Cost-effective Single Nucleotide Polymorphisms Genotyping Research Articles

Development of a cost-effective single nucleotide polymorphism genotyping array for management of greater yam germplasm collections.

Using genome‐wide single nucleotide polymorphism (SNP) discovery in greater yam (Discorea alata L.), 4,593 good quality SNPs were identified in 40 accessions. One hundred ninety six of these SNPs were selected to represent the overall dataset and used to design a competitive allele specific PCR array (KASPar). This array was validated on 141 accessions from the Tropical Plants Biological Resources Centre (CRB‐PT) and CIRAD collections that encompass worldwide D. alata diversity. Overall, 129 SNPs were successfully converted as cost‐effective genotyping tools. The results showed that the ploidy levels of accessions could be accurately estimated using this array. The rate of redundant accessions within the collections was high in agreement with the low genetic diversity of D. alata and its diversification by somatic clone selection. The overall diversity resulting from these 129 polymorphic SNPs was consistent with the findings of previously published studies. This KASPar array will be useful in collection management, ploidy level inference, while complementing accurate agro‐morphological descriptions.

Introduction of High Throughput and Cost Effective SNP Genotyping Platforms in Soybean

We presented here the application of two in-plate SNP (single nucleotide polymorphism) genotyping platforms for soybean plants [Glycine max (L.) Merr.], KASP® (Kompetitive Allele Specific PCR genotyping, LGC Genomics) and TaqMan® (Life Technologies) respectively. These two systems offer us an ability to determine the genotypes of 384 individual samples accurately and efficiently by allele specific PCR in a single plate using typical PCR conditions. Both of the systems require small quantity of genomic DNA obtained from a simple DNA extraction. The genomic sequences containing target SNPs can easily be used as a basic blueprint to design the probes and primers of KASP® and TaqMan® assays whether the sequences are obtained from the genome sequence of soybean William 82 (Wm82.a2.v1), Illumina Soy50k SNPs, or parallel resequencing. Moreover, we listed the pros and cons of the two systems and explained the principles behind the platforms. The high call rate and clear clustering separation of the SNPs can be readily obtained from these platforms without conducting any assay optimization processes. These platforms can routinely be performed on 96/384-well plate format with or without an automation procedure. Therefore, these platforms are especially suitable for the SNP genotyping on a particular trait with a large sample size, gene fine mapping, and marker assisted selection. Further, they require little hands-on experience and achieve per-site and per-individual costs below that of current SSR, AFLP, RFLP, and SNP chip technologies. The platforms can be used for genotyping on a wide range of organisms due to their simplicity and flexibility of handling. Meanwhile, we also especially presented some of the advantages using KASP® SNP genotyping pipeline, which was cost effective in the selection of allele specific assay and therefore, efficiently facilitated the soybean genotyping across large numbers (thousands or more) of individual lines for a great range of markers (hundreds to thousands) in our laboratory.

PCR-CTPP design for enzyme-free SNP genotyping using memetic algorithm.

In recent years, many single nucleotide polymorphisms (SNPs) have been successfully genotyped by polymerase chain reaction with confronting two-pair primers (PCR-CTPP). However, computation experiments of feasible CTPP primers are still challenging. The melting temperatures between four primers must be within a very narrow range, and many primer constraints need to be conformed to. PCR-CTPP is a simple, time- and cost-effective SNP genotyping method applied in molecular biology and biomedical fields. In this study, an MA (memetic algorithm)-based method is proposed to enable the design of feasible CTPP primer sets. Overall, 288 SNPs which exclude the deletion/insertion polymorphisms (DIPs) and multi-nucleotide polymorphisms (MNPs) in the SLC6A4 gene were tested in silico. The results were compared with a GA (genetic algorithm)-based method and indicate that the proposed method provides more feasible CTPP primers than the GA-based method. The MA-based CTPP primer design method provides critical melting temperatures and all kinds of evaluation of the common primer constraints. It could conceivably assist biologists and other researchers in obtaining feasible CTTP primer sets. The MA-CTPP algorithm is implemented in JAVA and a user-friendly input interface is freely available at http://bio.kuas.edu.tw/ma-ctpp/.

SNP genotyping using KASPar assays.

In a separate chapter we describe a simple method for single nucleotide polymorphism (SNP) discovery using genomic reduction. Here we describe a scalable and cost-effective SNP genotyping method based on KBioscience's competitive allele-specific PCR amplification of target sequences and endpoint fluorescence genotyping (KASPar™) using a FRET capable plate reader or Fluidigm's dynamic array high-throughput platform.

