Applying massively parallel sequencing to paternity testing on the Ion Torrent Personal Genome Machine.

Massively parallel sequencing (MPS) is a promising supplementary method for forensic genetics and has gradually been applied to forensic casework. In this study, we applied MPS to forensic casework on an Ion Torrent Personal Genome Machine to evaluate its performance in paternity testing with mismatched STR loci. A total of 15 samples from seven cases containing one mismatched locus by capillary electrophoresis typing were analyzed. Combined paternity index (CPI) and relative chance of paternity were calculated according to the International Society for Forensic Genetics guidelines and the Chinese national standards recommended for paternity testing. With simultaneous analysis of enough STR loci, the results support the certainty of paternity, and the mismatched alleles were considered to be mutations (CPI>10,000). With the detection of allele sequence structures, the origins of the mutations were inferred in some cases. Meanwhile, nine STRs (CSF1PO, D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D12S391, D21S11 and D4S2408) were found in an increased number of unique alleles and three new alleles in three STRs (D2S441, D21S11, and FGA) that have not been reported before were detected. Therefore, MPS can provide valuable information for forensic genetics research and play a promising role in paternity testing.

Genome-wide screening for highly discriminative SNPs for personal identification and their assessment in world populations.

The applications of DNA profiling aim to identify perpetrators, missing family members and disaster victims in forensic investigations. Single nucleotide polymorphisms (SNPs) based forensic applications are emerging rapidly with a potential to replace short tandem repeats (STRs) based panels which are now being used widely, and there is a need for a well-designed SNP panel to meet such challenge for this transition. Here we present a panel of 175 SNP markers (referred to as Fudan ID Panel or FID), selected from ∼3.6 million SNPs, for the application of personal identification. We optimized and validated FID panel using 729 Chinese individuals using a next generation sequencing (NGS) technology. We showed that the SNPs in the panel possess very high heterozygosity as well as low within- and among-continent differentiations, enabling FID panel exhibit discrimination power in both regional and worldwide populations, with the average match probabilities ranging from 4.77×10-71 to 1.06×10-64 across 54 world populations. With the advent of biomedical research, the SNPs connecting physical anthropological, physiological, behavioral and phenotypic traits will be eventually added to the forensic panels that will revolutionize criminal investigation.

Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification.

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. Here we combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Our results showed that complete single donor STR profiles could be successfully obtained from sperm/epithelial cell and sperm mixtures from two contributors. For unbalanced sperm/epithelial cells and sperm cells mixtures, sensitivity results revealed that target cells could be detected at as low as 1:32 and 1:8 mixed ratios, respectively. Although highly relies on cell number and blood types or secretor status of the individuals, this procedure would still be useful tools for forensic DNA analysis of multi-suspect sexual assault cases by the combined use of FACS and MACS based on sperm-specific AKAP3 antigen and human blood type antigen.

[Application of the Peak Area Ratio of STR Loci to Amelogenin Locus in the Estimation of DNA Degradation].

OBJECTIVE: To explore the change rules of peak area ratio of STR loci to Amelogenin (AMEL) locus (STR/AMEL), a sex-determining gene in DNA degradation, and to evaluate the application of STR/AMEL value in the estimation of DNA degradation degree. METHODS: DNA was extracted from iliopsoas, and the variations of STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) were analyzed after the artificial degradation was made by DNase I, and the changes of these three ratios of the iliopsoas naturally degraded in an outdoor environment were also analyzed. The regression curves were analyzed using the periods of DNA degradation and outside the body as the independent variable (x) and the STR/AMEL value as the dependent variable (y) and three curve equations under two conditions were established. RESULTS: Both under the conditions of artificial and natural degradation, STR/AMEL value had a negative relationship with the degradation time. The relationship between STR/AMEL and degradation time can be well simulated by the cubic function. R2 was over 0.99 under controlled degradation condition and over 0.86 under natural degradation condition. CONCLUSION: The STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) is negatively related with the DNA degradation degree, which follows mathematical regression models strictly, and it might be applied to evaluate the DNA degradation degree.

