Development,validation and genetic analysis of a large soybean SNP genotyping array

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under‐use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high‐density genetic mapping and genome‐wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome‐wide association analyses. More than four million high‐quality SNPs identified from high‐depth genome re‐sequencing of 16 soybean accessions and low‐depth genome re‐sequencing of 31 soybean accessions were used to select 180 961 SNPs for creation of the Axiom^® SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170 223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene‐rich chromosomal regions suggest that this array may be suitable for genome‐wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.     

Manual 768 or 384 well microplate gel 'dry' electrophoresis for PCR checking and SNP genotyping

Electrophoresis continues to be a mainstay in molecular genetic laboratories for checking, sizing and separating both PCR products, nucleic acids derived from in vivo or in vitro sources and nucleic acid-protein complexes. Many genomic and genetic applications demand high throughput, such as the checking of amplification products from many loci, from many clones, from many cell lines or from many individuals at once. These applications include microarray resource development and expression analysis, genome mapping, library and DNA bank screening, mutagenesis experiments and single nucleotide polymorphism (SNP) genotyping. PCR hardware compatible with industry standard 96 and 384 well microplates is commonplace. We have previously described a simple system for submerged horizontal 96 and 192 well polyacrylamide or agarose microplate array diagonal gel electrophoresis (MADGE) which is microplate compatible and suitable for PCR checking, SNP typing (restriction fragment length polymorphism or amplification refractory mutation system), microsatellite sizing and identification of unknown mutations. By substantial redesign of format and operations, we have derived an efficient 'dry' gel system that enables direct 96 pin manual transfer from PCR or other reactions in microplates, into 768 or 384 well gels. Combined with direct electrode contact in clamshell electrophoresis boxes which plug directly to contacts in a powered stacking frame and using 5-10 min electrophoresis times, it would be possible (given a sufficient supply of PCRs for examination) for 1 million gel tracks to be run per day for a minimal hardware investment and at minimal reagent costs. Applications of this system for PCR checking and SNP genotyping are illustrated.     

单核苷酸多态性基因分型技术原理与进展

在基因组规模了解遗传变异与生物功能之间的关系可望为生物学带来全新的深入认识。本从等位基因分型机理、反应形式和检测方法等三个方面讨论SNP分型方法的现状,并简要介绍了目前应用的一些分型方法。     

SNP discovery by amplicon sequencing and multiplex SNP genotyping in the allopolyploid species Brassica napus

Oilseed rape (Brassica napus) is an allotetraploid species consisting of two genomes, derived from B. rapa (A genome) and B. oleracea (C genome). The presence of these two genomes makes single nucleotide polymorphism (SNP) marker identification and SNP analysis more challenging than in diploid species, as for a given locus usually two versions of a DNA sequence (based on the two ancestral genomes) have to be analyzed simultaneously during SNP identification and analysis. One hundred amplicons derived from expressed sequence tag (ESTs) were analyzed to identify SNPs in a panel of oilseed rape varieties and within two sister species representing the ancestral genomes. A total of 604 SNPs were identified, averaging one SNP in every 42 bp. It was possible to clearly discriminate SNPs that are polymorphic between different plant varieties from SNPs differentiating the two ancestral genomes. To validate the identified SNPs for their use in genetic analysis, we have developed Illumina GoldenGate assays for some of the identified SNPs. Through the analysis of a number of oilseed rape varieties and mapping populations with GoldenGate assays, we were able to identify a number of different segregation patterns in allotetraploid oilseed rape. The majority of the identified SNP markers can be readily used for genetic mapping, showing that amplicon sequencing and Illumina GoldenGate assays can be used to reliably identify SNP markers in tetraploid oilseed rape and to convert them into successful SNP assays that can be used for genetic analysis.     

Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis

Allele-specific amplification (ASA) is a generally applicable technique for the detection of known single nucleotide polymorphisms (SNPs), deletions, insertions and other sequence variations. Conventionally, two reactions are required to determine the zygosity of DNA in a two-allele system, along with significant upstream optimisation to define the specific test conditions. Here, we combine single tube bi-directional ASA with a ‘matrix-based’ optimisation strategy, speeding up the whole process in a reduced reaction set. We use sickle cell anaemia as our model SNP system, a genetic disease that is currently screened using ASA methods. Discriminatory conditions were rapidly optimised enabling the unambiguous identification of DNA from homozygous sickle cell patients (HbS/S), heterozygous carriers (HbA/S) or normal DNA in a single tube. Simple downstream mathematical analyses based on product yield across the optimisation set allow an insight into the important aspects of priming competition and component interactions in this competitive PCR. This strategy can be applied to any polymorphism, defining specific conditions using a multifactorial approach. The inherent simplicity and low cost of this PCR-based method validates bi-directional ASA as an effective tool in future clinical screening and pharmacogenomic research where more expensive fluorescence-based approaches may not be desirable.     

