Research Reports
Improvements to bead-based oligonucleotide ligation SNP genotyping assays Shannon E. Bruse, Michael P. Moreau, Marco A. Azaro, Ray Zimmerman, and Linda M. Brzustowicz BioTechniques 45:559-571 (November 2008) doi 10.2144/000112960
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.
INTRODUCTION 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 genomewide 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) (9–12), 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) (13–18). 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 (19–22). 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.
Department of Genetics, Rutgers University, Piscataway, NJ, USA Vol. 45 ı No. 5 ı 2008
www.biotechniques.com ı BioTechniques ı 559
Research Reports 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 0.90. DISCUSSION We have presented a method for multiplexed SNP genotyping on the Luminex platform that is accurate, robust, and significantly less expensive than previously published methods. Differences between this method and previously published Luminex-based methods include the use of significantly fewer beads, use of a universal biotinylated oligonucleotide for signal quantification, and no wash or centrifugation steps. We assessed concordance with both direct sequencing and pyrosequencing to establish validity, and observed 100% and >99% concordance, respectively. Additional experiments with separate multiplexed assays demonstrated complete concordance across technical replicates and high call rates (>99%). Additionally, we presented a 19-plex assay in which 15 of 19 assays were converted. It should be noted that secondary SNPs within OLA probe binding sites can negatively impact conversion rates (17), and careful assay design requires knowledge of
secondary SNPs. Cross-hybridization between nucleic acids present within single-tube, multiplexed OLA reaction can also reduce conversion rates (data not shown). Bioinformatics approaches which predict unwanted nucleic acid interactions could be used to rationally partition multiplexed OLA reactions and increase conversion rate. The cost is approximately $0.085 per genotype when performing both the PCR and OLA reactions in a 15-plex format (Table 5). The cost of other SNP genotyping methods is difficult to estimate, as innovations continue to drive down the cost of established methods and individual users find ways to achieve economies of scale. For example, while service-based TaqMan SNP genotyping costs are approximately $0.50 per genotype, advances in microfluidics are likely to significantly drive down this cost (www.fluidigm.com). In general, per-genotype costs of $0.085 are on the low end of informal estimates of other medium throughput genotyping technologies, and our method should be useful to academic labs with limited budgets, as well as core facilities requiring flexibility in performing targeted genetic studies. While the PCR step of our assay is time consuming (5 h) due to specialized cycling parameters required for the multiplexed PCR reaction, workflows can be established that result in approximately 40,000 genotypes per day. In practice, we perform multiple PCR and OLA reactions in parallel, and can then analyze one plate per hour on the Luminex flow cytometer, with each Vol. 45 ı No. 5 ı 2008
Research Reports 96-well plate producing 4,800 genotypes when multiplexing at 50 SNP assays per well. For this study, a combination of k-means clustering, Silhouette scores, and manual inspection of raw data was used for genotype calling (outlined in Materials and Methods). While this provides a method for semi-automated genotype calling, k-means clustering requires manual inspection of the raw data to ensure that the algorithm arrives at a reasonable solution. In this study there were two SNPs for which k-means clustering resulted in genotype designations that clearly did not agree with visual inspection of scatterplots. It appeared in both cases that high-density genotype clusters were incorrectly divided, a problem for k-means clustering that has been noted elsewhere (30). In both cases, running the k-means algorithm multiple times eventually resulted in a visually “correct” solution. Alternatively, we
found that we could resolve the problem by splitting data from 750 samples into two separate data sets containing allelic ratios from 375 samples each. Neither of these solutions is ideal and refinements to the clustering algorithm, such as careful seeding of k centers, may prove to be useful (31). No reports directly assess the issue of bead count when performing SNP genotyping assays on the Luminex platform. One previous report has assessed the variability in measurements when counting different numbers of beads using limiting amounts of SA-PE or analyte, and concluded that, under the appropriate conditions, counting as few as 10 beads can distinguish two populations (32). The appropriate conditions included a high amount of SA-PE (or analyte) relative to bead number. The conditions of our assay utilize massively saturating amounts of SA-PE (equivalent to 1.5 × 108 molecules per bead) and
