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Molecular Diagnostics and Genetics |
1 Tm Bioscience Corporation, Toronto, Ontario, Canada.
2 Ottawa Health Research Institute, The Department of Medicine, University of Ottawa, Division of Hematology, Ottawa, Ontario, Canada.
aAddress correspondence to this author at: Tm Bioscience Corporation, 439 University Ave., Toronto, Ontario, Canada M5G 1Y8. Fax 416-593-1870; e-mail susanb{at}tmbioscience.com.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer sets, followed by multiplex allele-specific primer extension using 12 universally tagged genotyping primers. The products were then sorted on the Tag-It array and detected by use of the Luminex xMAPTM system. Genotypes were also determined by sequencing.
Results: Empirical validation of the universal array showed that the highest nonspecific signal was 3.7% of the specific signal. Patient genotypes showed 100% concordance with direct DNA sequencing data for 736 SNP determinations.
Conclusions: The Tag-It microsphere-based universal array platform is a highly accurate, multiplexed, high-throughput SNP-detection platform.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used universal, minimally cross-hybridizing tags combined with solution-phase kinetics characteristic of microsphere-based hybridization reactions to improve signal-to-noise ratios. We used microsphere-based arrays to avoid the quality control/quality assurance obstacles encountered by first-generation arrays in which "each" chip represents a new entity. Unlike first-generation arrays, the described assay may easily be modified to reflect the ever-changing panel of relevant mutations associated with a given condition. This report demonstrates the use of this microsphere-based universal array genotyping platform for the detection of six single-nucleotide polymorphisms (SNPs)1 believed to be associated with venous thromboembolism, a classic example of a complex, multifactorial disorder involving multiple genetic abnormalities (1)(2)(3)(4)(5).
Included in the Tag-It assay panel are the three SNPs most commonly associated with thrombophilia: factor V Leiden (G1691A), the most common inherited cause of venous thrombosis (6)(7); factor II (prothrombin) G20210A, the second most common mutation (8); and methylenetetrahydrofolate reductase (MTHFR) C677T, whose association with thrombosis remains controversial (9). Three additional SNPs, MTHFR A1298C, factor XIII val34leu, and tissue factor pathway inhibitor (TFPI) C536T, have been included in the panel (10)(11)(12). Individually, these additional polymorphisms are believed to have little or no independent effect on venous thrombosis (13)(14)(15)(16)(17)(18); they may, however, act synergistically with other genetic or acquired risk factors, producing a more than additive effect or, in the case of factor XIII val34leu, a protective effect. Furthermore, the complex pathogenesis of thrombosis suggests that the impact of a given polymorphism will be dependent on gene–environment interactions (19). Because the risk of thrombosis increases when multiple variant genes are present, comprehensive panel testing may be useful.
This report describes the empirical validation of the universal array and compares the accuracy of the Tag-It microsphere-based universal array genotyping platform with standard dideoxy DNA sequencing for 132 patient samples analyzed for the six SNPs comprising the thrombophilia panel.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) were unmodified and were purified by standard desalting procedures. Chimeric allele-specific primer extension (ASPE) primers, which consisted of a 24mer universal tag sequence 5' to the allele-specific (18–22mer) sequence, were also unmodified but were purified by polyacrylamide gel electrophoresis (Table 2
). Before use, all oligonucleotides were reconstituted in sterile distilled, deionized H2O, and their exact concentrations were determined spectrophotometrically based on extinction coefficients provided by the supplier. Reconstituted oligonucleotides were scanned between 200 and 800 nm, and the absorbance was measured at 260 nm to calculate concentration.
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reagents
Platinum Taq, Platinum Tsp, individual deoxyribonucleoside triphosphates, and biotin-dCTP were purchased from Invitrogen Corporation. Shrimp alkaline phosphatase and exonuclease I were obtained from USB Corporation. Carboxylated fluorescent microspheres were provided by Luminex Corporation. The cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Pierce. OmniPur reagents including MES, 100 g/L sodium dodecyl sulfate, NaCl, Tris, Triton X-100, Tween 20, and Tris-EDTA buffer were purchased from EM Science. The streptavidin-conjugated phycoerythrin (1 g/L) was obtained from Molecular Probes Inc.
universal array validation
Microsphere coupling.
