Progress in the 16S rRNA Gene Sequencing in Forensic Science.

Forensic microorganism is one of the hotspots of forensic science research. Due to its conservatism and specificity, the 16S rRNA gene is found to be an ideal marker for forensic identification. With the rapid development of high throughput sequencing technology, the research on microorganisms has been gradually applied to many fields such as environment and health care. In the field of forensic science, the results of forensic microbiology research, represented by 16S rRNA gene sequencing, are also gradually applied to forensic practice, such as biological samples identification, individual identification, postmortem interval estimation, and regional inference, which not only provide clues for the investigation of cases but also complement and assist traditional methods. This paper describes the research methods and related sequencing technologies of 16S rRNA gene sequencing, summarizes its research progress, and discusses the application value and potential of 16S rRNA in forensic science.

[Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGM™ Platform].

OBJECTIVES: To analyze and detect the whole genome sequence of human mitochondrial DNA （mtDNA） by Ion Torrent PGM™ platform and to study the differences of mtDNA sequence in different tissues. METHODS: Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer. Libraries were constructed with Ion Shear™ Plus Reagents kit and Ion Plus Fragment Library kit. Whole genome sequencing of mtDNA was performed using Ion Torrent PGM™ platform. Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HVⅠ region. RESULTS: The whole genome sequence of mtDNA from all samples were amplified successfully. Six unrelated individuals belonged to 6 different haplotypes. Different tissues in one individual had heteroplasmy difference. The heteroplasmy positions and the mutation positions on HVⅠ region were verified by Sanger sequencing. After a consistency check by the Kappa method, it was found that the results of mtDNA sequence had a high consistency in different tissues. CONCLUSIONS: The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues, which have good consistency. The results provide guidance for the further applications of mtDNA in forensic science.

Source Identification of Human Biological Materials and Its Prospect in Forensic Science.

Source identification of human biological materials in crime scene plays an important role in reconstructing the crime process. Searching specific genetic markers to identify the source of different human biological materials is the emphasis and difficulty of the research work of legal medical experts in recent years. This paper reviews the genetic markers which are used for identifying the source of human biological materials and studied widely, such as DNA methylation, mRNA, microRNA, microflora and protein, etc. By comparing the principles and methods of source identification of human biological materials using different kinds of genetic markers, different source of human biological material owns suitable marker types and can be identified by detecting single genetic marker or combined multiple genetic markers. Though there is no uniform standard and method for identifying the source of human biological materials in forensic laboratories at present, the research and development of a series of mature and reliable methods for distinguishing different human biological materials play the role as forensic evidence which will be the future development direction.

[Identification of Vaginal Fluid Using Microbial Signatures].

OBJECTIVES: To investigate the specific microbial signatures in vaginal fluid. METHODS: Vaginal fluid （16 samples）, saliva （16 samples）, feces （16 samples）, semen （8 samples）, peripheral blood （8 samples）, urine （5 samples）, and nasal secretion （4 samples） were collected respectively. The 16S rRNA genes of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, and Atopobium vaginae were amplified. PCR production was detected via a 3130xl Genetic Analyzer. RESULTS: The detected number of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, and Atopobium vaginae were 15, 5, 8, 14, and 3 in all vaginal fluid samples, respectively. Lactobacillus crispatus and Lactobacillus jensenii existed specifically in vaginal fluid. CONCLUSIONS: There is a potential application value to detect Lactobacillus crispatus and Lactobacillus jensenii for the identification of vaginal fluid.

[Research Progress on Gene Alterations of Amelogenin Locus in Gender Identification].

There are two kinds of amelogenin gene mutation, including mutation in primer-binding region of amelogenin gene and micro deletion of Y chromosome encompassing amelogenin gene, and the latter is more common. The mechanisms of mutation in primer-binding region of amelogenin gene is nucleotide point mutation and the mechanism of micro deletion of Y chromosome encompassing amelogenin gene maybe non-allelic homologous recombination or non-homologous end-joining. Among the population worldwide, there is a notably higher frequency of amelogenin gene mutations in Indian population, Sri Lanka population and Nepalese population which reside within the Indian subcontinent. Though amelogenin gene mutations have little impact on fertility and phenotype, they might cause incorrect result in gender identification. Using composite-amplification kit which including autosomal STR locus, amelogenin gene locus and multiple Y-STR locus, could avoid wrong gender identification caused by amelogenin gene mutation.