analyte, so it is not surprising that we can clearly and robustly distinguish two populations when counting as few as 20 beads (and perhaps fewer). Notably, run times on the Luminex flow cytometer are impacted by the number of input beads and the proportion of those input bead one attempts to count. A typical 96-well plate assay using 5000 input beads per well and counting 100 beads has an approximate 30-min runtime; using 200 input beads and counting 20 requires approximately 45 min. The degree of multiplexing does not dramatically affect run times. Issues of machine timeout begin to appear when trying to use less than 200 beads while still counting 20, or when trying to count more than 20 beads with only 200 input beads. Also, there may be a small percentage of well-towell bead carryover using the Luminex flow cytometer. Using the median, rather than the mean, ensures that this carryover will not significantly affect the MFI and
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Mutation Discovery Genotyping | Gene Expression
subsequent genotype calls when counting 20 beads. It is important to point out that we have only assessed using low bead counts for a qualitative, OLA SNP genotyping assay. In quantitative assays, where minimizing intra- and inter-well variance is paramount, it may not be appropriate to use bead counts as low as 20. Using low bead counts may also not be appropriate for assays utilizing the direct hybridization format, where the non-target DNA strand will be available to compete with the probe-coupled microsphere for annealing to the target strand. ACKNOWLEDGEMENTS
We thank Jared Hayter, Jaime Simone, and Irina Tereschenko for technical assistance. This work was supported by funding from grant nos. R01MH062440, R01MH070366, R01MH080429, R01MH080429, and U24MH068457 from the National Institute of Mental Health and NARSAD and the Staglin Family Music Festival Schizophrenia Research Award. This paper is subject to the NIH Access Policy. COMPETING INTEREST STATEMENT
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hsa hs hsa sa-le let-7 le let t-7 7a hsa-miR-107 hsa hsa-le hsa a--le le ett-7 t--7 -7b hsa-miR-122a hssa hsa sa-le --llettt-7 - c hsa-miR-126 h hsa sa-le le let-7 et-7 t 7d hsa-miR-127 hsa hs hsa a-le lett-7 let-7 7e 7e hsa hs h sa sa sa-miR-128a h hsssa-le hsa llett-7 7f hsa hssa hsa h a-mi miR mi R---1 R-1 12 28 28b 8b hsa hssa h hsa a-le let-7 le t-7 7g hs hssa-m hsa -mi miRmi R-1 R --1 12 29 9 hs hsa hs hsa hsa-le s -le lle ettt-7 -7i -7 hsa hsa h sa sa-mi -mi -m mR R--1 R-1 - 30 30 30a 0a a hsa h hssa h hsa sa-m -mi miiR m R---1 R-1 1 hsa h hs sa a--m -mi m miiR R-1 -13 30b 30 0b 0b hsa hsa a-mi -m miiR-1 m R--1 100 00 h -m hs hsa -mi miiR m R-1 R--1 132 32 hssa hs hsa a-m -m -mi miiR-1 R-1 R-103 103 03 hsa h a-mi -m miiRm iR-1 R---1 R 13 34 4 hsa h sa a--mi -m miR mi R-1 -105 -1 hssa hsa h sa-mi -miR-1 -m R-1 R-1 -13 35a 35 5 5a hs hsa hsa a-m --mi miiR m R-1 -1 -10 0a a hs hsa h hs ssa a-mi -m -m miRR--1 R-1 R-135 135 35b 35b 5b hhsa sa a-m a-m -mi miR-1 R-1 -10b 0b hssa hs hsa a-mi - R -m R-13 R-1 13 36 6 h hsa hs sa-mi sa sa-m -m miiR iR R-1 -1 -1 133 33 3 33a 3a hs hssa hs a-mi miR mi R--13 R-1 37 7 hs hsa h hs sa s -m -mi miiR m R-1 -1 13 33 33b 3b 3b hsa hsa hs h sa-mi sa --m miiR m R-1 R-14 40 0 hsa hsa h hs sa a-m miiR m R--1 R-1 -142 424 2--3p 2 3p h hsa h sa sa-mi --m miR R-1 - 41 -1 1 h hsa hsa hs sa-m miR mi R-1 R-1 15a 5a h hssa hsa h sa-mi -m miR mi R---1 R-1 143 4 hssa hsa h sa-m a-mi -m miiR-1 m R-15 R5b b hssa hsa hs a-miR R-1 R -1 -145 145 45 hsa h hs ssa a-m --mi miiR-1 m R 6 Rhssa-miR-1 -146 -146 -1 46 46a 6a a hsa hsa a--mi -m miiR R-182 R-1 82* 8 2** 2 hsa-miR-146 46b 46 4 6 6b b hsa hsa h saa-m -m miiR R-184 R-1 84 hsa-miR-147 hsa hssa hsa sa-mi -m miR-1 R--1 R-1 R 18a 8a hsa-mi miR-1 R-150 50 0 h hsa-miR-151 hsa hssa-mi hs sa a-mi miR mi iR R-1 R-1 18a 8a* 8 a** hsa-miR-152 h hsa hs h ssa a-m -mi miR mi R-1 18 18b 8b b h hssa hsa hs a-m -miR-1 miR R--1 -199a 9a a hsa-miR-153 h hsa hsa a-mi -miR-1 R-1 RR -1 199b 99b hsa-miR-154 hssa h hsa sa-mi -m miR-2 mi R-2 R-20a R-2 20a 0a hsa-miR-154* hsa hsa hs a-m a-m -mi miiR-2 m R 20b 0b 0 b hsa-miR-155 hsa h hs ssa a-mi -mi -m miR R-2 -2 21 14 4 hsa-miR-182 h hsssa-mi h sa a-mi miiR-2 m R-2 2Select 23 3a 3a hsa-miR-183 the miRNAs you want.
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Received 5 May 2008; accepted 24 August 2008. Address correspondence to Shannon E. Bruse, 145 Bevier Road, Department of Genetics, Rutgers University, Piscataway, NJ, USA, 08854. email: shannon.bruse@ mirusbio.com To purchase reprints of this article, contact:
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