Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse

We have developed a genotyping system for detecting genetic contamination in the laboratory mouse based on assaying single-nucleotide polymorphism (SNP) markers positioned on all autosomes and the X chromosome. This system provides a fast, reliable, and cost-effective way for genetic monitoring, while maintaining a very high degree of confidence. We describe the allelic distribution of 235 SNPs in 48 mouse strains, thereby creating a database of polymorphisms useful for genotyping purposes. The SNP markers used in this study were chosen from publicly available SNP databases. Four genotyping methods were evaluated, and dynamic two-tube allele-specific PCR assays were developed for each marker and tested on a set of 48 inbred mouse strains. The minimal number of assays sufficient to distinguish groups consisting of different numbers of mouse strains was estimated, and a panel of 28 SNPs sufficient to distinguish virtually all of the inbred strains tested was selected. Amplifluor SNP detection assays were developed for these markers and tested on an extended list of 96 strains. This panel was used as a genetic quality control approach to monitor the genotypes of nearly 300 inbred, wild-derived, congenic, consomic, and recombinant inbred strains maintained at The Jackson Laboratory. We have concluded that this marker panel is sufficient for genetic contamination monitoring in colonies containing a large number of genetically diverse mouse strains and that reduced versions of the panel could be implemented in facilities housing a lower number of strains.     

Mutagenic primer design for mismatch PCR-RFLP SNP genotyping using a genetic algorithm

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is useful in small-scale basic research studies of complex genetic diseases that are associated with single nucleotide polymorphism (SNP). Designing a feasible primer pair is an important work before performing PCR-RFLP for SNP genotyping. However, in many cases, restriction enzymes to discriminate the target SNP resulting in the primer design is not applicable. A mutagenic primer is introduced to solve this problem. GA-based Mismatch PCR-RFLP Primers Design (GAMPD) provides a method that uses a genetic algorithm to search for optimal mutagenic primers and available restriction enzymes from REBASE. In order to improve the efficiency of the proposed method, a mutagenic matrix is employed to judge whether a hypothetical mutagenic primer can discriminate the target SNP by digestion with available restriction enzymes. The available restriction enzymes for the target SNP are mined by the updated core of SNP-RFLPing. GAMPD has been used to simulate the SNPs in the human SLC6A4 gene under different parameter settings and compared with SNP Cutter for mismatch PCR-RFLP primer design. The in silico simulation of the proposed GAMPD program showed that it designs mismatch PCR-RFLP primers. The GAMPD program is implemented in JAVA and is freely available at http://bio.kuas.edu.tw/gampd/.     

Fine genetic characterization of elite maize germplasm using high-throughput SNP genotyping

To investigate the genetic structure of Chinese maize germplasm, the MaizeSNP50 BeadChip with 56,110 single nucleotide polymorphisms (SNPs) was used to genotype a collection of 367 inbred lines widely used in maize breeding of China. A total of 41,819 informative SNPs with minor allele number of more than 0.05 were used to estimate the genetic diversity, relatedness, and linkage disequilibrium (LD) decay. Totally 1,015 SNPs evenly distributed in the genome were selected randomly to evaluate the population structure of these accessions. Results showed that two main groups could be determined i.e., the introduced germplasm and the local germplasm. Further, five subgroups corresponding to different heterotic groups, that is, Reid Yellow Dent (Reid), Lancaster Sure Crop (Lancaster), P group (P), Tang Sipingtou (TSPT), and Tem-tropic I group (Tem-tropic I), were determined. The genetic diversity of within subgroups was highest in the Tem-Tropic I and lowest in the P. Most lines in this panel showed limited relatedness with each other. Comparisons of gene diversity showed that there existed some conservative genetic regions in specific subgroups across the ten chromosomes, i.e., seven in the Lancaster, seven in the Reid, six in the TSPT, five in the P, and two in the Tem-Tropical I. In addition, the results also revealed that there existed fifteen conservative regions transmitted from Huangzaosi, an important foundation parent, to its descendants. These are important for further studies since the outcomes may provide clues to understand why Huangzaosi could become a foundation parent in Chinese maize breeding. For the panel of 367 elite lines, average LD distance was 391 kb and varied among different chromosomes as well as in different genomic regions of one chromosome. This analysis uncovered a high natural genetic diversity in the elite maize inbred set, suggesting that the panel can be used in association study, esp. for temperate regions.     