Each of the 100 amino-modified anti-tag sequences was coupled to its corresponding population of carboxylated microspheres according to Luminex’s one-step carbodiimide coupling procedure (20). For each population, 5 x 106 microspheres were combined with 1 nmol of NH2-oligonucleotide in a final volume of 50 µL of 0.1 mol/L MES, pH 4.5. A 10 g/L EDC working solution was prepared just before use, and 2.5 µL was added to each microsphere mixture and incubated for 30 min. A second 2.5-µL aliquot of freshly prepared EDC solution was added, followed by an additional 30-min incubation. After washes in 0.2 mL/L Tween 20 and 1 g/L sodium dodecyl sulfate, each anti-tag-coupled microsphere population was resuspended in 100 µL of Tris-EDTA buffer [10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA] and enumerated by use of either a Beckman Coulter Z2 Particle Count and Size Analyzer or a Neubauer BrightLine Hemacytometer.
Universal array hybridization.
For each hybridization run of 100 biotinylated tags (targets) reacted individually against all 100 anti-tagged microsphere populations, a single "master" bead mixture (the Tag-It array) was prepared containing all 100 microsphere populations. Hybridization reactions were carried out in triplicate (30 targets/plate), and "no-target" controls were run in replicates of 6. Hybridization reactions were carried out at 37 °C for 30 min in a MJ Research PTC-100. Each hybridization reaction consisted of 2500 microspheres of each of the 100 populations and 50 fmol of a single target in 50 µL of hybridization buffer A [0.2 mol/L NaCl, 0.1 mol/L Tris (pH 8.0), and 0.8 mL/L Triton X-100]. We then added 15 µL of reporter solution (10 mg/L streptavidin-conjugated phycoerythrin in hybridization buffer A) directly to each reaction without removal of unbound target. Reactions were incubated at 37 °C for 15 min and then placed on the Luminex xMAPTM flow cytometer (also at 37 °C), with 100 events per bead population being read. The instrument’s gate setting was established before the samples were run and was maintained throughout the course of the study. Two hybridization runs were carried out in which 50 fmol of each of the 100 biotinylated tags were hybridized to the full 100-plex array.
genotyping on the TAG-IT platform
A generalized overview of the Tag-It platform is given in Fig. 1
.
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Patient samples.
Whole blood was collected from study participants after receipt of informed consent at the Ottawa Health Research Institute. Genomic DNA was isolated from whole blood by use of the Qiagen QIAamp Blood Kit. DNA samples were stored at –20 °C after isolation. Before analysis, genomic samples were quantified spectrophotometrically by measuring absorbance at 260 nm, diluted to 5 ng/µL, and stored at 4 °C.
Multiplex PCR (6-plex).
Multiplex PCR was carried out with 25 ng of genomic DNA in a final volume of 25 µL. A no-target PCR negative control was included with each assay run. The reaction consisted of 30 mM Tris-HCl (pH 8.4), 75 mM KCl, 2 mM MgCl2, 200 µM each deoxyribonucleoside triphosphate, and 1.25 U of Platinum Taq, with factor II primers each at 0.5 µM, factor V and TFPI primers each at 0.3 µM, and all others present at 0.2 µM. PCR primer sequences are given in Table 1
. Samples were cycled in a MJ Research PTC-200 thermocycler with cycling conditions set at 95 °C for 5 min followed by 30 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. Samples were then held at 72 °C for 5 min and kept at 4 °C until use.
ASPE.
Before the ASPE reaction, each PCR reaction was treated with shrimp alkaline phosphatase to inactivate any remaining nucleotides (particularly dCTP) so that biotin-dCTP could be efficiently incorporated during the primer extension reaction. Each PCR reaction was also treated with exonuclease I to degrade any remaining PCR primers to avoid any interference with the tagged ASPE primers and the extension reaction itself. To each 25-µL PCR reaction, we directly added 2 µL of shrimp alkaline phosphatase (2 U) and 0.5 µL of exonuclease I (5 U). Samples were then incubated at 37 °C for 30 min, followed by a 15-min incubation at 99 °C to inactivate the enzymes. Samples were then added directly to the ASPE reaction.
