Developmental validation of the Microreader™ Y Prime Plus ID System: An advanced Y-STR 38-plex system for forensic applications.

Y-STR is widely used in sexual assaults and familial searches of suspects. Here, we reported a novel 38-plex STR genotyping system designed for forensic applications. Microreader™ Y Prime Plus ID System (YPP) amplifies 38 loci in one reaction, including 29 loci from commonly used Yfiler® Plus PCR Amplification Kit & PowerPlex® Y23 System (DYS393, DYS570, DYS19, DYS392, DYS549, Y GATA H4, DYS460, DYS458, DYS481, DYS635, DYS448, DYS533, DYS449, DYS456, DYS389I, DYS390, DYS389Ⅱ, DYS438, DYS391, DYS439, DYS437, DYS385a/b, DYS643, DYS518, DYS576, DYF387S1a/b, and DYS627), 6 commonly used loci for the Y-STR database (DYS444, DYS447, DYS596, DYF404a/b, DYS527a/b, DYS557) and one Y-indel specific for the Chinese population. YPP is designed for different types of samples, such as blood card and swabs. In this work, YPP was validated following SWGDAM guidelines (2016) and guidelines from Ministry of Public Security of the People's Republic of China, including PCR-based, sensitivity, accuracy and precision, mixture, stability and inhibitor, and species specificity. The results indicate that the Microreader™ Y Prime Plus ID System is a powerful identification kit designed for forensic databases.

Validation of the Microreader™ 29Y Prime ID system for forensic use.

Currently, Y-short tandem repeat loci (Y-STRs) have been increasingly used in the forensic field, particularly in investigations of sexual assault, determination of paternity and male lineage studies because of the characteristics of male-only and paternal inheritance. The Microreader™ 29Y Prime ID system is a 29-plex Y-STR genotyping system that amplifies 17 widely used commercial loci (DYS570, DYS546, DYS460, DYS458, DYS635, DYS533, DYS448, DYS627, DYS456, DYS576, DYS449, DYS437, DYS643, DYS518, DYF387S1 a/b, and a sexual locus Y GATA H4), European recommended 7 single-copy "minimal haplotypes" (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, and DYS385a/b) and 2 additional loci (DYS438 and DYS439) recommended by The Scientific Working Group on DNA Analysis Methods (SWGDAM). The Microreader™ 29Y Prime ID system was validated according to the guidelines of "Validation Guidelines for DNA Analysis Methods (2016)" described by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including PCR-based, sensitivity, precision and accuracy evaluation, stutter percentage and peak height ratio, inhibitors, species specificity and DNA mixture studies. This study indicates that the Microreader™ 29Y Prime ID system is a useful tool for forensic cases and Y-STR genotyping.

Development of a massively parallel sequencing assay for investigating sequence polymorphisms of 15 short tandem repeats in a Chinese Northern Han population.

Massively parallel sequencing (MPS) has been used in forensic genetics in recent years owing to several advantages, e.g. MPS can provide precise descriptions of the repeat allele structure and variation in the repeat-flanking regions, increasing the discriminating power among loci and individuals. However, it cannot be fully utilized unless sufficient population data are available for all loci. Thus, there is a pressing need to perform population studies providing a basis for the introduction of MPS into forensic practice. Here, we constructed a multiplex PCR system with fusion primers for one-directional PCR for MPS of 15 commonly used forensic autosomal STRs and amelogenin. Samples from 554 unrelated Chinese Northern Han individuals were typed using this MPS assay. In total, 313 alleles obtained by MPS for all 15 STRs were observed, and the corresponding allele frequencies ranged between 0.0009 and 0.5162. Of all 15 loci, the number of alleles identified for 12 loci increased compared to capillary electrophoresis approaches, and for the following six loci more than double the number of alleles was found: D2S1338, D5S818, D21S11, D13S317, vWA, and D3S1358. Forensic parameters were calculated based on length and sequence-based alleles. D21S11 showed the highest heterozygosity (0.8791), discrimination power (0.9865), and paternity exclusion probability in trios (0.7529). The cumulative match probability for MPS was approximately 2.3157 × 10-20 .

[Consistency study of PowerPlex 21 kit and Goldeneye 20A kit and forensic application].