Monitoring of erythrocyte aggregate morphology under flow by computerized image analysis

The morphology of red blood cell (RBC) aggregates was studied by direct visualization of RBC aggregation at different flow conditions in a computerized image analyzer. The aggregate morphology is expressed by an Aggregate Shape Parameter (ASP), defined as the ratio of the aggregate projected area to its square perimeter. Aggregation was induced by either dextran-70 (m.w. 70,000) or dextran-500 (m.w. 500,000), and compared to that in plasma. It was found that the aggregate morphology is a characteristic of the aggregating agent-in dextran-500, the RBC form rouleau aggregates as in plasma, while in dextran-70, they form clusters. In each system, while maintaining the overall typical morphology, the ASP decreases (i.e., the aggregate becomes longer) as the aggregate size is increased. The distribution of the ASP as a function of the aggregate size remains unchanged when the aggregate size is changed by modulation of the dextran concentration or the shear stress. Stretching of a rouleau aggregate by application of shear stress is reflected by a corresponding change in the ASP. It is suggested that the ASP is a characteristic of intercellular interactions. A theoretical model is proposed for evaluation of the deviation of aggregate shape from that of rouleau structure.     

Automatic quantitative evaluation of autoradiographic band films by computerized image analysis

The present paper describes a new image processing method for automatic quantitative analysis of autoradiographic band films. It was developed in a specific image analysis environment (IBAS 2.0), but the algorithms and methods can be utilized elsewhere. The program is easy to use and presents some particularly useful features for evaluation of autoradiographic band films, such as the choice of whole film or single lane background determination; the possibility of evaluating bands with film scratch artifacts and the quantification in absolute terms or relative to reference values. The method was tested by comparison with laser-scanner densitometric quantifications of the same autoradiograms. The results show the full compatibility of the two methods and demonstrate the reliability and sensitivity of image analysis. The method can be used not only to evaluate autoradiographic band films, but to analyze any type of signal bands on other materials (e.g. electrophoresis gel, chromatographic paper, etc.).     

Ordered catenation of sequence-tagged sites and multiplexed SNP genotyping by sequencing

We describe a method for the efficient genotyping of SNPs, involving sequencing of ordered and catenated sequence-tagged sites (OCS). In OCS, short genomic segments, each containing an SNP, are amplified by PCR using primers that carry specially designed extra nucleotides at their 5′-ends. Amplification products are then combined and converted to a concatamer in a defined order by a second round of thermal cycling. The concatenation takes place because the 5′-ends of each amplicon are designed to be complementary to the ends of the presumptive neighboring amplicons. The primer sequences for OCS are chosen using newly developed dedicated software, OCS Optimizer. Using sets of SNPs, we show that at least 10 STSs can be concatenated in a predefined order and all SNPs in the STSs are accurately genotyped by one two-way sequencing reaction.     

SNP array analysis in constitutional and cancer genome diagnostics--copy number variants, genotyping and quality control

Array-based comparative genomic hybridization analysis of genomic DNA was first applied in postnatal diagnosis for patients with intellectual disability (ID) and/or congenital anomalies (CA). Genome-wide single-nucleotide polymorphism (SNP) array analysis was subsequently implemented as the first line diagnostic test for ID/CA patients in our laboratory in 2009, because its diagnostic yield is significantly higher than that of routine cytogenetic analysis. In addition to the detection of copy number variations, the genotype information obtained with SNP array analysis enables the detection of stretches of homozygosity and thereby the possible identification of recessive disease genes, mosaic aneuploidy, or uniparental disomy. Patient-parent (trio) information analysis is used to screen for the presence of any form of uniparental disomy in the patient and can determine the parental origin of a de novo copy number variation. Moreover, the outcome of a genotype analysis is used as a final quality control by ruling out potential sample mismatches due to non-paternity or sample mix-up. SNP array analysis is now also used in our laboratory for patients with disorders for which locus heterogeneity is known (homozygosity pre-screening), in prenatal diagnosis in case of structural ultrasound anomalies, and for patients with leukemia. In this report, we summarize our array findings and experiences in the various diagnostic applications and demonstrate the power of a SNP-based array platform for molecular karyotyping, because it not only significantly improves the diagnostic yield in both constitutional and cancer genome diagnostics, but it also enhances the quality of the diagnostic laboratory workflow.     

Rapid detection of quinolone-resistant Salmonella by real time SNP genotyping

A total of 63 isolates were screened for the gyrA mutation (87Asp-Tyr) in Salmonella enterica serovars using real time PCR. All of the isolates were successfully identified as resistant or susceptible, consistent with the MIC result of the agar dilution method and gyrA sequencing.     