Validation of Expressmarker mtDNA-SNP60: A mitochondrial SNP kit for forensic application.

Expressmarker mtDNA-SNP60 kit, a 3-dye fluorescence labeling forensic mitochondrial-SNP kit, was developed and modified based on previous work. In this study, it was validated through sensitivity, precision, accuracy, reproducibility, species specificity, mixture samples, PCR-based conditions, and inhibitor studies, according to the Scientific Working Group on DNA Analysis Methods guidelines. In the sensitivity study, full profile was obtained from template DNA with the quantity ≥ 4 pg. Consistent profiles were obtained at three different laboratories, which demonstrated the reproducibility of the kit. No peak was observed in the profiles of common animal species and microorganisms demonstrating high species specificity of the kit. In the mixture study, some loci (minor contributors) were missing at 19:1 and 9:1 ratios, whereas all loci could be profiled at other ratios (4:1, 2:1, and 1:1). PCR-based studies demonstrated that there recommended PCR conditions were optimized for the kit, and there was acceptable tolerance of error. The inhibitor study indicated that the kit has varying degrees of resistance to the presence of common inhibitors. Twelve random individuals were profiled by next-generation sequencing to confirm the allele-specific PCR assay, and a consistent result was obtained. Furthermore, 520 individuals from 260 families were profiled and the results showed that all loci of the confirmed mother and child were matched. In addition, forensic population genetics parameters, including random match probability, discrimination power, and haplotype diversity, were calculated (0.0474, 0.9526, and 0.9563, respectively). Our study demonstrates that the Expressmarker mtDNA-SNP60 kit can be a useful supplementary tool for forensic application.

Identification of vaginal fluid, saliva, and feces using microbial signatures in a Han Chinese population.

In recent years, forensic scientists have focused on the discrimination of body fluids using microbial signatures. In this study, we performed PCR-based detection of microbial signatures of vaginal fluid, saliva, and feces in a Han Chinese population. We investigated the 16S rRNA genes of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, and Atopobium vaginae in vaginal fluid, the 16S rRNA and the glucosyltransferase enzyme genes of Streptococcus salivarius and Streptococcus mutans in saliva, and the 16S rRNA genes of Enterococcus species, the RNA polymerase ß-subunit gene of Bacteroides uniformis and Bacteroides vulgatus, and the α-1-6 mannanase gene of Bacteroides thetaiotaomicron in feces. As a result, the detection proportions of L. crispatus, L. gasseri, L. jensenii, L. iners, and A. vaginae were 15/16, 5/16, 8/16, 14/16, and 3/16 in 16 vaginal fluid donors, respectively. L. crispatus and L. jensenii were specifically detected in vaginal fluid; L. gasseri, L. iners, and A. vaginae were also detected in non-vaginal fluid. S. salivarius and S. mutans were not specifically detected in saliva. The detection proportions of Enterococcus species, B. uniformis, B. vulgatus, and B. thetaiotaomicron in 16 feces samples were 16/16, 12/16, 15/16, and 11/16, respectively. B. uniformis and B. thetaiotaomicron were specifically detected in feces. In addition, DNA samples prepared for the identification of body fluid can also be used for individual identification by short tandem repeat typing. The mean detection sensitivities of L. crispatus and L. jensenii were 0.362 and 0.249 pg/uL, respectively. In conclusion, L. crispatus, L. jensenii, B. uniformis, and B. thetaiotaomicron can be used as effective markers for forensic identification of vaginal fluid and feces.

Mutation analysis of 19 autosomal short tandem repeats in Chinese Han population from Shanghai.

