Introduction of an single nucleodite polymorphism-based "Major Y-chromosome haplogroup typing kit" suitable for predicting the geographical origin of male lineages

The European Consortium "High-throughput analysis of single nucleotide polymorphisms for the forensic identification of persons--SNPforID", has performed a selection of candidate Y-chromosome single nucleotide polymorphisms (SNPs) for making inferences on the geographic origin of an unknown sample. From more than 200 SNPs compiled in the phylogenetic tree published by the Y-Chromosome Consortium, and looking at the population studies previously published, a package of 29 SNPs has been selected for the identification of major population haplogroups. A "Major Y-chromosome haplogroup typing kit" has been developed, which allows the multiplex amplification of all 29 SNPs in a single reaction. Allele genotyping was performed with a single base extension reaction (minisequencing) detected by CE. The validation of the multiplex was performed in a total of 1126 unrelated males distributed among 12 worldwide populations. The approach takes advantage of the specific geographic distribution of the Y-chromosome haplogroups and demonstrates the utility of binary polymorphisms to infer the origin of a male lineage.     

Developing a new nonbinary SNP fluorescent multiplex detection system for forensic application in China

Nonbinary single‐nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent‐labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis.     

Analysis of 17 Y‐STR loci haplotype and Y‐chromosome haplogroup distribution in five Chinese ethnic groups

To investigate genetic diversity in Chinese populations, 706 unrelated male individuals from five ethnic groups (Han, Korean, Hui, Mongolian, and Tibetan, respectively) were analyzed with 17 Y‐chromosomal STRs. The haplotype diversity was 0.99985 in the combined data. A total of 675 distinct haplotypes were observed, of which 649 were unique. Y‐chromosome haplogroups in the five groups were also predicted with Y‐STR haplotypes. Genetic distance between the five studied ethnic groups and other published groups was analyzed by analysis of molecular variance and visualized in a multidimensional scaling plot. In conclusion, the 17 Y‐STR loci are highly polymorphic markers in the five groups and hence are very useful in forensic application, population genetics, and human evolution studies.     

Multiplex genotyping assays for fine‐resolution subtyping of the major human Y‐chromosome haplogroups E,G, I,J, and R in anthropological,genealogical, and forensic investigations

Inherited DNA polymorphisms located within the nonrecombing portion of the human Y chromosome provide a powerful means of tracking the patrilineal ancestry of male individuals. Recently, we introduced an efficient genotyping method for the detection of the basal Y‐chromosome haplogroups A to T, as well as an additional method for the dissection of haplogroup O into its sublineages. To further extend the use of the Y chromosome as an evolutionary marker, we here introduce a set of genotyping assays for fine‐resolution subtyping of haplogroups E, G, I, J, and R, which make up the bulk of Western Eurasian and African Y chromosomes. The marker selection includes a total of 107 carefully selected bi‐allelic polymorphisms that were divided into eight hierarchically organized multiplex assays (two for haplogroup E, one for I, one for J, one for G, and three for R) based on the single‐base primer extension (SNaPshot) technology. Not only does our method allow for enhanced Y‐chromosome lineage discrimination, the more restricted geographic distribution of the subhaplogroups covered also enables more fine‐scaled estimations of patrilineal bio‐geographic origin. Supplementing our previous method for basal Y‐haplogroup detection, the currently introduced assays are thus expected to be of major relevance for future DNA studies targeting male‐specific ancestry for forensic, anthropological, and genealogical purposes.     

