We describe a bead-based, multiplexed, oligonucleotide ligation assay (OLA) performed on the Luminex flow cytometer. Differences between this method and those previously reported include the use of far fewer beads and the use of a universal oligonucleotide for signal detection. These innovations serve to significantly reduce the cost of the assay, while maintaining robustness and accuracy. Comparisons are made between the Luminex OLA and both pyrosequencing and direct sequencing. Experiments to assess conversion rates, call rates, and concordance across technical replicates are also presented.
Single-nucleotide polymorphisms (SNPs) represent a major source of genetic variation in human beings; the NCBI database dbSNP contains nearly 12 million unique SNPs, which correlates to an average of 1 SNP for every 250 base pairs. Due to their frequency, SNPs are an important type of marker used in association studies of human disease, and can themselves induce functional changes that contribute to disease and drug response variability (1,2). In a relatively short time, SNP genotyping technologies have evolved from rudimentary and simplexed gel-based assays to highly multiplexed arrays designed to simultaneously interrogate more than 1 million SNPs. Ultra-high throughput array–based genotyping technologies, such as those offered by Illumina (San Diego, CA, USA) (3) and Affymetrix (Santa Clara, CA, USA) (4), are utilized in genome-wide studies requiring the genotyping of hundreds of thousands of SNPs in hundreds (or low thousands) of samples. However, fine-mapping, candidate-gene, and other targeted genetic studies often require genotyping dozens or hundreds of SNPs in thousands of samples, and ultra-high throughput methods are not ideal for these applications. Though too numerous to list in their entirety, popular medium throughput commercial methods include technologies such as Biotage's pyrosequencing (Uppsala, Sweden) (5), Third Waves's Invader assay (Madison, WI, USA) (6), and Applied Biosystems’ TaqMan SNP assay (7) and SNPlex platform (8) (Foster City, CA, USA). Drawbacks of commercial genotyping platforms include equipment costs and the need to purchase proprietary reagents. The genotyping method presented here utilizes the Luminex flow cytometer (LX100 or LX200; Luminex Corporation, Austin, TX, USA), which is within the budget of many academic labs, and is a relatively open-source platform readily amenable to homebrew assays not requiring expensive and proprietary reagents. Additionally, unlike many SNP genotyping platforms, the Luminex flow cytometer is one of the few instruments capable of quantitating both proteins and nucleic acids (DNA, mRNA, and miRNA) (9,10,11,12), providing it with uses beyond the SNP genotyping assay presented here.
Our method uses the robust chemistry of the oligonucleotide ligation assay (OLA) in conjunction with the Luminex flow cytometry platform (Figure 1) (13,14,15,16,17,18). The assay is performed in 96-well plate format and has an upper multiplex capability of 50 SNPs per well, making it suitable for a moderate number of SNPs in a large number of samples. Two key modifications make this method less expensive and easier to perform than similar published methods. First, we use fewer beads than are traditionally used in quantitative Luminex assays (19,20,21,22). The most significant expense of typical Luminex-based assays is the polystyrene beads used. Typical recommendations for SNP typing assays are to use 2500 input beads while counting 100 beads, but our experience indicates that these numbers can be substantially reduced, with a significant savings in cost per genotype.
Second, a universal biotinylated oligonucleotide is employed for signal quantification. For a biallelic SNP, the OLA assay requires two allele-specific oligonucleotides and a common oligonucleotide. Traditionally, the common oligonucleotide is labeled (during synthesis) with a detector molecule (i.e., biotin), which allows for quantification of the OLA product. In contrast, we synthesize the common oligonucleotide to have an attached sequence which is complementary to a universal oligonucleotide double-labeled with biotin, thus saving on the cost of synthesizing biotin-labeled common oligonucleotides for each SNP assay. In addition to being less expensive, the method presented here does not employ typical wash or centrifugation steps, making it simple to perform.Materials and Methods Oligonucleotide primer and probe sets
The multiplex PCR primer selection program used in this study was an enhanced version of the program that was used to create >1000-plex PCRs for parallel genotyping (23). Amplicon size in multiplexed PCRs is generally <400 bp, though we have successfully genotyped from amplicons as large as 1200 bp. PCR primers were ordered in 96-well plate format (Integrated DNA Technologies, Coralville, IA, USA) and purified using standard desalting.
The OLA requires two allele-specific and one common probe for each SNP being assayed. Allele-specific probe pairs consist of a 5′ tag sequence and a 3′ locus specific portion which differs only at the terminal position of the probe. Each allele-specific probe contains a unique 24-base FlexMAP tag (Luminex Corporation) at the 5′ end to allow hybridization to a reverse complement anti-tag coupled to a unique FlexMAP microsphere. Common probes contain a locus-specific portion at the 5′ end, a universal capture sequence at the 3′ end, and are 5′-phosphorylated by the manufacturer (Integrated DNA Technologies). The universal capture sequence is the reverse complement of a doubly biotinylated universal oligonucleotide which is included in the bead hybridization step. For the universal capture sequence we employed the tag of FlexMAP bead 100. The universal oligonucleotide sequence is the anti-tag of bead 100; for this reason, FlexMAP bead 100 is not utilized in our assays. All OLA probe sets are designed so that the locus-specific portions of both the common and allele-specific probes have a melting temperature of approximately 64°C. Module 1 of the HyTher server was used to determine melting temperature (ozone3.chem.wayne. edu). OLA probe sets were ordered in 96-well plate format and purified using standard desalting. For primer and probe sets, see Supplementary Materials, available online at www.BioTechniques.com.