Analysis of DNA Mixture Using NGS Technologies (1/2)

 Abstract

Capillary electrophoresis(CE) of targeted DNA analysis has become routine test for individual identification of contemporary forensic science. However, mixtures of male and female DNA are considered challenging samples, sequence analysis of multiple source mixtures is not achievable by CE. The latest NGS (next generation sequencing) technology revolutionized DNA mixture interpretation as NGS reveals multiple single DNA molecules (massive parallel sequencing) in a single run, whereas CE examines gross DNA polynucleotides. Our study is to setup NGS approaches to resolve the issue of mixed DNA samples from real cases.

        Two-person mixtures of single-source human male and female DNA with different ratio were used to evaluate the CE and NGS abilities to deconvolute mixtures for STR, SNP and mitochondrial DNA(mtDNA) analysis. Our study shows that CE, due to its limitations, has higher allelic drop-out rates on STR comparing to NGS. Besides, CE-based analysis is unable to analyze multiple genetic polymorphisms in a single reaction using a single workflow. PCR process during STR analysis can produce stutter artifacts, it may be impossible to distinguish stutters from true alleles in DNA mixtures with CE and NGS sequencing. Results of mixed mitochondrial DNA sequence is also shown in our report, NGS analysis of mitochondrial HV1 and HV2 DNA sequence may further specify the minimum number and mixture proportion of contributors, the ratio of the mitochondrial polymorphism is proportional to the ratio of the DNA mixture.

        In addition to human mitochondrial sequencing, mitochondrial 12S rRNA, 16S rRNA and cytb gene for interspecies identification were also tested with NGS platform. Sheep-cat and human-cat DNA mixtures were then amplified by multiplex-PCR and processed to NGS sequencing. All of the three animal’s DNA sequence results were matched to their original derivations, with 98% identity or higher.

        In conclusion, NGS shows excellent performance on STR, SNP and sequence analysis of human DNA mixture prepared with commercial control DNA, NGS also exhibit capability to discriminate against species. At least 15 real investigate cases will be examined and reviewed to evaluate the NGS platform for future forensic studies and practices next year in this study.

Keywords: Next-Generation Sequencing, DNA mixture, Human identification, mitochondrial DNA, Short tandem repeat

Evaluation of the Correlation between Decomposition and DNA Degradation For Forensic Specimens- Body Fluid and Stain

Abstract

The essential information, which have effects on both cause of death determination and direction of case investigation, are post-mortem interval (PMI) and time of biological evidence degradation. It will be valuable deduction basis, if these information can be applied on forensic filed as quantification tools, such as DNA degradation index (DI) and DNA profile detection rate. Therefore, to evaluate the practicability of using the correlation between body fluid decomposition and DNA degradation on determining time of death and time period of biological evidence detached from body, we examined the decomposed body fluid samples for human DNA quantity, DI, and DNA profile detection rate. The body fluid samples, included blood, saliva, and urine, which were collected form volunteers, were analyzed on different timing of decomposed process. DNA quantity and ID were examined by DNA quantification kits: Quantifiler® Trio kit and Quantiplex Pro kit, when STR DNA profiles were analyzed by Globalfiler® kit. We also collected blood and buccal swab samples from unidentified persons for DNA quantity and DI analysis. The result shows that blood and saliva are decomposed faster at normal temperature. Although the urine samples can be preserved at 4℃ to slow down the DNA degradation, preservation time over two months might cause failure to detect DNA profile. This study established the influence of the timing of decomposition process in different environment on DNA quantity, DI and their correlation. By examining 54 bloods and 18 buccal swabs of unidentified persons, using average DI of blood samples as reference data, and combined the facts of cases, we can estimate the time of death and time period of biological evidence detached from body. Additionally, we compared three commercial DNA quantification kits: Quantifiler® kit, Quantiflier® Trio kit, and 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 Specimens;Biological Evidence; Post Mortem Interval; Degradation Index; DNA Quantification

The research of promoting the technology of semen identification (1/3)
Abstract

It is a hard challenge for forensic examiner to find sperm cells in sexual assault cases practically. The Christmas tree stain is a reliable method to label sperm cells used at the precent time. However, forensic examiner need to spend much time to find sperm cells because their head and tail quickly disconnect after ejaculation. Thus, development of an accurate and efficient staining technique for sperm labeling is an urgent need. We collected semen cells and buccal  swap epithelial cells in this study from three adult males and three adult females, respectively. These samples were then used to prepare the standard mixtures that were subsequently stored at -20℃, 4℃, and 25℃ for 1 day, 7 days, and 21 days, respectively, to mimic the decompositon of sperm cells to be in different temperatures and time periods. All of the mixtures were examined the sperm cells by using the Christmas tree stain, immunocytochemisty (ICC) stain, and immunofluroescent (IF) stain. The sperm specific protein acrosin, AKAP4, and Mospd3 were labeled by using anti-acrosin antibody, anti-AKAP4 antibody, and anti-Mospd3 antibody, respectively, for ICC, and IF stain. Acrosin was located at the head of sperms, whereas AKAP4 and Mospd3 were located at the flagella of sperms. Our results showed that, without antigen-antibody interaction, the Christmas tree stain presented a consistent result of sperm labeling, tolerated to protein degradation of sperms, and less affected by time and temperature changes so that it can be used to exam the samples which were stored for a long time . However, loss of sperm cells often happened because of washing steps in the process of Christmas tree stain. On the other hand, both ICC and IF, which depend on specific antigen-antibody interaction, indeed allow the forensic examiner to find sperm cells quickly and clearly, but are affected by protein stability. The signal of ICC and IF of AKAP4 was the most excellent, whereas that of acrosin was not as good as AKAP4 indicating that the stability of AKAP4 was the best, and that of acrosin was poor relatively . Taken together, Christmas tree stain is used for a long time. It is quick and simple, can be completed in 1 hour, and observed under a general optical microscope, but it lacks of specificity, so that it occasionally shows unclear results some time. It takes about 4 hours using ICC and IF, antibodies more expersive, and require a fluorescent microscopy for examination in IF. Therefore, we recommended that general laboratory can use Christmas tree stain combining with ICC to exam sperm cells. The rich-funding laboratory may set up IF staining method to increase the identification accuracy of sperm cells. The results of sperm cell examination are required to conclude the criminal facts that can greatly affect the human right of persons who involve in, give back justice to the innocents and victims, and persist in the glory of justice by forensic science.

Keywords: Semen identification; Christmas tree stain; immunofluorescent stain; Immunocytochemistry stain; sperms cells