Multiplex ASPE was carried out using 5 µL of treated PCR product in a final volume of 20 µL. Each reaction consisted of 20 mM Tris-HCl (pH 8.4); 50 mM KCl; 1.25 mM MgCl2; 5 µM biotin-dCTP; 5 µM each of dATP, dGTP, and dTTP; 1.5 U of Platinum Tsp; and 25 nM ASPE primer pool (i.e., each ASPE primer present at 500 fmol/reaction). The ASPE primer sequences are listed in Table 2
. The ASPE reactions were incubated at 96 °C for 2 min and then subjected to 40 cycles at 94 °C for 30 s, 54 °C for 30 s, and 74 °C for 60 s. Reactions were then held at 4 °C until use.
Universal array sorting.
Each hybridization reaction was carried out with 
2500 microspheres of each of the 12 anti-tag-bearing microsphere populations. Anti-tags were coupled to microspheres as described above in the section on universal array validation. The microspheres were combined in hybridization buffer B [0.22 mol/L NaCl, 0.11 mol/L Tris (pH 8.0), 0.88 mL/L Triton X-100], and 45 µL of the mixture was added to each well of a 96-well plate (MJ Research). A 5-µL aliquot of each ASPE reaction was then added directly to each well. The samples were then heated to 96 °C for 2 min in a PTC-200, followed by a 1-h incubation at 37 °C. After this incubation, samples were filtered through a 1.2 µm Durapore® membrane and washed once with hybridization buffer A [0.2 mol/L NaCl, 0.1 mol/L Tris (pH 8.0), 0.8 mL/L Triton X-100]. The microspheres were then resuspended in 150 µL of reporter solution (1 mg/L streptavidin-conjugated phycoerythrin in hybridization buffer A) and incubated for 15 min at ambient temperature. The reactions were read on the Luminex xMAP at ambient temperature. For each sample, instrument settings were set to read a minimum of 100 events per bead population with the gate setting being established before the samples were run and maintained throughout the course of the study.
genotyping by dna sequencing
For all samples used in the study, genotyping results obtained with the Tag-It assay were compared with genotyping results obtained with dideoxy dye-terminator sequencing chemistry. For each of the six SNPs, sequencing was performed in both the forward and reverse directions on PCR amplimers obtained using individual primer pairs. DNA sequencing reactions were performed at either the Ottawa Health Research Institute or Cortec DNA Service Laboratories.
data analysis and interpretation
DNA sequencing.
For all 132 patient samples in the study, forward and reverse DNA sequencing data for each of the six SNPs within a sample were analyzed by two individuals. For a sequencing call to be accepted for a particular SNP, a consensus between the two individuals was required. If a consensus was not obtained for a particular SNP, the call was determined to be ambiguous and was eliminated from the study. In addition, if the forward and reverse sequencing data for a particular SNP did not coincide, again the call was determined to be ambiguous and eliminated from the accuracy study.
Tag-It genotyping assay.
For each DNA sample tested with the Tag-It universal array platform, median fluorescence intensity (MFI) values were collected for each of the 12 microsphere populations corresponding to each allele within the assay. For each allele of a given sample, the NET MFI was set to be the larger of zero and the value obtained by subtracting the no-target (PCR negative control) MFI values from the respective MFI values of the sample. To exclude samples containing insufficient or degraded DNA or samples generating suboptimal results, acceptance criteria were defined such that, for each SNP within the assay, MFI units for at least one allele were required to be at least 10 times the no-target MFI for that allele and be at least 300. If these criteria were not met, none of the SNPs was called, and the sample was excluded from the accuracy study. For SNPs meeting the data requirements, the genotype was then determined based on the mutant allelic ratio where:
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The mutant allelic ratio represents the fraction of the total net MFI signal for a given SNP attributed to the presence of the mutant allele. Once threshold values were set, the allelic ratio was used to discriminate wild-type, mutant, and heterozygous SNP calls. Threshold values were empirically determined for each individual SNP. In this study, the mutant allelic ratio ranges were set to 0.00–0.10 for wild-type calls, 0.37–0.77 for heterozygous calls, and 0.90–1.00 for mutant calls. As with any hybridization-based assay, underlying polymorphisms occurring in the allele-specific primer-binding regions (other than those being queried) could affect the signals generated and consequently, the calls made.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Of the possible 9900 mismatch hybridization events that could have occurred when each of the 100 complementary tag sequences was hybridized individually to the pool of 100 bead-immobilized universal anti-tag sequences, only 6 events were observed in the first run (Fig. 2A
) and only 5 events were observed in the second run (Fig. 2B
). Four of the 11 total events were common to both runs. The highest nonspecific event generated a signal equivalent to 3.7% of the signal observed for the perfectly matched pair (i.e., specific hybridization event).
