System For Single Nucleotide Polymorphisms Research Articles

A microhap panel for kinship analysis through massively parallel sequencing technology.

It is widely recognized that microhaps are powerful markers for different forensic purposes, mainly due to their advantages of both short tandem repeats and single nucleotide polymorphisms, including multiple alleles, low mutation rate, and absence of stutter peaks. In the present study, a panel of 60 microhap loci was developed and utilized in forensic kinship analysis as a preliminary study. Genotyping of microhap was performed by massively parallel sequencing and haplotypes were directly achieved from sequence reads of 73 samples from Chinese Han population. We observed that 49 out of 60 loci have effective number of alleles greater than 3.0 and 10 out of 60 have values above 4.0, with an average value of 3.5598. The heterozygosity values were in a range from 0.5840 to 0.8546 with an average of 0.7268 and the cumulative power of exclusion value of the 60 loci is equal to 1-4.78 × 10-18 . Moreover, we demonstrated the applicability of this method by different relationship inference problems, including identification of single parent-offspring, full-sibling, and second-degree relative. The results indicated that the assembled microhap panel provided more power for relationship inference, than commonly used short tandem repeats or single nucleotide polymorphism system.

Genetic polymorphisms in Sox17 associated with intracranial aneurysm in Chinese Han people: a genotype-phenotype study.

PurposeGenetic factors play a vital role in intracranial aneurysm (IA) onset and development. Studying the relationship between IA and the Sox17 polymorphisms in diverse populations is essential for establishing credibility.Patients and methodsWe collected blood samples derived from a total of 596 sporadic IA patients and 600 individual controls in several medical institutes in China. We used the Sequenom MassArray system for single nucleotide polymorphisms (SNPs) genotyping after DNA extraction. The SNPs data was tested and analyzed in PLINK (version 1.9). Multiple-testing was performed in PLINK to make the statistics more rigorous and accurate.ResultsWe found that the allelic G of rs1072737 (OR=1.303, genomic-control corrected P-value =0.001032) is a risk allele, while the allelic G of rs9298506 (OR=0.7253, genomic-control corrected P-value =0.01559) is a protective allele in Chinese Han people.ConclusionThe allelic G of rs1072737 is a risk factor for IA, while the allelic G of rs9298506 serves as a protective factor for IA in Chinese Han people.

Implications of Targeted Next Generation Sequencing in Forensic Science

The outburst of next generation sequencing has outpaced the traditional capillary electrophoresis (CE) based forensic genomics in terms of throughput, scalability and allelic resolutions. The limitations of CE-based detection system for STRs (Short Tandem Repeats) and SNPs (Single Nucleotide Polymorphisms) markers associated with forensic DNA phenotyping have been greatly overcome through next generation sequencing (NGS). It offers simultaneous analysis of forensically relevant genetic markers including STRs, SNPs, mutations and transcripts to improve efficiency, capacity and resolution through massively parallel sequencing. The detailed sequence information in comparison with ever growing DNA databases across the world may aid mixture interpretation, and will ensure enhancement in statistical weight of the evidence. Nowadays, a total of 33 NGS machines with low to high throughput are available but two dedicated systems including MiSeq®FGx™ Forensic Genomics System (Illumina) and the HID-Ion Personal Genome Machine (PGM)™ (Thermo Fisher Scientific) offer the sequencing of customized forensically informative SNP panels. Ion torrent launches Ion S5 and Ion S5 XL (with additional feature of local computing which enhances its analysis speed) which include same core instrument. The future of forensic genomics lies with integrated high throughput approaches to solve the increasing number of cases or mass disaster studies where forensic specimens and samples are compromised and degraded. In future new real-time sequencing platforms will revolutionize the field of forensic genomics.

ABCB1 single nucleotide polymorphisms (1236C>T, 2677G>T, and 3435C>T) do not affect transport activity of human P-glycoprotein

P-glycoprotein (P-gp) is a multidrug efflux transporter that has a defined role in the absorption and disposition of drugs. Many studies have investigated the potential influence of ABCB1 polymorphisms on the disposition of its substrates. However, there remains significant controversy regarding the role of these polymorphisms. Our aim was to generate a P-gp expression system for single nucleotide polymorphisms (SNPs) and the reference sequence to assess the functional significance of these variants on transport. P-gp reference, a P-gp ATPase deficient mutant (G534D) and a triple SNP variant of P-gp (1236C>T, 2677G>T, and 3435C>T) were expressed in Xenopus laevis oocytes and used to assess the influence of these SNPs on transport of digoxin and imatinib. The inhibition of P-gp-mediated transport in Caco-2 cells and oocytes was also assessed. No effect of the triple SNP variant of P-gp on molecular transport of digoxin or imatinib was observed. The rank order of inhibition of P-gp in Caco-2 cells and ABCB1-injected oocytes was tariquidar>elacridar>PSC-833>laniquidar>cyclosporine>verapamil>dipyridamole. These data suggest there is no functional consequence of these SNPs for molecular transport of model substrates or inhibition by model inhibitors for P-gp. Transporter-injected oocytes may be a useful tool for probing the mechanism for unexplained drug-drug interactions or to characterize therapeutic transport inhibitors.

