27个常染色体AIM-SNP的筛选和验证

目的构建27个常染色体AIM-SNP组合用于未知个体种族来源推断。方法通过对Hap Map数据库中描述祖先遗传信息标记的908个AIMs位点(非洲、欧洲、东亚人群)筛选,选出27个AIMs位点组合,利用相关软件同时与数据库908个AIMs不同子集合的分析进行对比,验证其推断祖先来源的可行性。结果应用本研究27个AIMs的SNP多态性分析方法可以正确推断未知样品祖先起源,估算祖先成分比例。结论本研究建立的常染色体27个AIMs的SNP多态性分析方法可准确推断来自于欧洲、非洲、东亚3大祖先血统个体的祖先来源,是SNP多态性分析用于个体种族来源推断的一种有效方法,在法医实践中可以用于DNA检验中未知DNA供者洲际人群祖先来源推断。     

The ForAPP: Forensic Ancestry Prediction Pipeline for the interpretation of ancestry informative markers

Interpretation of ancestry informative markers (AIMs) in a forensic case can be time-consuming as different tools are used that need to be initiated separately. We develop the ForAPP; an open-source pipeline (running offline) that initiates multiple ancestry prediction analyses and summarizes results in an interactive interface for interpretation of autosomal ancestry-informative markers.     

Analysis of eight mtDNA coding region polymorphisms for characterization of the female lineages ancestry in Alagoas,Brazil

In this work we evaluated eight mitochondrial DNA coding region polymorphisms (8281–8289d, 1736, 13263, 4883, 3594, 10873, 10400, and 12705) in order to identify lineages of African, Amerindian and European origin in Alagoas, Northeastern Brazil. Seven of these polymorphisms were detected by multiplex minisequencing with SnaPshot. For validation purposes a sample previously characterized by sequencing of the HVI and HVII regions was analyzed. Good agreement was observed between the phylogenetic results obtained in this work and the HVI and HVII regions sequencing. The results obtained suggest that the set of markers evaluated in this study can be used in ancestry studies of Brazilian populations.     

Inference of human geographic origins using Alu insertion polymorphisms

The inference of an individual's geographic ancestry or origin can be critical in narrowing the field of potential suspects in a criminal investigation. Most current technologies rely on single nucleotide polymorphism (SNP) genotypes to accomplish this task. However, SNPs can introduce homoplasy into an analysis since they can be identical-by-state. We introduce the use of insertion polymorphisms based on short interspersed elements (SINEs) as a potential alternative to SNPs. SINE polymorphisms are identical-by-descent, essentially homoplasy-free, and inexpensive to genotype using a variety of approaches. Herein, we present results of a blind study using 100 Alu insertion polymorphisms to infer the geographic ancestry of 18 unknown individuals from a variety of geographic locations. Using a Structure analysis of the Alu insertion polymorphism-based genotypes, we were able to correctly infer the geographic affiliation of all 18 unknown human individuals with high levels of confidence. This technique to infer the geographic affiliation of unknown human DNA samples will be a useful tool in forensic genomics.     

A LDR‐PCR Approach for Multiplex Polymorphisms Genotyping of Severely Degraded DNA with Fragment Sizes <100 bp*

Abstract: Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini‐short tandem repeat‐polymerase chain reaction (PCR) and mini‐sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small‐scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.     

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot™ assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.     

"Biological identikit": Development of a SNPs-panel for the analysis of forensic DNA phenotyping and ancestry

Personal identification in mass disasters and in crimes is essential for humanitarian, ethical and legal reasons. In these contexts, when individuals cannot be identified by standard forensic DNA analysis, the Forensic DNA Phenotyping and the analysis of the biogeographical ancestry could help. The aim of this study was to evaluate the potential of a new panel of 891 SNPs in predicting phenotypic traits and biogeographical origin to create a “biological identikit”. In addition to fresh biological material, old evidence found at the crime scene or extracted and long-term stored DNA were tested with 41 SNPs for phenotyping and 850 SNPs for ancestry. All the SNPs were successfully incorporated into a single two-step multiplex PCR reaction using the IonAmpliSeq ™ Library Plus and applied for massive parallel sequencing with the Ion S5 platform using up to 0.05 ng/µL of DNA. The analysis of the results was carried out with an in-house predictive algorithm and consulting 20 population databases. By comparing the results obtained with identikit or video-photographic surveys, it was possible to predict phenotype and ancestry with an accuracy greater than 90%. While these new markers cannot identify a specific individual, they can be a valuable investigative tool.     