UNLABELLED: To ensure the consistency of genotype results for PowerPlex 21 kit and Goldeneye 20A kit. METHODS: The STR loci were amplified in DNA samples from 205 unrelated individuals in Beijing Han population. And consistency of 19 overlap STR loci typing were observed. The genetic polymorphism of D1S1656 locus was obtained. RESULTS: All 19 overlap loci typing showed consistent. The proportion of peak height of heterozygous loci in two kits showed no statistical difference (P > 0.05). The observed heterozygosis of D1S1656 was 0.878. The discrimination power was 0.949. The excluding probability of paternity of triplet was 0.751. The excluding probability of paternity of diploid was 0.506. The polymorphism information content was 0.810. CONCLUSION: PowerPlex 21 kit and Goldeneye 20A kit present a good consistency. The primer design is reasonable. The polymorphism of D1S1656 is good. The two kits can be used for human genetic analysis, paternity test, and individual identification in forensic practice.

[Genetic polymorphisms of 16 non-CODIS STR loci in Beijing Han population].

OBJECTIVE: To investigate the genetic polymorphisms of 16 non-CODIS loci (D6S477, D22-GATA198B05, D15S659, D8S1132, D3S3045, D17S1290, D14S608, D2S441, D18S535, D13S325, D10S1435, DlS2368, DIS1656, D7S3048, D10S1248 and D19S253) in Beijing Han population. METHODS: The DNA of 300 unrelated individuals in Beijing Han population were PCR amplified using GoldeneyeM DNA identification system 18NC kit, and the PCR products were analyzed by electrophoresis through 3130XL genetic analyzer. The fragment sizes of alleles were taken subsequently by GeneMapper v3.2. RESULTS: The distributions of genotype frequencies of 16 non-CODIS STR loci in Beijing Han population satisfied the Hardy-Weinberg equilibration. The population genetic parameters were obtained as followings: heterozygosity was 0.677-0.873; discrimination power, 0.890-0.967; probability of paternity exclusion, 0.393-0.741; and polymorphism information content, 0.706-0.853. CONCLUSION: These 16 non-CODIS STR loci show great genetic polymorphisms in Beijing Han population, and are useful for the research of population genetics and forensic application.

[Modified TRIzol method for RNA and DNA co-extraction from blood].

OBJECTIVE: To establish a new method for RNA and DNA co-extraction from the same sample by TRIzol reagent. METHODS: After the aqueous phase which contained total RNA was removed by traditional TRIzol method, the values of pH of the interphase phase and organic phase were adjusted. The DNA was precipitated with ethanol and purified with DNA IQ system. The purified DNA was measured in quality and quantity. As the template, it was amplified and typed by PCR-STR. The data was compared with that extracted by traditional TRIzol method. RESULTS: The DNA extracted by this modified method showed a better result of quality and quantity than that by traditional TRIzol method and a good STR typing. CONCLUSION: The modified TRIzol method is advisable and reliable to simultaneously extract both DNA and RNA from the same sample. It could be used for individual identification and paternity testing to satisfy the need of forensic science.

Assessment of application value of 19 autosomal short tandem repeat loci of GoldenEye 20A kit in forensic paternity testing.

This study was carried out to assess the application value of 19 autosomal short tandem repeat (STR) loci of GoldenEye 20A kit, in which 13 combined DNA index system core STR loci and PentaE, PentaD, D2S1338, D19S433, D12S391, and D6S1043 of six STR loci could be used in forensic paternity testing in Chinese population. We amplified the genomic DNA from blood samples on FTA paper of 289 paternity testing cases by using the GoldenEye 20A kit. The amplified products were detected by capillary electrophoresis, and then the genotypes of 20 genetic markers including 19 STR loci as well as Amelogenin for sex determination were analyzed by GeneMapper v3.2 and GeneMarker HID Software. The results of genotypes were compared to the three commonly used commercial kits including AmpFℓSTR Identifiler, PowerPlex16, and AmpFℓSTR Sinofiler kits. Compared to the three other common commercial kits, the GoldenEye 20A kit had higher value of combined paternity index in certainty of paternity or non-exclusion paternity cases, and more numbers of STR loci were excluded in exclusionary paternity cases. Our data in this study showed that the GoldenEye 20A kit has a higher application value in forensic paternity testing and will be of help for kinship analysis.

[A new method of identifying the peripheral blood and the menstrual blood].

OBJECTIVE: To explore the tissue-specific gene expressions of the peripheral blood and the menstrual blood, and to search some specific factors to establish an effective method for identifying the peripheral blood and the menstrual blood. METHODS: The specific products of the peripheral blood and the menstrual blood were detected by RT-PCR and separated by electrophoretic technology. RESULTS: Beta-spectrin (SPTB) as one specific marker of peripheral blood and 18S rRNA as a kind of the housekeeping gene were expressed in both the peripheral blood and the menstrual blood. However, matrix metalloproteinase 7 (MMP7) as one specific marker of menstrual blood and human beta defensin 1 (HBD1) as one specific marker of vaginal discharge were only found in the menstrual blood. CONCLUSION: There are differences of specific gene expressions between the peripheral blood and the menstrual blood. They could be accurately distinguished from each other by using the combination of fluorescence technology and RT-PCR to detect the specific identification of mRNA.

