay was evaluated using the southern Chinese Han population from Hubei province. All markers were proved to be highly polymorphic with an average observed heterozygosity (Hobs) of 0.4885. The combined power of discrimination (CPD) and the combined probability of paternity exclusion (CPE) were 0.999999999999966 and 0.9982, respectively. Further validation studies indicated that the 32-plex InDels assay was highly sensitive to DNA template amounts as low as 31.25 pg and was effective for degraded DNA. The obtained allele frequencies from this study were compared with published data of other populations.

An unbalanced tri-allelic pattern at the D10S1248 locus: Characteristics, genetic basis and transmission.

To address issues related to unbalanced tri-allelic patterns, an example at the D10S1248 locus characterized by the sum of heights of alleles 13 and 15 approximately equal to allele 14 was intensively investigated. The coexistence of these three alleles was confirmed by profiling the rs2246512-D10S1248 marker using fluorescently labelled primers and allelic sequencing. Multi-tissue genotyping revealed that this pattern had chimeric characteristics, and pedigree analysis found that allele 13 or allele 14 were inherited to offspring independently of the other two alleles. These evidences suggest that the pattern should stem from a somatic mutation in early embryonic development and that peak height ratio is not an accurate indicator of mutation time point and direction. The rs2246538-D10S1248-rs2246512 marker was subsequently used to determine the mutation from allele 15 to allele 13. Notably, allele 13 with the lowest peak height was misidentified as a stutter peak when genotyped using next generation sequencing.

Targeted capture and sequencing of 1245 SNPs for forensic applications.

Next generation sequencing (NGS) technologies have enabled the possibility of analyzing a large number of SNPs simultaneously from multiple samples in a single experiment, for complementing the shortcomings of STR based methods. To efficiently genotype the desired SNPs, it is critical to optimize the library construction procedures. In this study, we formulated a strategy combining the molecular inversion probe (MIP) based target region capture method and NGS for genotyping 1245 SNPs. All the SNPs we selected exhibited high heterozygosity (minor allele frequency (MAF) > 0.3) according to 1000 genomes data. We applied the method to genotype a population of 210 unrelated individuals from the Hubei province of China and assessed the allele frequencies, Hardy-Weinberg equilibrium and linkage disequilibrium. The MAFs of more than 95% of the SNPs were ≥0.2, and no significant deviation or strong linkage was observed for 98% of the SNPs. The data indicated that, even within a relatively confined region, our SNP panel is suitable for individual identifications. Furthermore, we performed paternity test for 7 trio families using low quality DNA samples. The conclusions are in total agreement with these of STR-based analyses, with higher confidence indexes. Finally, we evaluated the performance of the MIP-NGS method with mock degraded DNA samples. We were able to genotype most of the SNPs even when the genomic DNA was sonicated to ˜100 bp range. In summary, we established a highly accurate and cost-effective method of SNP genotyping, which is potentially capable of solving complex issues encountered in forensic practices.

Differences of microRNA expression profiles between monozygotic twins' blood samples.

Monozygotic (MZ) twins are widely regarded as genetically identical, and traditional DNA typing methods are insufficient in identifying MZ twins. So the discrimination of MZ twins become a forensic problem. MicroRNAs (miRNAs) are a class of small, endogenous, non-protein-coding RNA molecules of approximately 22 nucleotides in length, and exist extensively in a variety of eukaryotic cells. MiRNAs regulate gene expression and play fundamental roles in multiple biological processes, including cell differentiation, proliferation and apoptosis as well as aging and disease processes. The goal of this study is to explore the differential expression of miRNAs within MZ twin pairs, and aimed to find new biomarkers for distinguishing MZ twins. Thus, the miRNA expression profiles of seven pairs of healthy MZ twins of different sex and age were analyzed by miRNA microarray. A total of 545 miRNAs were found to be differentially expressed in these MZ twin pairs, and 2, 5, 22, 53 and 132 differentially expressed miRNAs were shared across six, five, four, three and two pairs of MZ twins respectively. These findings had been confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays on select miRNAs, including miR-151a-3p, miR-3653-3p, miR-142-3p, miR-4325, miR-16-5p, let-7i-5p, miR-222-3p, miR-550b-3p, miR-4791 and miR-27a-3p. The results demonstrated that there are differences in the expression of miRNAs within MZ twin pairs, suggesting a role of miRNAs in identifying MZ twins.

Microdeletion at 8q24.13 rather than multistep microsatellite mutation resulting in the genetic inconsistency at the D8S1179 locus in a true trio.

When using microsatellite loci for DNA paternity testing, genetic inconsistencies sometimes occur in true trios and duos and may be erroneously attributed to germline mutations of microsatellite alleles. Here, we reported a typical case and discussed the issue of how to find out the cause of a genetic inconsistency. In our case, a genetic inconsistency in a true trio was observed at the D8S1179 locus, where the father has only allele 10 as compared to only allele 16 of his son. A set of tests were then performed. The results showed that the inconsistency was not result from the germline mutation of allele 10 to allele 16, or from the presence of null alleles due to primer binding site mutations, but from the microdeletion at 8q24.13, about 2.99 to 49.76 kb, detected in both the father and his son, which revealed by deletion mapping using short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). In conclusion, genetic inconsistencies observed in true trios or duos cannot be rashly attributed to germline STR mutations, especially multistep mutations, in the absence of verification or specification; otherwise, the reliability of the genetic proofs established will be challenged.

A novel multiplex assay of SNP-STR markers for forensic purpose.

Like DIP-STR markers (deletion/insertion polymorphism-short tandem repeat combinations), SNP-STR markers (single nucleotide polymorphism-STR combinations) are also valuable in forensic DNA mixture analysis. In this study, eight SNP-STRs were selected, and a stable and sensitive multiplex polymerase chain reaction (PCR) assay was developed for amplifying these SNP-STRs and the Amelogenin gender marker according to the principle of amplification refractory mutation system (ARMS). This novel multiplex set allows detection of the minor DNA contributor in a DNA mixture of any gender and cellular origin with high resolution (beyond a DNA ratio of 1:20). In addition, SNP-STR haplotype frequencies were estimated based on a survey of 350 unrelated individuals from Chinese Han population, and the combined power of discrimination (PD) and power of exclusion (PE) of the eight SNP-STRs were calculated as 0.99999999965 and 0.9996, which were obviously higher than that of the eight STR loci: 0.9999999954 and 0.9989 respectively. The results indicated that the SNP-STR compound markers have higher application value in forensic identification compared to standard autosomal STRs, especially in the analysis of imbalanced DNA mixtures.

Comparison of different methods for repairing damaged DNA from buffered and unbuffered formalin-fixed tissues.

Formalin fixation is considered an important process for preservation of human tissue samples for long periods. However, this process not only results in cross-linking complicating isolation of nucleic acid but also introduces polymerase "blocks" during polymerase chain reaction (PCR). At present, many protocols have already been developed aiming at extracting high amounts of amplifiable DNA from formalin-fixed tissues (FFTs). However, there are few methods for repairing formalin-damaged DNA. In this study, we compared the effectiveness of several post-extraction enzymatic repair techniques, including Taq DNA polymerase, DNA polymerase I and T4 DNA ligase, the PreCR™ Repair Mix and Restorase® DNA Polymerase, in restoring STR profiles from formalin-damaged DNA. Our results indicated that formalin-damaged DNA may be repaired partly with Taq DNA polymerase and the Restorase® DNA Polymerase, and lost alleles may be restored and STR peak heights may increase upon repair with them. Moreover, the repair ability of the protocol 2 with Taq DNA polymerase surpasses the Restorase® DNA Polymerase.