Analysis of DNA Mixture Using NGS Technologies (2/2) 

Abstract

Capillary electrophoresis (CE) has been used for routine DNA analysis in the forensic science laboratory. However, this technology still has a limitation of human identification. The majority of DNA mixture is low quantity or degraded DNA that is why it is a great challenge by CE. The Next-generation sequencing (NGS) can increase the coverage to overcome this difficult problem of low quantity DNA. In addition, single nucleotide polymorphism (SNP) and mitochondrial DNA (mtDNA) of NGS analysis can assist to distinguish multiple sources of DNA.

In this study, a total of 28 forensic autopsy cases was collected, analyzed by CE and NGS, including human STR, human mtDNA (HV1 and 2) and animal mtDNA (12S rRNA, 16S rRNA, and Cytb sequences). A comparison of results obtained with two methods was used to validate the accuracy and reliability of NGS technologies. Besides, DNA sequencing of NGS result also can aid to identify sources of DNA mixture. Among 15 DNA mixtures were analyzed STRs and Y-STRs loci and four mixed DNA were examined human mtDNA of HV1 and 2 regions. Moreover, nine animal cases were analyzed mtDNA 12S rRNA, 16S rRNA, and Cytb sequences.

The research results were divided into the following three sections: (a) 15 cases of the accuracy of human STR: CE and NGS were 15, 13 cases, respectively. The NGS STR results of two specimens were invalid because of lower than 1ng DNA for analysis. However, the other 13 cases were demonstrated that NGS was showed excellent performance than CE. The research was observed that NGS approaches can increase the coverage of samples or using SNP data to assist with human identification of mixture samples. (b) Four cases of human mtDNA sequence: NGS results of mitochondrial DNA HV1 and HV2 sequence may further calculate mitochondrial polymorphism proportions of the mixture. CE results only showed heteroplasmy of sequence. (c) Animal species identification of nine cases: CE and NGS can identify five, eight cases, respectively. These results were illustrated that NGS method is more sensitive than traditional CE method.

In conclusion, the NGS method can successfully utilize to the analysis of complex forensic DNA mixtures for identification and interpretation of forensic case in the future.

Keywords: Next-Generation Sequencing (NGS), DNA mixture, Human identification, Mitochondrial DNA, Species identification

Evaluation of the Correlation between Decomposition and DNA Degradation For Forensic Specimens- Skeleton and Muscle

Abstract

The essential information, which has effects on both cause of death determination and direction of case investigation, are post-mortem interval (PMI) and degradation time of biological evidence. It will be valuable deduction basis, if these information can be applied on forensic filed as quantification tools, such as human DNA quantity, DNA degradation index (DI) and DNA profile detection rate. Therefore, to evaluate the practicability of using the correlation between skeleton and muscle decomposition and DNA degradation on determining PMI and time period of biological evidence detached from body, we examined the decomposed skeleton and muscle samples for human DNA quantity, DI, and DNA profile detection rate. The fresh skeleton and muscle samples, were collected form unidentified persons, were analyzed on different timing of decomposed process. DNA quantity and DI were examined by DNA quantification kit Investigator Quantiplex Pro kit, when STR DNA profiles were analyzed by Globalfiler® kit. The result shows that the strong positive linear relationship (R2 as 0.8970) between DI and timing of decomposed process can be found, when muscle was preserved at room temperature within 7 months. Similar correlation can also be seen on skeleton which were stored at room temperature or buried underground within 8 months, as the R2 is 0.8776 and 0.9537 respectively. These correlations can be as a tool for estimating the PMI or time period of biological evidence detached from body; however, after 8 to 12 months of storage, due to the decreased DI, the relationship failing to adhere to a linear form, and leads to decline sharply. Nevertheless the observed DI variation can be offering to judge the time of decomposition from a sample which was left over 9 months. In order to estimate PMI or time period of biological evidence detached from body, based on the averaged DI which is established in this study, the DNA quantity, DNA profile detection rate, and details of case should all be considered. It can be applied to sampling, preservation, and delivery of biological specimen with establishment of the timing of decomposition process in different environment to improve the quality of identifying the decomposed skeleton and muscle samples. Additionally, we compared two commercial DNA quantification kits: Quantifiler® kit and Investigator Quantiplex Pro kit to find out the appropriate DNA quantification kit for decomposed specimens as a reference data to conduct further PCR experiments, and achieve the goal of accuracy identification.

Keywords：Forensic Specimen; Skeleton; Muscle; Post-Mortem Interval; Degradation Index; DNA Quantification

The research of promoting the technology of semen identification (2/3) 

Abstract

In many sexual crime cases, it has always been the focus of forensic investigation how to effectively detect male sperm cells in the evidence. In this study, we aimed to utilize the magnetic activated cell sorting (MASC) by immunolabeling micromagnetic beads (IBM) to separate sperm cells and epithelial cells. First, the surface proteins (surface antigens) of sperm cells were interacted to their specific antibodies which were biotinylated, and then the sperm-antibody complex were interacted with streptavidin which covalently bound to the micromagnetic beads, and formed an immune complex containing sperms and magnetic beads. The magnetic beads were attracted by the magnetic base, and, as a result, the sperm cells were effectively separated and isolated from the cell mixture to conducing subsequent researches and DNA extraction. We further improved the purity of isolated sperm cells by DNase 1 and EDTA reagents to obtain a single male DNA type. The results indicated that the amount of sperm cells needed to reach 103 cells/mL or more to obtain the whole 15 groups of STR types. Among antibodies we used, the anti-CD52 antibody presented the best affinity to sperm cells, and the best effect to detect the single male STR DNA type. In this study, we used three different methods: IBM, Erase kit, and traditional differential extraction (DE) to isolate sperm DNA, and compared the results of the male STR DNA typing of standardized samples which were prepared with the conditions of storing at -20 °C, 4 °C, or 25 °C for 1 day, 7 days, or 21 days. The results showed that the DE method could not remove a large number of free female epithelial cells, resulting in interference of sperm cell DNA type. Both the IBM and Erase kit methods can detect male 15 groups of STR types in a single male. However, the sperm cells are lost due to the IBM method of separation, suggesting that the IBM method is not recommended for the actual forensic case where the number of sperm cells is small, unless intact sperm cells are required for further researches. In addition, both IBM and Erase kit methods were also applied to the mixed semen plaques of five forensic cases in 2018. There were respectively three and four cases detecting male 15 groups of STR types by the IBM method and the Erase kit method. Therefore, our results suggest that the Erase kit method is more effective and time-saving than the IBM method for the purpose of detecting suspect DNA.

Keywords: semen identification; Immunomagnetic beads method; differential extraction method; Erase kit extraction method ; sperms cells