Whole genome genotyping with the single base extension assay

We describe an efficient, accurate and robust whole-genome genotyping ( WGG ) assay based on a two-color , single-base extension ( SBE ), single-nucleotide polymorphism ( SNP )-scoring step. For quality control, at least two Illumina control DNA samples were genotyped in parallel with every experimental DNA samples we provided. Pyrosequencing is based on single-base extension chemistry, and the detection of the added nucleotide is achieved with the help of the firefly enzyme , luciferase.

This enzyme generates luminescence upon the release of the natural byproduct of DNA synthesis, pyrophosphate , when a base attaches to the primer ( Pyrosequencer developed by Biotage). Using high-throughput next-generation sequencing (NGS) and microarray technologies, researchers can obtain a deeper understanding of the genome , providing insight into the functional consequences of genetic variation.

Probes on the 27k array target regions of the human genome to measure methylation levels at 25CpG dinucleotides in 14genes. The assay uses hapten-labelled nucleotides in a primer extension reaction. SNPstream utilizes Single Base extension assay (sbe) and tag array technology. It is a multistep assay encompassing in brief, a multiplexed PCR reaction (up to SNP-specific PCR assays ) followed by a clean-up step and a primer extension reaction using tagged primers and labelled ddNTPS as terminators (Biodipy-Fluorescein and TAMRA). This high-value solution for parentage testing of multiple beef and dairy cattle breeds includes genotyping of relevant traits in a single assay.

SNP density for robust genome-association studies in cattle. Single base extension is followed by fluorescent staining, signal amplification, scanning, and analysis using the Genome Studio software.

I present argyle, an R package for analysis of genotyping array data tailored to Illumina arrays. Genotyping microarrays are an important and widely-used tool in genetics. The goal of the argyle package is to provide simple, expressive tools for nonexpert users to perform quality checks and exploratory analyses of genotyping data.

In the iPLEX assay , an oligonucleotide primer anneals immediately upstream of the SNP site being genotyped. The primer extension then takes place which is a single complementary mass-modified base. The mass of the extended products is determined by using MALDI-TOF mass spectrometry. Infinium II Assay Single Base Extension. These platforms have been widely exploited for dozens of major studies in human genetics including whole genome association studies, population genetic analyses, and copy number variation investigations.

There is a demand for technologies that allow the interrogation of large numbers of SNP polymorphisms, both in whole - genome panels and in smaller custom designed sets, to attempt to elucidate the nature of complex disease through linkage and association studies. The Illumina BeadArray technology offers a flexible platform. UPD) and mitotic recombination10.

This comprehensive genome -wide bovine genotyping array kit features over 770SNPs, and is compatible with any breed of beef or dairy cattle. The combination of proven assays, high-density arrays, and integrated software enables the analysis of LOH and copy number changes in both single and paired (i.e., matched) samples with high precision. Following single -tube, whole - genome amplification (no PCR or ligation steps), samples are hybridized to 50-mer bead-based probes.

After a labeled extension step, the BeadChip is imaged on Illumina.

MassARRAY SNP genotyping can analysis multiple SNP sites in one reaction. Enzymatic-based genotyping assays such as for 1SNP assays (1probe pairs, allele A and allele B), 3nonpolymorphic primer extension and ligation have previously been used directly on assays (match only), nonpolymorphic discrimination assays (probe pairs, array surfaces to achieve high levels of discrimination in genotyping match and mismatch), yeast loci (probe pairs, match and mismatch) and captured PCR products28–32. Steemers FJ, Chang W, Lee G, Barker DL,Shen R, et al. Most SNPs are binary, meaning that the process of genotyping a single SNP typically consists of determining which one of two nucleotide bases is present at the SNP locus. Methods for making that determination are diverse, and include array-based hybridization, PCR, and sequencing.

Overview of the whole - genome sampling assay The SNP Assay is based on a whole - genome sampling assay. In this assay , genomic DNA (5ng) is digested with Nsp I and Sty I restriction enzymes and ligated to adaptors that recognize the cohesive bp overhangs. Assays are processed with a single sample per array and perform both SNP genotyping and copy-number interrogation.

Single - base extension Single - base - extension reactions use a primer that binds to a region of interest and follow this with an extension reaction that allows the incorporation of a single base after the primer. The chemistry is based on the dideoxy single-base extension of an unlabeled oligonucleotide primer (or primers). Each primer binds to a complementary template in the presence of fluorescently labeled ddNTPs and DNA polymerase.

The polymerase extends the primer by one nucleotide, adding a single ddNTP to its 3´ end. The need for multiplexed methods for SNP genotyping has rapidly increased during the last decade. We present here a flexible system that combines highly specific genotyping by minisequencing single - base extension with the advantages of a microarray format that allows highly multiplexed and parallel analysis of any custom selected SNPs. The ability to simultaneously genotype hundreds of thousands of single‐nucleotide polymorphisms (SNPs) in a single assay has recently become feasible due to innovative combinations of assay and array platform multiplexing.

Whole Genome SNP Genotyping.