Development and application of a multiplex SNP system to evaluate the mating dynamics of Pinus thunbergii clonal seed orchards

To provide an efficient tool for the identification of individuals and paternity testing in the breeding of Pinus thunbergii Parl., a battery of 18 single nucleotide polymorphism (SNP) markers was developed. Multiplexed single nucleotide primer extension was utilized as the assay system, which facilitated automated high-throughput analysis. The expected heterozygosity and polymorphic information content were 0.342–0.500 (average 0.434), and 0.283–0.375 (average 0.339), respectively. The cumulative confusion probability and discrimination power were 3.40 × 10−8 and 1 − 3.40 × 10−8, respectively, whereas the probabilities of paternity exclusion when only one parent was genotyped and when the genotypes of both suspected parents were known were 0.965 and 0.996, respectively. The assay system was applied to investigate the mating dynamics of four clonal seed orchards. By genotyping the 18 nuclear SNPs in both the biparentally derived diploid embryo and the corresponding maternally derived haploid megagametophyte, the maternity and paternity of 1,915 seeds could be assigned accurately. The paternity analysis indicated that, for 64.5 % of the seeds, both parents were found in the orchard. The analysis of relative maternal contribution revealed that, in all the seed orchards, one clone of the 15 in the orchards did not act as a mother. The analysis of relative paternal contribution showed that all clones functioned as fathers; however, the degree of contribution differed considerably among the clones. The SNP markers, in combination with separate analysis of two seed tissues (embryo and megagametophyte), were an effective means of investigating mating dynamics in P. thunbergii.

A Fully Integrated and Automated Detection System for Single Nucleotide Polymorphisms of UGT1A1 and CYP2C19

The need for examinations of single nucleotide polymorphisms (SNPs) on drug metabolizing enzymes is accelerating. Especially, SNPs of UTG1A1 and CYP2C19 are important for patients who are treated with irinotecan and proton pump inhibitors, respectively. Thus, a method for the rapid, fully automated, and accurate measurement of these SNPs is desired. We genotyped 109 Japanese volunteers for UGT1A1*6, UGT1A1*28, CYP2C19*2, and CYP2C19*3 with the quenching probe (QP) method. Only 90 min after whole blood was applied, QP method enabled to detect these SNPs automatically. The results obtained by QP method were absolutely identical to those examined by the conventional direct sequencing. These findings indicate that the QP method will enable point-of-care testing in clinical laboratories and patient-oriented therapy with its convenience and speed for patients who are treated with irinotecan or proton pump inhibitors.

Development of novel SNP system for individual and pedigree control in a Japanese Black cattle population using whole‐genome genotyping assay

Individual identification and parentage analysis using DNA markers are essential for assuring food identity and managing livestock population. The objective of this study was to develop a single nucleotide polymorphism (SNP) panel system for individual effective identification and parentage testing in a Japanese Black cattle population using BovineSNP50 BeadChip. On the basis of SNP frequencies, 60 unlinked informative SNPs were finally selected as candidate markers. The allelic frequencies for each SNP were estimated using additional individuals by PCR-RFLP (restriction fragment length polymorphism). A total of 87 SNP markers added in conjunction with previously developed 27 SNPs were evaluated to assess the utility of the test. The estimated identity power was 2.01 x 10(-34). Parentage exclusion probabilities, when both suspected parents' genotypes were known and when only one suspected parent was genotyped, were estimated as 0.99999997 and 0.99998010, respectively. This developed SNP panel was quite powerful and could successfully exclude false sires with a probability of >0.9999 even if the dam's genotype information was not obtained. The SNP system would contribute to management of the beef industry in Japan.

Detection of single nucleotide polymorphism in the FUT2 gene by temperature gradient gel electrophoresis

A simple and rapid analysis system for single nucleotide polymorphisms (SNPs) was investigated for the FUT2 gene using the temperature gradient gel electrophoresis (TGGE) method. The 426-bp or 259-bp FUT2 fragments were amplified from heterozygous samples using primers, and the heteroduplex and homoduplex bands were detected by TGGE. The FUT2 fragments amplified from homozygous samples were denatured and re-annealed with a known sequence fragment, forming heteroduplex bands which were analyzed by TGGE. The fragment patterns of homoduplex and heteroduplex bands in TGGE were specific to the genotypes, and more specifically, the number of heteroduplex bands and the migration of the homoduplex bands corresponded to the number of nucleotide substitutions and the change in GC content due to the substitutions, respectively. Thus, the TGGE technique can act as a high-throughput method for the detection and the presumption of sequences of known and unknown SNPs in the FUT2 gene.