Automatic quantitation of cell colonies on petri dishes by computerized image analysis

Clonogenic assay is one of the most sensitive assays, widely used to evaluate the effects of antineoplastic agentsin vitro. A computer program was developed on an IBAS 2.0 Image Analysis System for automated quantiation of cell colonies and clone area on Petri dishes. The sensitivity of the clonogenic assay can be greatly increased by evaluating the mean area of the clones. The program gives an objective, accurate and fast evaluation of large samples. It is simple to use and offers a high degree of flexibility. Special algorithms and techniques have been implemented for good quantitation of both connected and well-separated colonies and to reduce the background noise and the general error rate. The principles and solutions presented are applicable to any other image analysis system.Abbreviations FBS fetal bovine serum - ATCC American Type Culture Collection - DDATHF 5,10-dideazatetrahydrofolic acid - PBS phosphate buffered saline     

Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.     

SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis

We have compared the accuracy, efficiency and robustness of three methods of genotyping single nucleotide polymorphisms on pooled DNAs. We conclude that (i) the frequencies of the two alleles in pools should be corrected with a factor for unequal allelic amplification, which should be estimated from the mean ratio of a set of heterozygotes (k); (ii) the repeatability of an assay is more important than pinpoint accuracy when estimating allele frequencies, and assays should therefore be optimised to increase the repeatability; and (iii) the size of a pool has a relatively small effect on the accuracy of allele frequency estimation. We therefore recommend that large pools are genotyped and replicated a minimum of four times. In addition, we describe statistical approaches to allow rigorous comparison of DNA pool results. Finally, we describe an extension to our ACeDB database that facilitates management and analysis of the data generated by association studies.     

SNP discovery and population structure analysis in Lassi and Marecha camel breeds using a genotyping by sequencing method

Pakistani camels have been classified socio-geographically into 20 breeds, but they have not yet been subjected to substantial selective pressures and the genetic basis for these breeds is not understood. However, it should be possible to distinguish them by use of molecular data. This study investigated the genetic diversity and population structure within and between two major Pakistani camel breeds, Marecha and Lassi. As no SNP array is currently available, we first identified 63 619 SNPs using a genotyping by sequencing approach. After quality control, a panel of 36 926 SNPs was used in the analysis. Population structure was investigated with a principal coordinate analysis as well as a cluster analysis using NetView , and multilocus heterozygosity analysis to explore between- and within-breed genetic variation. In addition, between-breed variation was explored using the fixation index, F_ST. We also compared relationship matrices computed using the VanRaden SNP-based method and a method developed specifically for genotyping by sequencing data. Among the two camel breeds, Lassi showed a lower level of genetic diversity whereas Marecha showed a higher level. As a genotyping platform has not yet been developed for the camel, the SNPs discovered in this study will be useful in future genetic studies in camels.     

High-resolution analysis of chromosomal imbalances using the Affymetrix 10K SNP genotyping chip

Array-based comparative genome hybridization is a powerful tool for detecting chromosomal imbalances at high resolution. However, the design and setup of such arrays are time consuming and expensive and thus worthwhile only when large numbers of arrays will be processed. To provide a feasible solution, we have developed an algorithm that renders the publicly available Affymetrix 10K SNP genotyping chip useful for high-resolution analysis of chromosomal imbalances. We have used our newly developed algorithm to analyze data from Affymetrix 10K chips that were hybridized with DNA probes from a variety of different sources, such as primary tumors, cell lines, and blood from patients with unbalanced translocations. In summary, we were able to (i) demonstrate the capability of our method by reproduction of published and unpublished data obtained with alternative methods and (ii) identify novel imbalances that were not shown before.     

Quantification of angiogenesis by a computerized image analysis system in renal cell carcinoma

OBJECTIVE: To ascertain whether tumor angiogenesis quantitated by a computerized image analysis system correlates with clinical outcome in renal cell carcinoma. STUDY DESIGN: Microvessels were immunohistochemically labeled with antibodies to CD34 in sections from 62 cases of renal cell carcinoma. Computerized image analysis was used to evaluate the mean microvessel count (MMC) and mean percentage microvessel area (MPMA). RESULTS: MMC ranged from 19.3 to 315.0, while MPMA was 0.6-17.9%. There was a highly significant correlation between MMC and MPMA (r = .867, P < .01). Although MMC and MPMA decreased with increasing nuclear grade and TNM stage, this difference failed to achieve statistical significance. No statistically significant differences in survival were found for MMC or MPMA. CONCLUSION: Our results indicate that computerized image analysis can evaluate accurately tumor angiogenesis, but tumor angiogenesis in renal cell carcinoma does not provide significant prognostic information in renal cell carcinoma.