Development a multiplex panel of AISNPs,multi-allelic InDels,microhaplotypes, and Y-SNP/InDel loci for multiple forensic purposes via the NGS

Recently, next generation sequencing has shown the promising application value in forensic research. In this study, we constructed a multiplex amplification system of different molecular genetic markers based on the previous selected ancestry informative single nucleotide polymorphisms (SNPs), multi-allelic insertion/deletion (InDel) polymorphisms, microhaplotypes, and Y-chromosomal SNP/InDel loci, and evaluated forensic efficiencies of the system in Chinese Shaanxi Han, Chinese Hui, and Chinese Mongolian groups via the next generation sequencing platform. Ancestry information analyses of Shaanxi Han, Hui, and Mongolian groups revealed that most Mongolian individuals could be differentiated from Shaanxi Hans and Huis based on the selected ancestry informative SNPs. Multi-allelic InDels and microhaplotypes showed the multiple allele variations and possessed relatively high genetic polymorphisms in these three groups, indicating these loci could provide higher forensic efficiencies for individual identification and paternity testing. Based on Y-chromosomal SNPs, different haplogroup distributions were observed among Shaanxi Han, Hui, and Mongolian groups. In conclusion, the self-developed system could be used to simultaneously carry out the individual identification, paternity analysis, mixture deconvolution, forensic ancestry information analysis, and Y-chromosomal haplogroup inference, which could provide more valuable investigative clues in forensic practices.     

Exploring of tri-allelic SNPs using pyrosequencing and the SNaPshot methods for forensic application

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.     

Use of multi‐InDels as novel markers to analyze 13 X‐chromosome haplotype loci for forensic purposes

Many studies have been proposed to identify insertion/deletion (InDel) polymorphisms in humans for forensic genetic studies. However, the discriminatory power of InDels is limited by the poor polymorphisms of diallelic markers. To improve their discriminatory power, we developed multi‐InDel, a novel autosomal marker comprising more than two InDel loci that are tightly linked by their physical position and combined into a specific marker by a pair of PCR primers. This strategy gives at least three haplotypes for each multi‐InDel marker. Such markers can be potentially very useful in forensic applications. In this study, we focused on multi‐InDel markers located on X chromosome (ChrX). A multiplex system with 13 multi‐InDel markers, including 28 InDel loci in ChrX, was developed. To validate the multi‐InDel panel, the haplotype distribution in a population sample and in a set of pedigrees was investigated. This study demonstrates usefulness of these markers for individual identification and relationship studies. We highlight the fact that the multi‐InDel markers located on ChrX can provide new supporting information for complex kinship testing.     

Detection of single-nucleotide polymorphisms coding for three ovine prion protein variants by primer extension assay and capillary electrophoresis

An alternative method is described for the determination of ovine prion protein allelic variants at codon 136, 154, and 171. The four mutations responsible for amino acid changes are typed simultaneously. The technique utilizes dideoxy chain termination reaction using fluorescently labeled dideoxy nucleotides. The single-base extended primers are resolved on a capillary electrophoresis instrument. Data obtained by our approach are presented according to genotype distribution in some breeds as a part of the validation procedure.     

Development of the 16 X‐STR loci typing system and genetic analysis in a Shanghai Han population from China

This study developed a new multiplex PCR system that simultaneously amplifies 16 X‐STR loci in the same PCR reaction, and the polymorphism and mutation rates of these 16 X‐STR loci were explored in a Shanghai Han population from China. These loci included DXS10134, DXS10159, DXS6789, DXS6795, DXS6800, DXS6803, DXS6807, DXS6810, DXS7132, DXS7424, DXS8378, DXS9902, GATA165B12, GATA172D05, GATA31E08, and HPRTB. Samples from 591 unrelated individuals (293 males and 298 females) and 400 two‐generation families were successfully analyzed using this multiplex system. Allele frequencies and mutation rates of the 16 loci were investigated, with the comparison of allele frequency distributions among different populations performed. Polymorphism information contents of these loci were all >0.6440 except the locus DXS6800 (0.4706). Nine cases of mutations were detected in the 16 loci from the investigation of 9232 meioses. Pairwise comparisons of allele frequency distributions showed significant differences for most loci among populations from different countries and ethnic groups but not among the Han population living in other areas of China. These results suggest that the 16 X‐STR loci system provides highly informative polymorphic data for paternity testing and forensic identification in the Han population in Shanghai, China, as a complementary tool.     