[Isolating sperm cells by laser capture microdissection technique from mixture sample].

OBJECTIVE: To assess the application value of laser capture microdissection (LCM) technique for isolating a small number of sperm cells from mixture sample. METHODS: Mixture samples were prepared with sperm cells and vaginal epithelia at different concentrations. Both LCM technique and the differential lysis method were employed to obtain sperm cells from the mixture samples, and DNA was extracted by magnetic beads method. STR genotyping was determined using Identifiler kit. RESULTS: The successful STR genotype rate of sperm cells isolated from mixture samples with LCM technique was 92.86% (13/14). The rate of differential lysis method was 7.14% (1/14). The successful rates between the two methods were statistically different (P < 0.05). CONCLUSION: LCM technique can effectively exclude the interference of female cell component and isolate a small number of sperm cells to obtain a single male STR genotyping. LCM technique is obviously better than the differential lysis method.

A multiplex PCR for 4 X chromosome STR markers and population data from Beijing Han ethnic group.

STR multiplex is a practical and simple method to obtain large amounts of important information in forensic and population genetic studies. The present work describes a new multiplex system that allows the simultaneous analysis of 4 X-STR markers, namely DXS9902, DXS6800, DXS6799 and DXS7132, as the tool of approach for X-STR studies. In addition, this work presents the genotyping results obtained for a sample 400 individuals (200 males and 200 females) from Beijing Han ethnic group in China.

Genetic polymorphisms for 17 Y-chromosomal STRs haplotypes in Chinese Hui population.

We have co-amplified 17 Y-chromosomal STRs (including DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a,b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y GATA H4) for samples of 143 unrelated male individuals of Chinese Hui Ethnic Minority Group. We obtained allelic frequency distribution and haplotype diversity of 17 Y-chromosomal STR for Hui population. A total of 136 different haplotypes were identified in 143 individuals, among which 129 were found only once, and seven haplotypes were found twice. The gene diversity values of STR loci ranged from 0.4161 (DYS391) to 0.9571 (DYS385a,b). The overall haplotype diversity for the 17 Y-STR loci was 0.9933, and the discrimination capacity was 0.9511.

Allele frequencies of mitochondrial D-loop (CA)n repeat polymorphism in six Chinese ethnic groups.

Allele frequencies of mitochondrial D-loop (CA)(n) repeat was carried out in six Chinese ethnic groups: Han ethnic group in Beijing, Uygur ethnic group in Kashi of Xinjiang province, Li ethnic group in Hainan province, Yao ethnic group in Junan of Guangxi province, Tibet ethnic group in Lasa of Xizang province and Yi ethnic group in Honghe of Yunnan province. A total of 542 individuals were typed. Comparative analyses between our population data and other populations gathered from the literature were performed.

Genetic polymorphisms of 17 Y-STRs haplotypes in Chinese Han population residing in Shandong province of China.

We have co-amplified (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a,b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y GATA H4) in a single PCR using the AmpFLSTR Yfiler PCR Amplification system. Allelic frequency distribution and haplotype diversity of 17 Y-chromosomal STR from a sample of 131 unrelated individuals of Chinese Han population living in Shandong province of China were obtained. A total of 129 haplotypes were observed in the 131 individuals studied, of which 127 were unique and two were found in two individuals. The gene diversity values ranged from 0.3560 (DYS391) to 0.9675 (DYS385a,b), The overall haplotype diversity for the 17 Y-STR loci was 0.9998, and the discrimination capacity was 0.9695. These results are compared with those observed in worldwide populations at both the locus and the haplotype level.

Genetic polymorphisms of 17 Y-STRs haplotypes in Tibetan ethnic minority group of China.

Haplotypes and allele frequencies for the 17 Y-chromosomal STRs loci, namely DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a,b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438, DYS448 were determined in a population sample of 112 healthy unrelated autochthonous Tibetan ethnic male individuals from Tibet of China. No shared haplotypes were observed. The gene diversity values for the Y-STRs loci ranged from 0.2052(DYS391) to 0.9301(DYS385a,b). The results demonstrate that these loci will be very useful for human identification in forensic cases and paternity tests in Tibetan population of China.