A comparison between SNaPshot, pyrosequencing, and biplex invader SNP genotyping methods: accuracy, cost, and throughput

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Abstract

Three methods of Single Nucleotide Polymorphism (SNP) detection: SNaPshot®, Pyrosequencing® and Biplex Invader®, with two different chemistries were investigated to compare, (1) accuracy, (2) ease of use, (3) throughput capability, and (4) cost. We genotyped 192 human DNA samples across 24 SNPs (minor allele frequencies above 30%), of which seven SNPs were genotyped with all three methods. We show that the Biplex Invader® genotyping method was found to be the most accurate and easiest to use with lowest cost, although Pyrosequencing® provided similar results at a low cost. With little optimization, the accuracy of the SNaPshot® method was also comparable to these two methods with a higher cost, if only singleplex reactions are used.

Introduction

Single nucleotide polymorphisms (SNPs) are the most frequent forms of DNA sequence variation in the human genome and are the markers of choice for association studies of human complex traits. A large number of SNPs have been identified and more than 9.5 million entries are currently in the National Center for Biotechnology Information (NCBI) SNP database (http://www.ncbi.nlm.nih.gov/SNP). In some cases a single SNP is responsible for genetic disease [1]. Accurate and fast genotyping of SNPs is very important in exploring the relationship between genome structure and function [2], [3], [4]. The emerging role of SNPs in disease has led to the development of several elegant methods of genotyping, or determining the SNP allele in a sample. Despite the many methodologies available for genotyping, no technology for scoring SNPs has become a widely accepted standard, and the task of choosing a method is difficult [5], [6], [7], [8], [9], [10], [11], [12]. Most of these methods are expensive and often beyond the reach of the typical low throughput academic laboratory. Many laboratories are now pursuing candidate gene approaches rather than a genome scan, where the extent of markers that need to be typed can be prohibitive. Small to medium sized academic laboratories are therefore looking for a suitable method that provides a simple, highly accurate and cost-effective technology for a modest number of SNPs typed on hundreds of individuals or on pooled samples for a candidate gene region. Genotyping with a suitable technology as well as pooling into an effective sample size are the most cost and time efficient [13], [14], [15], [16], [17].

In this study, we tested 24 SNPs (minor allele frequency above 30%) on 192 samples (from 48 nuclear families with two sibs) with three easily implementable genotyping methods (Table 1). Currently popular methods include Pyrosequencing, SNaPshot, Read It, Biplex Invader assay, TaqMan and Molecular Beacon. We chose three: SNaPshot (ABI) and Pyrosequencing (Pyrosequencing AB) using allele-specific primer extension, and Biplex Invader assays (Third Wave Technologies) using structure-specific endonucleolytic cleavage. We chose these three methods based on the criteria that these methods were all non-radioactive, gel-free and able to genotype both alleles in a single reaction. Although TaqMan and Molecular Beacon technologies are also non-radioactive and gel-free, the use of fluorescent oligonucleotide probes in these two methods make them much more expensive than SNaPShot and Pyrosequencing methods which use only unlabelled primers. The Biplex Invader method, however, does use a Fluorescence Resonance Energy Transfer (FRET) probe, but the cost is much less than TaqMan and Molecular Beacon. The Read IT method was also not included, because it requires two separate reactions to interrogate both alleles. All three chosen methods provide a practical solution for tackling medium-throughput genotyping. The aim of this investigation was to compare the ability of each method in terms of accuracy, ease of use, throughput capabilities and relative cost.