Y-SNP typing of U.S. African American and Caucasian samples using allele-specific hybridization and primer extension

Multiplex analysis of genetic markers has become increasingly important in a number of fields, including DNA diagnostics and human identity testing. Two methods for examination of single nucleotide polymorphisms (SNPs) with a potential for a high degree of multiplex analysis of markers are primer extension with fluorescence detection, and allele-specific hybridization using flow cytometry. In this paper, we examined 50 different SNPs on the Y-chromosome using three primer extension multiplexes and five hybridization multiplex assays. For certain loci, the allele-specific hybridization method exhibited sizable background signal from the absent alternate allele. However, 100% concordance (>2000 alleles) was observed in ten markers that were typed using both methods. A total of 18 unique haplogroups out of a possible 45 were observed in a group of 229 U.S. African American and Caucasian males with the majority of samples being assigned into 2 of the 18 haplogroups.     

GENETIC PORTRAIT OF THE PUNJABI POPULATION FROM PAKISTAN USING THE PRECISION ID ANCESTRY PANEL

Prediction of geographical ancestry using genetic markers has a great potential in forensic genetics and may be used as an investigative lead in crime casework or missing person identification. Exploration of AIMs in Pakistan is interesting due to the distinct subpopulations with multidirectional ancestry from different groups. In the current study, 87 individuals from the Punjabi population from Pakistan were investigated using the Precision ID Ancestry Panel (Thermo Fisher Scientific) to assess whether it was possible to diff ;erentiate Punjabi individuals from other populations. With this panel, it is revealed that Punjabis are admixed and cannot be distinguished from other populations in South Central Asia and the Middle East.     

Inferring ancestral origin using a single multiplex assay of ancestry-informative marker SNPs

Tests that infer the ancestral origin of a DNA sample have considerable potential in the development of forensic tools that can help to guide crime investigation. We have developed a single-tube 34-plex SNP assay for the assignment of ancestral origin by choosing ancestry-informative markers (AIMs) exhibiting highly contrasting allele frequency distributions between the three major population-groups. To predict ancestral origin from the profiles obtained, a classification algorithm was developed based on maximum likelihood. Sampling of two populations each from African, European and East Asian groups provided training sets for the algorithm and this was tested using the CEPH Human Genome Diversity Panel. We detected negligible theoretical and practical error for assignments to one of the three groups analyzed with consistently high classification probabilities, even when using reduced subsets of SNPs. This study shows that by choosing SNPs exhibiting marked allele frequency differences between population-groups a practical forensic test for assigning the most likely ancestry can be achieved from a single multiplexed assay.     

A classifier for the SNP-based inference of ancestry

Ancestral inference from DNA could serve as an important adjunct for both standard and future human identity testing procedures. However, current STR methods for the inference of ancestral affiliation have inherent statistical and technical limitations. In an effort to identify bi-allelic markers that can be used to infer ancestral affiliation from DNA, we screened 211 SNPs in the human pigmentation and xenobiotic metabolism genes. Allele frequencies of 56 SNPs (most from pigmentation genes) were dramatically different between groups of unrelated individuals of Asian, African, and European descent, and both observed and simulated log likelihood ratios revealed that the markers were of exceptional value for ancestral inference. Log likelihood ratios of the multilocus estimates of biological ancestry (EAE/EBA) ranged from 7 to 10, which are on par with the best of the STR batteries yet described. A linear classification method was developed for incorporating these SNPs into a classifier model that was 99, 98, and 100% accurate for identifying individuals of European, African, and Asian descent, respectively. The methods and markers we describe are therefore an important first step for the development of a practical multiplex test for the inference of ancestry in a forensics setting.     

Multiplex high resolution melt (HRM) messenger RNA profiling assays for body fluid identification

Traditional body fluid identification methods use a variety of technologically diverse techniques that do not permit the identification of all body fluids. Definitive identification of the biological material present can be crucial to a fuller understanding of the circumstances pertaining to a crime. Thus definitive molecular based strategies for the conclusive identification of forensically relevant biological fluids need to be developed. Messenger (mRNA) profiling is an example of such a molecular based approach.Current mRNA body fluid identification assays typically involve either capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the time required for analysis. For qRT-PCR assays, only 3 or 4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays and to reduce the time and cost of analysis, we have developed multiplex high resolution melt (HRM) assays that provide an identification of all forensically relevant biological fluids and tissues.     