Development of a SNP genotyping panel for detecting polymorphisms in Oryza glaberrima/O. sativa interspecific crosses

Oryza glaberrima accessions harbor genes for tolerance to abiotic stresses such as mineral deficiency in problem soils. This genetic potential could be exploited in interspecific crosses with Oryza sativa, as in the case of the ‘New Rice for Africa’ (NERICA) varieties; however, to attain this goal it would be desirable to develop a high-throughput marker system to specifically detect O. glaberrima introgressions in an O. sativa background. Therefore, a single nucleotide polymorphism (SNP) genotyping analysis of an O. glaberrima accession (CG14) with two O. sativa lines (WAB56-104 and WAB181-18) was performed on a genome-wide basis. Comparison of CG14 and the WAB lines resulted in a set of 9,523 polymorphic SNPs which would be suitable to detect O. glaberrima introgressions in upland NERICAs. In addition, a set of 1,540 polymorphic SNPs between O. glaberrima versus O. sativa was identified. A subset of SNPs which were evenly distributed in the genome was then used to design a flexible and cost-effective SNP genotyping panel using the Competitive Allele-Specific PCR technology (KASP). This SNP genotyping panel consists of 2,015 SNPs successfully converted into KASP markers, providing 745 polymorphic SNPs for the parents O. glaberrima CG14/O. sativa WAB56-104 (upland NERICA), and 752 for O. glaberrima TOG5681/O. sativa IR64 (lowland NERICA). KASP markers were successfully validated by mapping O. glaberrima introgressions in NERICA-derived breeding lines. This new SNP genotyping panel will be useful in modern breeding applications such as QTL mapping and/or marker-assisted selection.

Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ∼50% to ∼80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (∼100 ng to ∼0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.

Confronting two-pair primer design for enzyme-free SNP genotyping based on a genetic algorithm

BackgroundPolymerase chain reaction with confronting two-pair primers (PCR-CTPP) method produces allele-specific DNA bands of different lengths by adding four designed primers and it achieves the single nucleotide polymorphism (SNP) genotyping by electrophoresis without further steps. It is a time- and cost-effective SNP genotyping method that has the advantage of simplicity. However, computation of feasible CTPP primers is still challenging.ResultsIn this study, we propose a GA (genetic algorithm)-based method to design a feasible CTPP primer set to perform a reliable PCR experiment. The SLC6A4 gene was tested with 288 SNPs for dry dock experiments which indicated that the proposed algorithm provides CTPP primers satisfied most primer constraints. One SNP rs12449783 in the SLC6A4 gene was taken as an example for the genotyping experiments using electrophoresis which validated the GA-based design method as providing reliable CTPP primer sets for SNP genotyping.ConclusionsThe GA-based CTPP primer design method provides all forms of estimation for the common primer constraints of PCR-CTPP. The GA-CTPP program is implemented in JAVA and a user-friendly input interface is freely available at http://bio.kuas.edu.tw/ga-ctpp/.

Application of High-Resolution Melting to Large-Scale, High-Throughput SNP Genotyping: A Comparison with the TaqMan® Method

Because of the wide use of single-nucleotide polymorphisms (SNPs) as markers of genetic variation, several high-throughput genotyping methods have been developed and applied during the past decades. High-resolution melting (HRM) is a very attractive, advanced, fast, and cost-effective SNP genotyping technology based on the analysis of the melting profile of PCR products, using intercalating fluorescent dyes to monitor the transition from unmelted to melted DNA. The authors used HRM for genotyping 215 human DNA samples for SNPs in the ABCB1, NQO1, and SLC19A1 genes and 96 samples for SNPs in the IL1A and IL12B genes with the aim of assessing HRM sensitivity and accuracy in comparisons with the TaqMan((R)) assay in view of large-scale, high-throughput SNP-typing applications. The potential effect of PCR product size, T(M), GC content, and SNP position on HRM performances was explored with amplicons that were heterogeneous for these factors. Discrimination power ranged from 91.4% to 98.4%, being significantly lower only when the number of rare homozygotes dropped to 1 or few units. The availability of specific and validated assays, in addition to a better standardization of HRM experimental conditions, can considerably reduce time and costs of large-scale genotyping studies with a negligible risk of failure or misclassification.

Prim-SNPing: A Primer Designer for Cost-Effective SNP Genotyping

Many kinds of primer design (PD) software tools have been developed, but most of them lack a single nucleotide polymorphism (SNP) genotyping service. Here, we introduce the web-based freeware "Prim-SNPing," which, in addition to general PD, provides three kinds of primer design functions for cost-effective SNP genotyping: natural PD, mutagenic PD, and confronting two-pair primers (CTPP) PD. The natural PD and mutagenic PD provide primers and restriction enzyme mining for polymerase chain reaction-restriction fragment of length polymorphism (PCR-RFLP), while CTPP PD provides primers for restriction enzyme-free SNP genotyping. The PCR specificity and efficiency of the designed primers are improved by BLAST searching and evaluating secondary structure (such as GC clamps, dimers, and hairpins), respectively. The length pattern of PCR-RFLP using natural PD is user-adjustable, and the restriction sites of the RFLP enzymes provided by Prim-SNPing are confirmed to be absent within the generated PCR product. In CTPP PD, the need for a separate digestion step in RFLP is eliminated, thus making it faster and cheaper. The output of Prim-SNPing includes the primer list, melting temperature (Tm) value, GC percentage, and amplicon size with enzyme digestion information. The reference SNP (refSNP, or rs) clusters from the Single Nucleotide Polymorphism database (dbSNP) at the National Center for Biotechnology Information (NCBI), and multiple other formats of human, mouse, and rat SNP sequences are acceptable input. In summary, Prim-SNPing provides interactive, user-friendly and cost-effective primer design for SNP genotyping. It is freely available at http://bio.kuas.edu.tw/prim-snping.