Arrayed primer extension in the "array of arrays" format: a rational approach for microarray-based SNP genotyping

This study provides a new version of the arrayed primer extension (APEX) protocol adapted to the 'array of arrays' platform using an instrumental setup for microarray processing not previously described. The primary aim of the study is to implement a system for rational cost-efficient genotyping where multiple singlenucleotide polymorphisms (SNPs) and individuals are genotyped on each microarray slide. Genotyping results are collected across 185 healthy Danish subjects and 76 SNPs on chromosome 3q13.31, because linkage to atopic disease phenotypes have been suggested in the Danish population. Linkage disequilibrium (LD) results from the experimental data are used in a novel comparison to baseline data defined by the international HapMap SNP database. Comparison on the LD results reveals a strong linear correlation irrespective of LD measure considered: R2 (D') = 0.73 and R2(r2) = 0.54. In conclusion, our results show that this setup is strong enough to support high-throughput genotyping, and these observations support that the HapMap genotype resource is important for defining SNP panels aimed at gene mapping in local subpopulations from Europe.     

Biophysical characterization of double-stranded oligonucleotides using ETBR and isothermal fluorescence spectroscopy: implication for SNP genotyping

The UV-absorption, fluorescence and CD spectra of aps 23 bp oligoduplexes were performed for potential diagnostic purpose. These oligonucleotide sequences were mimicked from natural mutations (mitochondrial genome) of human population (unpublished). This work was designed on the basis of hybridization of non-self complementary oligoduplexes (aps) containing no mismatch, one-mismatch and two-mismatches. Since melting temperature™ is dependent on concentration of the oligoduplex, various concentrations were used in this study protocol. The thermal spectra profiles (UV absorbance and fluorescence) of these oligoduplexes (aps) are different for a particular concentration, and can be implicated for mutations. − dF/dT (or dA/dT) vs T, lnK (or RlnK) vs T_M, ΔG vs T_M, ΔS vs T_M and ΔH vs T_M are also variable for those sequences. All these thermodynamic data were calculated from absorbance (at 260 nm) data. On the contrary to the 23 bp oligoduplexes (aps), the PCR products of 97 bp and 256 bp length were genotyped with ETBR (excitation 530 nm, emission 600 nm) fluorimetrically. But our attempts to genotype these PCR sequences with isothermal UV absorbance spectroscopy were unsuccessful. Isothermal UV absorbance spectra has a limitation of sequence length. However, the structural conformation (all B-type) of the oligoduplexes (aps) was determined using CD. The minor discrepancy in CD spectra of these oligoduplexes are not significant for mutational analysis. 97 bp nested PCR product was an amplicon having either GcT or AcC mutation of mitochondria of normal human population, whereas 256 bp PCR product was an amplicon of human BRCA2 gene (NCBI Accession No. AY151039) of chromosome 13 having either A or G mutation at position − 26.     

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.     

Microarray-based method for genotyping of functional single nucleotide polymorphisms using dual-color fluorescence hybridization

Screening disease-related single nucleotide polymorphism (SNP) markers in the whole genome has great potential in complex disease genetics and pharmacogenetics researches. It has led to a requirement for high-throughput genotyping platforms that can maximize the efficient screening functional SNPs with respect to accuracy, speed and cost. In this study, we attempted to develop a microarray-based method for scoring a number of genomic DNA in parallel for one or more molecular markers on a glass slide. Two SNP markers localized to the methylenetetrahydrofolate reductase gene (MTHFR) were selected as the investigated targets. Amplified PCR products from nine genomic DNA specimens were spotted and immobilized onto a poly-l-lysine coated glass slide to fabricate a microarray, then interrogated by hybridization with dual-color probes to determine the SNP genotype of each sample. The results indicated that the microarray-based method could determine the genotype of 677 and 1298 MTHFR polymorphisms. Sequencing was performed to validate these results. Our experiments successfully demonstrate that PCR products subjected to dual-color hybridization on a microarray could be applied as a useful and a high-throughput tool to analyze molecular markers.     

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.     

Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping

DNA sequencing has markedly changed the nature of biomedical research, identifying millions of polymorphisms along the human genome that now require further analysis to study the genetic basis of human diseases. Among the DNA-sequencing platforms available, Pyrosequencing has become a useful tool for medium-throughput single nucleotide polymorphism (SNP) genotyping, mutation detection, copy-number studies and DNA methylation analysis. Its 96-well genotyping format allows reliable results to be obtained at reasonable costs in a few minutes. However, a specific biotinylated primer is usually required for each SNP under study to allow the capture of single-stranded DNA template for the Pyrosequencing assay. Here, we present an alternative to the standard labeling of PCR products for analysis by Pyrosequencing that circumvents the requirement of specific biotinylated primers for each SNP of interest. This protocol uses a single biotinylated primer that is simultaneously incorporated into all M13-tagged PCR products during the amplification reaction. The protocol covers all steps from the PCR amplification and capture of single-stranded template, its preparation, and the Pyrosequencing assay itself. Once the correct primer stoichiometry has been determined, the assay takes around 2 h for PCR amplification, followed by 15-20 min (per plate) to obtain the genotypes.     