17 to 23: A novel complementary mini Y‐STR panel to extend the Y‐STR databases from 17 to 23 markers for forensic purposes

A Y‐STR multiplex system has been developed with the purpose of complementing the widely used 17 Y‐STR haplotyping (AmpFlSTR Y Filer® PCR Amplification kit) routinely employed in forensic and population genetic studies. This new multiplex system includes six additional STR loci (DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643) to reach the 23 Y‐STR of the PowerPlex® Y23 System. In addition, this kit includes the DYS456 and DYS385 loci for traceability purposes. Male samples from 625 individuals from ten worldwide populations were genotyped, including three sample sets from populations previously published with the 17 Y‐STR system to expand their current data. Validation studies demonstrated good performance of the panel set in terms of concordance, sensitivity, and stability in the presence of inhibitors and artificially degraded DNA. The results obtained for haplotype diversity and discrimination capacity with this multiplex system were considerably high, providing further evidences of the suitability of this novel Y‐STR system for forensic purposes. Thus, the use of this multiplex for samples previously genotyped with 17 Y‐STRs will be an efficient and low‐cost alternative to complete the set of 23 Y‐STRs and improve allele databases for population and forensic purposes.     

Applications of MALDI‐TOF MS to large‐scale human mtDNA population‐based studies

Analysis of the mitochondrial DNA variation in populations is commonly carried out in many fields of biomedical research. We propose the analysis of mitochondrial DNA coding region SNP (mtSNP) variation to a high level of phylogenetic resolution based on MALDI‐TOF MS. The African phylogeny has been chosen to test the applicability of the technique but any other part of the worldwide phylogeny (or any other mtSNP panel) could be equally suitable for MALDI‐TOF MS genotyping. SNP selection thus aimed to fully cover all the mtSNPs defining major and minor branches of the known African tree, including, macro‐haplogroup L, and haplogroups M1, and U6. A total of 230 mtSNPs were finally selected. We used tests samples collected from two different African locations, namely, Mozambique and Chad Basin. Different internal genotyping controls and other indirect approaches (e.g. phylogenetic checking coupled with automatic sequencing) were used in order to evaluate the reproducibility of the technique, which resulted to be 100% using samples previously subjected to whole genome amplification. The advantages of the MALDI‐TOF MS are also discussed in comparison with other popular methods such as minisequencing, highlighting its high‐throughput nature, which is particularly suitable for case–control medical studies, forensic databasing or population and anthropological studies.     

SNaPshot minisequencing to resolve mitochondrial macro‐haplogroups found in Africa

African mitochondrial DNA (mtDNA) haplogroups are divided into seven macro‐haplogroups (L0′1′2′3′4′5′6), while the rest of the world's lineages are classified as subgroups of macro‐haplogroups M, N and R. The most common approach to characterizing mtDNA variation is the sequencing of hypervariable segments I and II of the non‐coding control region of the molecule. Given the higher mutation rate within the control region compared with the coding regions of the molecule, recurrent mutations in the former can sometimes hide possible phylogenetic structure. The incorporation of haplogroup‐defining coding region mutations has helped in overcoming this limitation. By judiciously selecting 14 coding region SNPs and incorporating them into a multiplex minisequencing assay we were able to resolve mtDNA sequences from some sub‐Saharan African populations into ten macro‐haplogroups (L0–L6, M, N and R). We tested the efficacy of the panel by screening 699 individuals, consisting mostly of Khoe‐San, Bantu speakers and individuals with mixed ancestries (Coloreds) and found no inconsistencies compared with hypervariable segment sequencing results. The panel provided a fast and efficient means of classifying mtDNA into the ten mitochondrial macro‐haplogroups and provided a reliable screening to distinguish African from non‐African‐derived mtDNA lineages.     

A new set of DIP‐SNP markers for detection of unbalanced and degraded DNA mixtures

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP‐SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP‐SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP‐SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP‐SNPs in a Chinese Han population. The DIP‐SNPs were capable of detecting the minor contributor's allele in home‐made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1 –10 ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP‐SNPs may serve as a valuable tool in detection of UDM in forensic medicine.     

Exploring of tri-allelic SNPs using pyrosequencing and the SNaPshot methods for forensic application

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.     

