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Objective To genotype mixed samples with next generation sequencing and evaluate its prospects in forensic DNA application. Methods Three mixed biological samples from rapes cases and their reference samples were collected. DNA was extracted using the MagAttract M48 DNA Manual Kit(200). The ForenSeqTMDNA Signature Prep Kit was used for library preparation, and next generation sequencing was performed on the MiSeq FGx system. The ForenSeqTMUniversal Analysis v1.2.1 software was used for data analysis. NGS-based STR results were compared with CE-based genotypes. Results A single length polymorphic STR allele in the mixed profile could be recognized as two sequence polymorphic STR alleles from different donors, which would assist mixed profile analysis. Such phenomenon was observed in D3S1358, D9S1122 and D13S317 in this work. Conclusion Our results suggested that precision STR genotyping of mixed samples based on NGS can provide more information and hints for mixed STR profile separation.
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Objective To establish a SYBR Green real-time PCR detection method with tissue-specific miRNAs and explore a novel approach for forensic body fluid identification. Methods The frequently reported 6 standard miRNAs were synthesized to establish a SYBR Green method, and verify with body fluid. The relative expression data for the 6 miRNAs were obtained using SYBR Green real-time PCR method in peripheral blood, menstrual blood, saliva and semen. Results The assays showed that miRNA205 permitted the unequivocal identification among different fluids. miRNA451 and miRNA144 could be used to distinguish blood from non-blood. Menstrual blood or peripheral blood could be identified through miRNA214. miRNA888 and miRNA891 was highly expressed in semen. Conclusion The results of this study indicate that miRNA SYBR Green profiling may provide a feasible and effective approach to body fluid identification for forensic casework.
ABSTRACT
Capillary electrophoresis-based STR genotyping is accepted as the gold standard for human individual identification. Next generation sequencing (NGS) allows for the full resolution of STR base composition, and has the potential to be widely used in the field of forensics. Compared with length polymorphism, STR sequencing could provide more information, and quantitatively calculating the forensic parameters is necessary. In this paper, we established simple models for length-based and sequence-based STRs, and calculated the forensic parameters for both models. The results showed that for a single STR locus, compared with length polymorphism, STR sequence polymorphism could provide higher power of discrimination and power of exclusion, indicating sequence-based STR marker have stronger ability for identifying unrelated individuals and exclude non biological father. By combining 15 non-linkage loci for forensic DNA analysis, the cumulative matching probability values for length-based and sequence-based STR models are at 10-18and 10-26levels, respectively. Only 10 non-linkage sequence-based STR is required to reach a cumulative matching probability of as high as 15 length-based STR loci. It is hoped that these simulated models and calculations can provide a reference for the forensic application of NGS-based STR genotyping.
ABSTRACT
Next generation sequencing (NGS),also known as massively parallel sequencing (MPS),with significant advantages such as high throughput,rapidness,integration and low cost,has been widely used in research and clinical diagnosis fields and promises bright future for forensic applications.Compared with traditional DNA sequencing methods,NGS technologies allow the simultaneous detection of various forensic genetic markers.The classical CE-based STR genotyping method only can differentiate alleles with length polymorphism.However,there are occasions like DNA base substitution and repeat sequence variations,which give different alleles with the same size of amplicons.These alleles are heritable from parents to off-springs,and could be of great importance for solving criminal cases.NGS-based STR genotyping can be compatible with current STR data output mode,and allows for the full resolution of STR loci by providing additional sequencing polymorphism information.In this article,the authors focused on the forensic STR genetic markers and next generation sequencing,and reviewed recent progresses on NGS-based STR genotyping.Perspectives of NGS-based full resolution STR genotyping were presented and possible challenges were discussed in the hope of providing a reference for related studies and applications.
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DNA extracted from 100 unrelated Chinese were detected by using the Polymerase Chain Reaction (PCR), mini polyacryl gel vertical electrophorese and silver staining techniques at the p33. 4 locus. Among 100 unrelated individuals, 8 alleles were detected,Foster-Freeman BIOTRAC system indicated that the number of copies of the tandem repeats in different allele was 7, 10 to 15 respectively.There was a rare allele whose copy number was more than 13 but less than 14. The allele fragmehtlength varied from 603 to 1115bp,the allele freqency 0.5 % ~ 53. 5 %,heterozygosity 64 %,DP value84. 5 %. Pedigree analysis indicated that alleles of p33. 4 locus obey Mendel's Laws. Successful typingof various tissues and single hair folicle had confirmed that this technique is suitable for personal identification. In addition, by using Chelex we had established an alterntive method for extracting DNAfrom biological materials,whlch is rapid and easy to perform.
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The polymorphisms of minisatellite p33.6 (D1S111) locus were typed rapidly and accurately us- ing polymerase chain reaction (PCR). The amplified fragments were analyzed by mini- polyacrylamide gel electrophoresis followed by silver stain. The distribution of allele frequencies among 100 unrelated Chinese is reported. 14 alleles, containing 9 to 22 repeat units, had been detected with a heterozygosity of 76%. The allele frenquencies were from 0.5 %to 35.5 %. The size of amplified fragments ranged from 435 bp to 925bp. The discrimination power of p33.6 locus was 0. 916. The Amp --FLP was inherited according to the Mendelian Law. The results showed that the polymorphisms of p33.6 locus can be used for individual identification and paternity test.
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A region of the D17S30(pYNZ22) locus was amplified in DNA from 120 unrelated chinese individuals was carried out. The amplified products were analysed by mini-polyacrylamide gel electrophoresis followed by silver stain. 11 alleles, ranging from 170bp to 870bp in size and from 0. 4 % to 30. 4 % in frequency, was detected. The heterozygosity was 73%, Dp value was 0.938. The family study showed that the Amp-FLP in pYNZ22 locus is inherited according to the Mendelian law. The correct genotyping results can be obtained from very little amount of biological material, such as mixed stains, blood stains, single hair and saliva.
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Objective To study genetic alterations in 9 STR loci and the Amelogenin locus in various tumor tissues. Methods twenty cancer tissues taken from 20 different unrelated individuals and their blood specimens were examined with Chelex-100 extraction of DNA, Profiler Plus PCR amplification and 310 Genetic Analyzer. Results All of the 10 STR loci exist genetic alterations. The genetic alterations occurred in 6out of 20 cases. The rate of genetic alteration was 30%. Six genetic alterations were found in one tumor tissue. Conclusion The forensic community has to take be cautious not to use the tumor tissue for personnel identification.