Constructing universal multiplex PCR systems for comparative genotyping

Analysis of length polymorphisms at STR loci in the human genome has become a standard approach for comparative genotyping in many areas including disease research and diagnostics, parentage assessment, investigations of human diversity, and forensic science. The simultaneous analysis of multiple STR loci through multiplex PCR and multicolor fluorescence detection offers sample conservation, high throughput, and automated genetic analysis. Careful design and optimization of tetranucleotide STR multiplexes has led to reliable, standardized systems that powerfully differentiate and distinguish individual human DNA profiles. The development of these multiplex systems involved a rigorous experimental strategy that included careful selection of PCR primer sequences (for yield, specificity, and multiplex compatability), along with optimization of PCR component concentrations, thermal cycling parameters, and fluorescence detection conditions. This developmental approach rendered well-characterized DNA typing systems that are high performing (sensitive, specific, and balanced), optimized to universal parameters (same reaction conditions), resilient to fluctuations in reaction conditions, and simple to implement and use routinely.     

Accurate STR allele designations at the FGA and vWA loci despite primer site polymorphisms

Polymerase chain reaction (PCR)-based STR DNA typing systems are used extensively in the field of human identification. Under optimal PCR conditions, the amplicon yield from both alleles of an STR locus is expected to be approximately equivalent. However, it is reasonable to expect that rare genomic sequence polymorphisms will co-localize with well-designed primer sets and induce allele imbalance or "dropouts". Two samples were identified in the course of genotyping thousands of individuals with AmpF/STR Profiler Plus that showed strong disparity in amplitude peak height of heterozygous peaks at the loci vWA and FGA. These samples were reamplified at reduced annealing temperature in an attempt to balance the peak heights. Nucleotide sequencing documented polymorphisms at the PCR primer binding sites of the affected alleles. The results indicate that reducing the annealing temperature to improve primer-binding efficiency at the mismatch and employing an alternative multiplex enhanced the data from both samples. Reducing annealing temperatures could provide a simple general solution to improving data quality for samples where polymorphisms are suspected to cause allele imbalance. Finally, we report on additional polymorphisms surrounding the vWA locus in a genetically diverse population.     

基于38-plex InDels的青海地区三个族群遗传推断

目的 基于38-plex InDels族群推断体系研究青海地区汉族、回族及撒拉族的族群成分与遗传结构.方法 使用38-plex InDels复合扩增体系检测3个族群的220份样本并获取InDels位点分型,利用主成分分析、STRUCTURE聚类分析及系统发育树综合分析族群之间的遗传关系,使用族群推断软件DAA v1.0...     

Forensic typing of autosomal SNPs with a 29 SNP-multiplex—Results of a collaborative EDNAP exercise

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.     

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA

Six multiplex PCR systems using single‐base extension reactions to analyze 46 mitochondrial DNA (mtDNA)‐coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.     

A multiplexed system for quantification of human DNA and human male DNA and detection of PCR inhibitors in biological samples

Forensic analysts routinely encounter samples containing DNA mixtures from male and female contributors. To obtain interpretable Short Tandem Repeat (STR) profiles and select the appropriate STR analysis methodology, it is desirable to determine relative quantities of male and female DNA, and detect PCR inhibitors. We describe a multiplex assay for simultaneous quantification of human and human male DNA using the ribonuclease P RNA component H1 (RPPH1) human target and the sex determining region Y (SRY) male-specific target. A synthetic oligonucleotide sequence was co-amplified as an internal PCR control. Standard curves were generated using human male genomic DNA. The SRY and RPPH1 assays demonstrated human specificity with minimal cross-reactivity to DNA from other species. Reproducible DNA concentrations were obtained within a range of 0.023-50 ng/μl. The assay was highly sensitive, detecting as little as 25 pg/μl of human male DNA in the presence of a thousand-fold excess of human female DNA. The ability of the assay to predict PCR inhibition was demonstrated by shifted IPC Ct values in the presence of increasing quantities of hematin and humic acid. We also demonstrate the correlation between the multiplex assay quantification results and the strength of STR profiles generated using the AmpF?STR^®PCR Amplification kits.