Confirmation that SNPs in the high mobility group‐A2 gene (HMGA2) are associated with adult height in the Japanese population; wide‐ranging population survey of height‐related SNPs in HMGA2

Adult height is a highly heritable trait in that multiple genes are involved. Recent genome‐wide association studies have identified a novel single‐nucleotide polymorphism (SNP) rs1042725 in the high mobility group‐A2 gene (HMGA2) and shown it to be associated with human height in Caucasian populations. We performed a replication study to examine the associations between SNPs in HMGA2 and adult height in the Japanese population based on autopsy cases. Although we could not confirm a significant association between rs1042725 in HMGA2 and adult height, another SNP, rs7968902, in the gene achieved significance for its association in the same populations, and the effect was the same as that documented previously. These findings permit us to conclude that the SNPs in HMGA2 are common variants influencing human height across different populations. Moreover, a worldwide population study of these SNPs using 14 different populations including Asians, Africans and Caucasians demonstrated that both haplotypes and genotypes for three height‐related SNPs (rs1042725, rs7968682 and rs7968902) in HMGA2 were distributed in an ethnicity‐dependent manner. This information will be useful for clarifying the genetic basis of human height.     

Genetic diversity and haplotype structure of 24 Y‐chromosomal STR in Chinese Hui ethnic group and its genetic relationships with other populations

In the present study, 24 Y‐chromosomal short tandem repeat (Y‐STR) loci were analyzed in 115 unrelated Hui male individuals from Haiyuan county or Tongxin county, Ningxia Hui Autonomous Region, China, to evaluate the forensic application of the 24 STR loci and to analyze interpopulation differentiations by making comparisons between the Hui group data and previously published data of other 13 populations. A total of 115 different haplotypes were observed on these 24 Y‐STR loci. The gene diversities ranged from 0.4049 (DYS437) to 0.9729 (DYS385a, b). The overall haplotype diversity was 1 at AGCU 24 Y‐STR loci level, while the values were reduced to 0.999237, 0.996949, and 0.996644 at the Y‐filer 17 loci, 11 Y‐STR loci of extended haplotype and 9 Y‐STR loci of minimal haplotype levels, respectively; whereas, haplotype diversity for additional 7 loci (not included in Y‐filer 17 loci) was 0.995271. The pairwise F_ST, multidimensional scaling plot and neighbor‐joining tree indicated the Hui group had the closest genetic relationship with Sala in the paternal lineage in the present study. In summary, the results in our study indicated the 24 Y‐STRs had a high level of polymorphism in Hui group and hence could be a powerful tool for forensic application and population genetic study.     

Pyrosequencing‐based strategy for a successful SNP detection in two hypervariable regions: HV‐I/HV‐II of the human mitochondrial displacement loop

Forensic analysis of mitochondrial displacement loop (D‐loop) sequences using Sanger sequencing or SNP detection by minisequencing is well established. Pyrosequencing has become an important alternative because it enables high‐throughput analysis and the quantification of individual mitochondrial DNAs (mtDNAs) in samples originating from more than one individual. DNA typing of the mitochondrial D‐loop region is usually the method of choice if STR analysis fails because of trace amounts of DNA and/or extensive degradation. The main aim of the present work was to optimize the efficiency of pyrosequencing. To do this, 31 SNPs within the hypervariable regions I and II of the D‐loop of human mtDNA were simultaneously analyzed. As a novel approach, we applied two sets of amplification primers for the multiplexing assay. These went in combination with four sequencing primers for pyrosequencing. This method was compared with conventional sequencing of mtDNA from blood and biological trace materials.     

A SNaPshot assay for genotyping 44 individual identification single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs), which have relatively low mutation rates and can be genotyped after PCR with shorter amplicons compared with short tandem repeats (STRs), are being considered as potentially useful markers in forensic DNA analysis. Those SNPs with high heterozygosity and low Fst (F-statistics) in human populations are described as individual identification SNPs, which perform the same function as STRs used in forensic routine work. In the present study, we developed a multiplex typing method for analyzing 44 selected individual identification SNPs simultaneously by using multiplex PCR reaction in association with fluorescent labeled single base extension (SBE) technique. PCR primers were designed and the lengths of the amplicons ranged from 69 to 125?bp. The population genetics data of 79 unrelated Chinese individuals for the 44 SNP loci were investigated and a series of experiments were performed to validate the characteristic of the SNP multiplex typing assay, such as sensitivity, species specificity and the performance in paternity testing and analysis of highly degraded samples. The results showed that the 44-SNPs multiplex typing assay could be applied in forensic routine work and provide supplementary data when STRs analysis was partial or failed.     

