DNA methylation-based age estimation from semen: Genome-wide marker identification and model development.

DNA methylation at age-related CpG (AR-CpG) sites holds significant promise for forensic age estimation. However, somatic models perform poorly in semen due to unique methylation dynamics during spermatogenesis, and current studies are constrained by the limited coverage of methylation microarrays. This study aimed to identify novel semen-specific AR-CpG sites using double-enzyme reduced representation bisulfite sequencing (dRRBS) and validate these markers, alongside previously reported sites and neighboring CpGs, using bisulfite amplicon sequencing (BSAS) to develop robust age estimation models.

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.

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.

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.

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.

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.

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

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).

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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 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).

An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

Background: Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine.

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).

Multiplex assay development and mutation rate analysis for 13 RM Y-STRs in Chinese Han population.

In this study, a novel multiplex polymerase chain reaction (PCR) assay was developed for amplifying the newly introduced 13 rapidly mutating Y-STR markers (RM Y-STRs) including DYF387S1, DYF399S1, DYF403S1a/b, DYF404S1, DYS449, DYS518, DYS526b, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627. In addition, a survey for mutation rates of the 13 RM Y-STRs in Chinese Han population was performed to make sure of the mutation characteristic and application in Chinese group. With 14,476 allele transfers in 1034 father-son pairs, 221 mutation events occurred, of which 215 were one-step mutations and 6 were two-step mutations.

Haplotype diversity of 13 RM Y-STRs in Chinese Han population and an update on the allele designation of DYF403S1.

Rapidly mutating Y-STRs (RM Y-STRs) have been paid much attention in recent years. The 13 RM Y-STRs have been proved to have substantially higher haplotype diversity and discrimination capacity than conventionally used Y-STRs, indicating the considerable power in paternal lineage differentiation. To investigate the haplotype diversity in Chinese Han population, we collected 252 unrelated male samples and tested the genotype of the 13 RM Y-STRs.