The SNaPshot method first requires target amplification from genomic DNA, followed by purification of the amplified DNA. The SNaPshot primer targets a sequence immediately upstream of the SNP site and is extended by a single base in the presence of all four fluorescently labelled dideoxy nucleotides (ddNTPs). Each fluorescent ddNTP emits a different wavelength, which is translated into a specific color for each base. The size of the product is the size of the initial probe plus one fluorescent base. The reactions are run on an ABI 3700 and genotypes are determined by the color and location of the peak that is generated from the emitted fluorescence. Data are then analyzed with the ABI Gene Scan™ software package using size standards for verification of the peaks. Primer design and DNA template purification can significantly affect genotyping accuracy. Failure to remove unincorporated ddNTPs can yield extraneous fluorescence. This can prevent a sample from being genotyped, or cause it to be genotyped incorrectly. Genemapper™ software is available to automatically determine sample genotypes.

Pyrosequencing is a recently developed DNA sequencing method that also requires target amplification from genomic DNA, followed by purification. This method is based on detecting the formation of pyrophosphate, the by-product of DNA polymerization. In a number of enzymatic steps, pyrophosphate is converted to ATP, which fuels a luciferase reaction and converts luciferin to oxyluciferin. Light is generated with every addition of a nucleotide in the growing DNA chain [18], [19]. The intensity of light generated is proportional to the amount of nucleotide incorporation. Scores are determined by computer-automated comparison of predicted SNP patterns with raw data. Samples generally do not require manual interpretation, which provides reliability and accuracy in scoring.

The Biplex Invader Assay can work with genomic DNA without the need for PCR amplification or with PCR product if genomic DNA is limited. It is a fluorescent method, based on the ability of a thermostable flap endonuclease to cleave a structure formed by the hybridization of two overlapping oligonucleotide probes to a target sequence. Probe (for allele 1 or allele 2) and Invader® oligonucleotides hybridize in tandem to a specific region of DNA. They cover a polymorphism at the 5′ end and generate a structure recognized by the Cleavase×enzyme. Once the proper structure is formed the 5′ flap is released by cleavage as the target specific product. Both FRET cassettes (fluorescein and Invader red dye) are in the same reaction, and each released allele-specific flap invades its FRET cassette, causing a fluorescein or Invader red dye (depending on genotype) to be freed from its quencher. Since each released flap can in turn invade multiple FRET cassettes, this produces an amplified fluorescent signal. The fluorescent signals generated are detected at an arbitrary end time point with a traditional fluorescence plate reader.

Section snippets

Materials and methods

Genomic DNA from a total of 192 individuals comprising 48 families was kindly provided by Dr. John Todd's laboratory, Cambridge, UK. DNA was quantitated using the PicoGreen assay (Molecular Probes, USA). Out of 90 SNPs generated by comparing genomic sequence information, we randomly selected 24 with minor allele frequency >30%. PCR primers were generated using Primer3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and IDT's OligoAnalyzer 2.5 program (http://www.idtdna.com

Results

Here, we define a few terms that have been used in this study: (1) Failure—We could not make a call on genotype. (2) Cross Platform Error Rate (CPER)—In the seven control SNPs, the number of incorrect genotypes for a method when compared to sequencing data. Internal Error Rate (IER)—Discrepancy of a genotype in two runs with same method. This IER was based on the number of successful samples that happened to be repeated during the course of any failed sample repeat. Therefore, the number of

Discussion

Over the past few years, several new methods have been developed that are an improvement over the previously existing methods of genotyping. In our study, we have compared SNP genotyping methods for labs pursuing candidate gene approaches rather than a genome scan, where the extent of markers that need to be typed can be prohibitive. These types of studies still require accurate, cost-effective genotyping methods with medium-to-high throughput. We have found that the methods adopted here are

Acknowledgements

We thank Dr. John Todd, Cambridge, UK, for providing us 192 DNA samples. We thank Eric Christensen (Pyrosequencing) and Dr. David Grimm (Abbott laboratories), for helping us to use the Pyrosequencing Instrument and analyse the data in Abbott laboratories. We thank Dr. Martin Hessner for reviewing this manuscript. We also thank The Max McGee National Research Center for Juvenile Diabetes, Medical College of Wisconsin and Children's Hospital of Wisconsin for funding this study.

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