A method for non-invasive genotyping of APCmin/+ mice using fecal samples

The APC^min/+ mouse is commonly used in cancer research and is just one of many genetically altered models that is currently being developed. With high numbers of breeding programs, it is important to have a simple method that can be used to genotype the mice non-invasively. Here we report a reproducible method for genotyping mice with DNA extracted from fecal samples. Comparison of fecal results with those obtained from intestinal tissue DNA and clinical outcome (presence/absence of tumors) showed this technique to have 100% accuracy. This non-invasive method of genotyping may be applied to other transgenic mouse models.     

Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay

We have developed a new method using the Qbead system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral 'barcodes' are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein-protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.     

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.     

Accuracy of genotyping for single nucleotide polymorphisms by a microarray-based single nucleotide polymorphism typing method involving hybridization of short allele-specific oligonucleotides.

Advances in technologies for identifying genetic polymorphisms rapidly and accurately will dramatically accelerate the discovery of disease-related genes. Among a variety of newly described methods for rapid typing of single-nucleotide polymorphisms (SNPs), gene detection using DNA microarrays is gradually achieving widespread use. This method involves the use of short (11- to 13-mer) allele-specific oligonucleotides. This method allows simultaneous analysis of many SNPs in DNAs from a large number of individuals, in a single experiment. In this work, we evaluated the accuracy of a new microarray-based short allele-specific oligonucleotide (ASO) hybridization method. There is a 96-well formatted array on a single plate, in which up to 256 spots are included in each well. Fluorescent probes for our experiments were produced by multiplex PCR amplification often target SNP-containing regions. We genotyped 192 individuals across a panel of ten single base variations, which included an insertion/deletion polymorphism. For comparison, we genotyped the same individuals for the same SNPs by the method of single-base extension with fluorescence detection. The typing accuracies of the microarray-based PCR-ASO and single-base extension methods were calculated as 99.9% and 99.1%, respectively, on the basis of genotyping results determined by direct sequencing. We conclude that the microarray-based hybridization method using short ASO probes represents a potential breakthrough technology for typing large numbers of SNPs rapidly and efficiently.     

Construction of a genetic map based on high-throughput SNP genotyping and genetic mapping of a TuMV resistance locus in Brassica rapa

Brassica rapa is a member of the Brassicaceae family and includes vegetables and oil crops that are cultivated worldwide. The introduction of durable resistance against turnip mosaic virus (TuMV) into agronomically important cultivars has been a significant challenge for genetic and horticultural breeding studies of B. rapa. Based on our previous genome-wide analysis of DNA polymorphisms between the TuMV-resistant doubled haploid (DH) line VC40 and the TuMV-susceptible DH line SR5, we constructed a core genetic map of the VCS-13M DH population, which is composed of 83 individuals derived from microspore cultures of a F₁ cross between VC40 and SR5, by analyzing the segregation of 314 sequence-characterized genetic markers. The genetic markers correspond to 221 SNPs and 31 InDels of genes as well as 62 SSRs, covering 1,115.9 cM with an average distance of 3.6 cM between the adjacent marker loci. The alignment and orientation of the constructed map showed good agreement with the draft genome sequence of Chiifu, thus providing an efficient strategy to map genic sequences. Using the genetic map, a novel dominant TuMV resistance locus (TuMV-R) in the VCS-13M DH population was identified as a 0.34 Mb region in the short arm of chromosome A6 in which four CC–NBS–LRR resistance genes and two pathogenesis-related-1 genes reside. The genetic map developed in this study can play an important role in the genetic study of TuMV resistance and the molecular breeding of B. rapa.     

MARA: a novel approach for highly multiplexed locus-specific SNP genotyping using high-density DNA oligonucleotide arrays

We have developed a locus-specific DNA target preparation method for highly multiplexed single nucleotide polymorphism (SNP) genotyping called MARA (Multiplexed Anchored Runoff Amplification). The approach uses a single primer per SNP in conjunction with restriction enzyme digested, adapter-ligated human genomic DNA. Each primer is composed of common sequence at the 5′ end followed by locus-specific sequence at the 3′ end. Following a primary reaction in which locus-specific products are generated, a secondary universal amplification is carried out using a generic primer pair corresponding to the oligonucleotide and genomic DNA adapter sequences. Allele discrimination is achieved by hybridization to high-density DNA oligonucleotide arrays. Initial multiplex reactions containing either 250 primers or 750 primers across nine DNA samples demonstrated an average sample call rate of ~95% for 250- and 750-plex MARA. We have also evaluated >1000- and 4000-primer plex MARA to genotype SNPs from human chromosome 21. We have identified a subset of SNPs corresponding to a primer conversion rate of ~75%, which show an average call rate over 95% and concordance >99% across seven DNA samples. Thus, MARA may potentially improve the throughput of SNP genotyping when coupled with allele discrimination on high-density arrays by allowing levels of multiplexing during target generation that far exceed the capacity of traditional multiplex PCR.