A microfluidic chip‐based fluorescent biosensor for the sensitive and specific detection of label‐free single‐base mismatch via magnetic beads‐based “sandwich” hybridization strategy

A novel microfluidic chip‐based fluorescent DNA biosensor, which utilized the electrophoretic driving mode and magnetic beads‐based “sandwich” hybridization strategy, was developed for the sensitive and ultra‐specific detection of single‐base mismatch DNA in this study. In comparison with previous biosensors, the proposed DNA biosensor has much more robust resistibility to the complex matrix of real saliva and serum samples, shorter analysis time, and much higher discrimination ability for the detection of single‐base mismatch. These features, as well as its easiness of fabrication, operation convenience, stability, better reusability, and low cost, make it a promising alternative to the SNPs genotyping/detection in clinical diagnosis. By using the biosensor, we have successfully determined oral cancer‐related DNA in saliva and serum samples without sample labeling and any preseparation or dilution with a detection limit of 5.6 × 10^?11 M, a RSD (n = 5) < 5% and a discrimination factor of 3.58–4.54 for one‐base mismatch.     

Sampling strategies for the analysis of glass fragments by LA-ICP-MS Part I. Micro-homogeneity study of glass and its application to the interpretation of forensic evidence

The authors have previously reported the use of laser ablation ICP-MS as a powerful analytical tool for elemental analysis of glass. LA is a simpler, faster and less intrusive sample introduction method than the conventional solution ICP-MS. Due to the minute amount of material removed in LA (∼300 ng, 50 μm crater size), the analyst should be aware of special sampling considerations such as characterization of the glass fragments originating from the “known” source, fragment size and selection of the area and surface of ablation.The purpose of this work was to evaluate the micro-homogeneity of the elemental composition of glass samples commonly found in crime scenes like containers, architectural windows and windshields. The set of glasses under study was comprised of 56 fragments originated from glass containers, 28 fragments from automobile windshields and 20 fragments from architectural windowpanes. All fragments were selected with a size smaller than 2 mm² in order to simulate the typical glass fragments transferred from the crime scenarios. A Nd:YAG laser, 266 nm, flat top beam profile was used in single point mode sampling 50 μm spot size for 50 s at 10 Hz (500 shots). In this study, ²⁹Si was used as an internal standard and the standard reference material, SRM NIST 612, was used as a single point external calibrator. In addition, SRM 621 was used as another control standard for the containers set and SRM 1831 for the automobile and architectural window sets due to their very similar matrix with the samples of interest. For each set of glasses, the mean values and standard deviation of 10 replicates (n = 10) of a single fragment were compared with the values obtained from 10 (n = 10) different fragments of glass within the area of interest in order to evaluate whether or not the variation within a glass was bigger than the variation due to the method. In addition, a subset of tempered glasses was evaluated to perform an elemental composition profile within different depths of the fragments. Single shot (one laser pulse per analysis) was also evaluated and its limitations for the forensic analysis of glass are also presented. The results show that float glass is homogenous even at the micro-range level allowing LA-ICP-MS as an alternative technique to perform elemental analysis of glass. However, the variation of elemental composition of headlamps and containers is larger over the source than the instrumental variation due to inherent heterogeneity and therefore specific statistical methods are recommended to compare the glass samples.     

A mixed film composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) brushes to enhance selectivity for detection of single nucleotide polymorphisms

Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (ΔT_m) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1-0.6 M). Increases of ΔT_m when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1 bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.