The mutation of short tandem repeat (STR) loci is affected by several factors, such as sex, age, and DNA architectures. Previous studies have shown a different profile of mutation rates at autosomal STR loci among populations. It is important to provide population data and reveal underlying factors influencing the evaluation of STR mutation rates. In this study, we performed a comprehensive analysis on the mutation of 19 autosomal STR loci through 124,773 parent-child allelic transfers from 5846 paternity testing cases. A total of 197 mutations were observed including 187 single-step mutations. The observed mutation rates ranged from 0.15 × 10-3 (TH01) to 4.57 × 10-3 (FGA), and the average mutation rate across all the 19 loci was 1.58 × 10-3. Furthermore, the average mutation rate of STR loci increases with the paternal conception ages and remains relatively stable in different maternal age groups, which suggest the profile of paternal conception ages as a potential factor influencing the evaluation of STR mutation rates and the ratio of paternal versus maternal mutation rate in populations. Multidimensional scaling analysis (MDS) shows a difference in the profile of mutation rates at 13 CODIS STR loci among ethnical groups. Based on our data, our results support that short alleles are biased towards expansion mutation and longer alleles favor contraction mutation. In conclusion, our results provide useful information for further investigation on STR mutation in forensic genetics and population genetics.

Developmental validation of forensic DNA-STR kits: Expressmarker 16+10Y and expressmarker 16+18Y.

DNA-STR analysis is widely used in the forensic science field and obtaining results in shorter time is highly demanded. The developed forensic STR Kit, referred to as Expressmarker 16+10Y (EX16+10Y) and Expressmarker 16+18Y (EX16+18Y), could amplify the common autosomal and Y chromosome STR loci simultaneously. The kits are validated by a series of tests, including DNA mixtures, stutter ratios, PCR based studies, species specificities, inhibitors, sensitivity, sizing precision, reproducibility and parallel tests. The results demonstrated that EX16+10Y and EX16+18Y were useful tools for rapid criminal investigation.

Multiplex Y-STRs analysis using the ion torrent personal genome machine (PGM).

Massively parallel sequencing (MPS) technologies allow parallel sequencing analyses of many targeted regions of multiple samples at desirable depth of coverage. Routine use of MPS for forensic genetics is on the horizon. In this study, we explore the application of MPS technology in forensic Y-STR analysis. We designed a multiplex assay with 13 Y-STR loci (DYS19, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS437, DYS438, DYS439, DYS448, DYS456, DYS635, GATA-H4) for the purpose of MPS. The multiplex Y-STR assay was amplified in 42 unrelated male individuals and amplicons were sequenced simultaneously using the ion torrent personal genome machine (PGM) system. All loci were detected successfully, except for DYS389 II that exhibited a failure rate of 1.8% due to the relatively long amplicon sizes. We observed 7, 3, 2, 6 and 5 new alleles, respectively in DYS389 II, DYS390, DYS437, DYS448 and DYS635 due to the presence of sub-repeat composition differences, and a new allele in DYS438 because of nucleotide substitution. One allele of DYS390 was inconsistent with allele call from conventional capillary electrophoresis (CE) because of 4 bp deletions upstream of the core repeat unit. This study demonstrates that Y-STR typing by MPS can provide more genetic information, holding the promise for high discriminatory power.

[Application of Ion Torrent PGM™ System in Detection of Fetal DNA in Maternal Plasma].

OBJECTIVE: To explore the feasibility of detecting of Y-STR of fetal DNA in maternal plasma using Ion Torrent PGM™ System. METHODS: A total of 16 fetal DNA samples from maternal plasmas (8 cases from 38 weeks gestational age and 8 ones from 12 weeks) were prepared and a multiplex assay with 7 STR loci (DYS390, DYS391, DYS393, DYS438, DYS437, DYS456, DYS635) was designed for multiplex-PCR amplification. Using Ion Torrent PGM™ System, the results of Y-STR sequences and capillary electrophoresis were obtained and compared. RESULTS: Y-STR specific alleles were detected in the maternal plasma of all the pregnant women having male babies of second and third trimester, which were higher than that detected by capillary electrophoresis. Consistent Y-STR genotypes were observed between fetal DNA from maternal plasma and genomic DNA from the newborn babies. CONCLUSION: Based on Ion Torrent PGM™ System, the prenatal Y-STR detection method may provide a high-sensitive and high-throughput choice for prenatal STR detection in forensic testing.