Development of an automation system for single nucleotide polymorphisms genotyping using bio-strand, a new three-dimensional microarray

Previously, we developed a novel three-dimensional microarray system called Bio-Strand, which may be used in various applications including single nucleotide polymorphisms genotyping. In Bio-Strand, samples for detection are immobilized on a one-dimensional thread, which is wound around a cylinder-shaped core to form a three-dimensional thread-and-core structure. The thread-and-core structure is then inserted into a plastic pipette tip, where hybridization and detection are performed. In this study, we have developed an automation system, NIAGALA Bio-Station SDx, which enables automated hybridization and detection during the genotyping procedure using Bio-Strand. Using this system, we have performed the single nucleotide polymorphism (SNP) genotyping of CYP2C, one of the important human cytochrome P450 genes and the results were completely consistent with the genotyping results determined by the TaqMan method.

A new 39-plex analysis method for SNPs including 15 blood group loci

A novel 39-plex typing system for single nucleotide polymorphisms (SNPs) has been developed. This multiplex approach has the advantage of being able to type 38 autosomal SNPs and one sex-discriminating base exchange site on the X and Y chromosomes rapidly and simultaneously. The SNP loci on the autosomes, which we examined, contain 15 loci distributed on blood type genes: three on RhCE, two each on Km and Gc, and one each on Duffy, AcP1, Tf, MN, GPT, EsD, PI, and Kidd genes. Thirty-seven genomic DNA fragments containing a total of 38 SNPs and one sex-discriminating site were amplified in one multiplex PCR reaction. Following the reaction, single nucleotide primer extension reaction was performed by dividing these SNP loci into five groups. The SNP type of each of the 39 loci was determined at one time by capillary electrophoresis using the newly designed multi-injection method. The combined PD (power of discrimination) of this typing system was (1–1.1)×10 −14, and the MEC (mean exclusion chance) was 0.9990. We applied this system to forensic cases, including 16 paternity testing cases (13 non-exclusion and three exclusion cases) and one personal identification case. For the paternity testing cases, the highest Essen–Möller’s W-value was 0.9999995. The pM (matching probability) of the personal identification case was 2.22×10 −17. These data showed that this system was an excellent tool for use in forensic cases of paternity testing and personal identification.

Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system.

We selected 125 candidate single nucleotide polymorphisms (SNPs) in genes belonging to the human type 1 interferon (IFN) gene family and the genes coding for proteins in the main type 1 IFN signalling pathway by screening databases and by in silico comparison of DNA sequences. Using quantitative analysis of pooled DNA samples by solid-phase mini-sequencing, we found that only 20% of the candidate SNPs were polymorphic in the Finnish and Swedish populations. To allow more effective validation of candidate SNPs, we developed a four-colour microarray-based mini-sequencing assay for multiplex, quantitative allele frequency determination in pooled DNA samples. We used cyclic mini-sequencing reactions with primers carrying 5'-tag sequences, followed by capture of the products on microarrays by hybridisation to complementary tag oligonucleotides. Standard curves prepared from mixtures of known amounts of SNP alleles demonstrate the applicability of the system to quantitative analysis, and showed that for about half of the tested SNPs the limit of detection for the minority allele was below 5%. The microarray-based genotyping system established here is universally applicable for genotyping and quantification of any SNP, and the validated system for SNPs in type 1 IFN-related genes should find many applications in genetic studies of this important immunoregulatory pathway.

A single base extension technique for the analysis of known mutations utilizing capillary gel electrophoreisis with electrochemical detection.

A novel single nucleotide polymorphism (SNP) detection system is described in which the accuracy of DNA polymerase and advantages of electrochemical detection are demonstrated. A model SNP system is presented to illustrate the potential advantages in coupling the single base extension (SBE) technique to capillary gel electrophoresis (CGE) with electrochemical detection. An electrochemically labeled primer, with a ferrocene acetate covalently attached to its 5' end, is used in the extension reaction. When the Watson-Crick complementary ddNTP is added to the SBE reaction, the primer is extended by a single nucleotide. The reaction mixture is subsequently separated by CGE, and the ferrocene-tagged fragments are detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first single base resolution separation of DNA coupled with electrochemical detection. The unextended primer (20-mer) and the 21-mer extension product are separated with a resolution of 0.8.

Common, Rare, and Individual Variations of Mitochondrial DNA Associated with Diseases or Longevity.

The human nuclear genome is now being extensively analyzed in search for single nucleotide polymorphisms (SNPs) that are useful for predicting susceptibility to various diseases. To identify SNPs associated with diseases or longevity in the human mitochondrial genome, we have started analyzing the entire sequences of mitochondrial DNA (mtDNA) from 576 individuals, including 96 centenarians, 96 patients with Parkinson's disease, 192 patients with diabetes mellitus with or without severe vascular complications, and 192 young adults with or without obesity. We are going to establish a database for SNPs in the human mitochondrial genome based on these analyses. We assume that the SNPs in the mitochondrial genome are functionally as important as those in the nuclear genome. We hypothesize that the functional property of mitochondria of an individual is genetically influenced by a combination of common, rare, and individual variations in their mtDNA. The construction of human mtDNA database will provide important information for developing an efficient multiplex analysis system for SNPs to predict longevity or susceptibility to age-associated diseases.