Nucleic acid and SNP detection via template‐directed native chemical ligation and inductively coupled plasma mass spectrometry

Detection of nucleic acids and single nucleotide polymorphisms (SNPs) is of pivotal importance in biology and medicine. Given that the biological effect of SNPs often is enhanced in combination with other SNPs, multiplexed SNP detection is desirable. We show proof of concept of the multiplexed detection of SNPs based on the template‐directed native chemical ligation (NCL) of PNA‐probes carrying a metal tag allowing detection using ICP‐MS. For the detection of ssDNA oligonucleotides (30 bases), two probes, one carrying the metal tag and a second one carrying biotin for purification, are covalently ligated. The methodological limit of detection is of 29 pM with RSD of 6.7% at 50 pM (n = 5). Detection of SNPs is performed with the combination of two sets of reporter probes. The first probe set targets the SNP, and its yield is compared with a second set of probes targeting a neighboring sequence. The assay was used to simultaneously differentiate between alleles of three SNPs at 5‐nM concentration.     

SNPlexing the human Y-chromosome: a single-assay system for major haplogroup screening

SNPs are one of the main sources of DNA variation among humans. Their unique properties make them useful polymorphic markers for a wide range of fields, such as medicine, forensics, and population genetics. Although several high-throughput techniques have been (and are being) developed for the vast typing of SNPs in the medical context, population genetic studies involve the typing of few and select SNPs for targeted research. This results in SNPs having to be typed in multiple reactions, consuming large amounts of time and of DNA. In order to improve the current situation in the area of human Y-chromosome diversity studies, we decided to employ a system based on a multiplex oligo ligation assay/PCR (OLA/PCR) followed by CE to create a Y multiplex capable of distinguishing, in a single reaction, all the major haplogroups and as many subhaplogroups on the Y-chromosome phylogeny as possible. Our efforts resulted in the creation of a robust and accurate 35plex (35 SNPs in a single reaction) that when tested on 165 human DNA samples from different geographic areas, proved capable of assigning samples to their corresponding haplogroup.     

Human Y‐chromosome SNP characterization by multiplex amplified product‐length polymorphism analysis

We designed an allele‐specific amplification protocol to optimize Y‐chromosome SNP typing, which is an unavoidable step for defining the phylogenetic status of paternal lineages. It allows the simultaneous highly specific definition of up to six mutations in a single reaction by amplification fragment length polymorphism (AFLP) without the need of specialized equipment, at a considerably lower cost than that based on single‐base primer extension (SNaPshot?) technology or PCR‐RFLP systems, requiring as little as 0.5 ng DNA and compatible with the small fragments characteristic of low‐quality DNA. By designation of two primers recognizing the derived and ancestral state for each SNP, which can be differentiated by size by the addition of a noncomplementary nucleotide tail, we could define major Y clades E, F, K, R, Q, and subhaplogroups R1, R1a, R1b, R1b1b, R1b1c, J1, J2, G1, G2, I1, Q1a3, and Q1a3a1 through amplification fragments that ranged between 60 and 158bp.     