[Development of a Forensic Multiplex Amplification STR Kit for 15 Autosomal STR Loci and 10 Y-STR Loci].

OBJECTIVE: To establish a multiplex STR genotyping method for autosomal STR and Y-STR loci in forensic biological practice. METHODS: Widely used autosomal STR loci and Y-STR loci were selected. A set of PCR primers was designed, and a 5-dye fluorescent labeled STR multiplex PCR reagent kit was developed. RESULTS: A kit was developed which can simultaneously detect 15 autosomal STR loci, 10 Y-STR loci, and an Amelogenin. CONCLUSION: The 15 autosomal STR plus 10 Y-STR kit in combination with capillary electrophoresis method was used to STR genotyping with accurate and reliable results. The new one-step testing kit can potentially be widely used in forensic cases and DNA databank in the future.

[Progress in Association between Genetic Correlation and Human Violent Behavior].

Human violent behavior is a complex behavior which is influenced by genetic and environmental factors. There is a trend in investigating the mechanism of violent behavior by using the genetic methods. This article reviews several candidate genes and advances in epigenetics which are associated with violent behavior. The prospects and significance of violent behavior research from the view of gene polymorphism and epigenetics are also discussed.

Copy number variations and genetic admixtures in three Xinjiang ethnic minority groups.

Xinjiang is geographically located in central Asia, and it has played an important historical role in connecting eastern Eurasian (EEA) and western Eurasian (WEA) people. However, human population genomic studies in this region have been largely underrepresented, especially with respect to studies of copy number variations (CNVs). Here we constructed the first CNV map of the three major ethnic minority groups, the Uyghur, Kazakh and Kirgiz, using Affymetrix Genome-Wide Human SNP Array 6.0. We systematically compared the properties of CNVs we identified in the three groups with the data from representatives of EEA and WEA. The analyses indicated a typical genetic admixture pattern in all three groups with ancestries from both EEA and WEA. We also identified several CNV regions showing significant deviation of allele frequency from the expected genome-wide distribution, which might be associated with population-specific phenotypes. Our study provides the first genome-wide perspective on the CNVs of three major Xinjiang ethnic minority groups and has implications for both evolutionary and medical studies.

[Allele-specific PCR and its application in forensic science].

Allele-specific polymerase chain reaction (AS-PCR) is a technique based on allele-specific primers, which can be used to analyze single nucleotide polymorphism (SNP) effectively including the transition, transversion and insertion/deletion polymorphism and has been exploited in the study of diseases research, molecular diagnosis, and forensic biological evidence. The article systematically reviews the principle, the detection methods, improvement of AS-PCR, and its research updates in the fields of autosome, Y chromosome and mitochondrial SNP, as well as its application in forensic science.

Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification.

The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.

[AS-PCR assay for 20 mtDNA SNP typing and haplotype frequency].

OBJECTIVE: To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluorescence labeling for mitochondrial DNA (mtDNA) SNP typing. METHODS: Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided into 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. RESULTS: Distinct electropherograms of 200 blood samples were obtained successfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10 microL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. CONCLUSION: AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.

[Establishment of the multiplex genotyping system for 16 SNP loci on mtDNA].

OBJECTIVE: To establish a multiplex genotyping system of mtDNA SNP. METHODS: A multiplex analysis system of 16-plex mtDNA SNP loci was established with allele specific PCR and capillary electrophoresis genotyping technology. Fifty samples from unrelated Chinese Han individuals were typed with the multiplex system. The multiplex assay was validated by comparing with the direct sequencing method. RESULTS: The genotypes of all 50 samples were correctly determined by the multiplex system. The optimal genotypic graphs were obtained with an input DNA of 0.5-10 pg, and the typing results were completely consistent with those by direct sequencing method. CONCLUSION: The established multiplex system by allele specific PCR has high sensitivity, operational simplicity and high accuracy. It provides an effective and high output method for mtDNA SNP typing.