Analysis of four PCR/SNaPshot multiplex assays analyzing 52 SNPforID markers

The SNPfor ID consortium identified a panel of 52 SNPs for forensic analysis that has been used by several laboratories worldwide. The original analysis of the 52 SNPs was based on a single multiplex reaction followed by two single‐base‐extension (SBE) reactions each of which was analyzed using capillary electrophoresis. The SBE assays were designed for high throughput genetic analyzers and were difficult to use on the single capillary ABI PRISM 310 Genetic Analyzer and the latest generation 3500 Genetic Analyzer, as sensitivity on the 310 was low and separation of products on the 3500 with POP‐7™ was poor. We have modified the original assay and split it into four multiplex reactions, each followed by an SBE assay. These multiplex assays were analyzed using polymer POP‐4™ on ABI 310 PRISM® and polymers POP‐4™, POP‐6™ and POP‐7™ on the 3500 Genetic Analyzer. The assays were sensitive and reproducible with input DNA as low as 60 pg using both the ABI 310 and 3500. In addition, we found that POP‐6™ was most effective with the 3500, based on the parameters that we assessed, achieving better separation of the small SBE products; this conflicted with the recommended use of POP‐7™ by the instrument manufacturer. To support the use of the SNP panel in casework in Malaysia we have created an allele frequency database from 325 individuals, representing the major population groups within Malaysia. Population and forensic parameters were estimated for all populations and its efficacy evaluated using 51 forensic samples from challenging casework.     

Genetic and expression analysis of all non‐synonymous single nucleotide polymorphisms in the human deoxyribonuclease I‐like 1 and 2 genes

Members of the human DNase I family, DNase I‐like 1 and 2 (DNases 1L1 and 1L2), with physiological role(s) other than those of DNase I, possess three and one non‐synonymous SNPs in the genes, respectively. However, only limited population data are available, and the effect of these SNPs on the catalytic activity of the enzyme remains unknown. Genotyping of all the non‐synonymous SNPs was performed in three ethnic groups including six different populations using the PCR‐RFLP method newly developed. Asian and African groups including Japanese, Koreans, Ghanaians and Ovambos were typed as a single genotype at each SNP, but polymorphism at only SNP V122I in DNase 1L1 was found in Caucasian groups including Germans and Turks; thus a Caucasian‐specific allele was identified. The DNase 1L1 and 1L2 genes show relatively low genetic diversity with regard to these non‐synonymous SNPs. The level of activity derived from the V122I, Q170H and D227A substituted DNase 1L1 corresponding to SNPs was similar to that of the wild‐type, whereas replacement of the Asp residue at position 197 in the DNase 1L2 protein with Ala, corresponding to SNP D197A, reduced its activity greatly. Thus, SNP V122I in DNase 1L1 exhibiting polymorphism exerts no effect on the catalytic activity, and furthermore SNP D197A in DNase 1L2, affecting its catalytic activity, shows no polymorphism. These findings permit us to postulate that the non‐synonymous SNPs identified in the DNase 1L1 and 1L2 genes may exert no influence on the activity levels of DNases 1L1 and 1L2 in human populations.     

Expansion of a SNaPshot assay to a 55‐SNP multiplex: Assay enhancements,validation, and power in forensic science

A previously developed multiplex assay with 44 individual identification SNPs was expanded to a 55plex assay. Fifty‐four highly informative SNPs and an amelogenin sex marker were amplified in one PCR reaction and then detected with two SNaPshot reactions using CE. PCR primers for four loci, 28 single‐base extension primers, and the reaction conditions were altered to improve the robustness of the method. A detailed approach for allele calling was developed to guide analysis of the electropherogram. One hundred and eighty unrelated individuals and 100 father‐child‐mother trios of the Han population in Hebei, China were analyzed. No mutation was found in the SNP loci. The combined mean match probability and cumulative probability of exclusion were 1.327 × 10^?22 and 0.999932, respectively. Analysis of the 54 SNPs and 26 STRs (included in the AmpFLSTR Identifiler and Investigator HDplex kits) showed no significant linkage disequilibriums. Our research shows that the expanded SNP multiplex assay is an easily performed and valuable method to supplement STR analysis.     

A multiplex SNP genotyping by allele‐specificspecific PCR based on stem‐loop and universal fluorescent primers of Chr1daxin mice

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele‐specific PCR amplification of genomic DNA with two stem‐loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome‐wide association study, pharmacogenetics, and medical diagnostics.