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genotyping assay optimization
Multiplex PCR conditions were established in a series of preliminary experiments (data not shown). PCR products generated under optimized conditions were analyzed by gel electrophoresis using the Helixx SuperGel150 system, which is capable of resolving single base pair differences within products. Fig. 3
shows six clearly resolved bands ranging in size from 97 to 154 bp for six different patient samples amplified under optimum PCR conditions. The PCR negative control showed no evidence of primer-dimer formation or spurious amplification products.
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Multiplex ASPE was optimized for several factors affecting specificity and signal output. Conditions examined included cycling conditions, annealing temperature, ASPE primer concentrations, and PCR reaction volume added to the ASPE reaction. To assess specificity at the ASPE reaction level, ASPE reactions were carried out on individual amplimers representative of each of the 12 alleles in the presence of all ASPE primers. The results are shown in Fig. 4
. Hybridization conditions used in the Tag-It genotyping assay differed slightly from those used during validation of the full 100-plex array.
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patient study
A total of 132 patient DNA samples were genotyped by both the Tag-It assay and DNA dideoxy sequencing for each of the following six SNPs: factor V Leiden G1691A, factor II G20210A (prothrombin), MTHFR C677T, MTHFR A1298C, factor XIII val34leu, and TFPI C536T. The DNA samples were selected from consecutive patients with venous thrombosis and from age-, sex-, and ethnicity-matched friend controls.
Overall, the Tag-It universal microsphere-based genotyping assay was able to generate calls for 779 of the 792 SNP calls possible for the 132 patient samples analyzed for each of the six SNPs. A total of 13 calls resulting from three individual samples could not be made because minimum signal requirements or allelic ratio cutoffs were not met. More specifically, two samples failed completely (i.e., 12 SNP calls were not made) because the signals for both TFPI alleles were less than 10 times the MFI of their no-target counterparts. These two samples were repeated twice with comparable results, indicating that the DNA quality or quantity was suboptimal. A thirteenth "no call" was generated by a third sample for which the mutant allelic ratio for MTHFR 1298 was 0.18, which is outside the allowable range (0.00–0.10). Again, the sample was repeated twice with equivalent results, indicating a sample-related issue. Sequencing data were obtained for 749 SNP calls. The 43 calls not made by sequencing were ambiguous (as defined in the Results section) and resulted from 38 samples. To assess the accuracy within the study, as determined by concordance, the 56 calls that could not be made by either the Tag-It assay (13 calls) or sequencing (43 calls) were excluded from the calculation. Of the remaining 736 SNP calls, 14 calls initially showed discordance between the Tag-It universal genotyping assay and DNA sequencing. The 14 discordant SNPs were resequenced and were found to correspond with the results obtained with the Tag-It universal genotyping assay. The error was attributed to sample mix-up during the preparation of sequencing amplimers. Thus, after sample re-runs, the Tag-It assay showed 100% concordance for all calls made compared with sequencing. The ability of the Tag-It assay to discriminate among the three possible calls (wild-type, heterozygote, and mutant) is illustrated in Fig. 5
. The genotype distribution of the results of the patient study is summarized in Table 3
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) is composed of four basic steps: multiplex target amplification by PCR, genotyping by multiplex ASPE chemistry, universal array sorting by hybridization, and detection on the Luminex xMAP flow cytometer (21)(22)(23). The first two steps are specific to the SNP panel being analyzed, whereas the universal array sorting step remains constant and can be adapted to any SNP/mutation panel. Universal arrays are advantageous in that once the optimal tag/anti-tag hybridization conditions have been established, no further assay-specific hybridization optimization is required. In this sense, the Tag-It array represents a "universal" component of any DNA-based test regardless of the application. In designing any diagnostic test, specificity is a primary consideration. This is particularly relevant to multiplexed DNA-based tests, in which the numerous sequences present in the reaction mixture, most of which are noncomplementary, may interact nonspecifically depending on the reaction conditions. Analytical specificity was assessed at multiple points within the Tag-It universal genotyping assay, including PCR, ASPE, and universal tag sequence sorting, both individually and within the fully integrated assay.
The Tag-It universal array was empirically validated to assess the specificity attainable in a multiplex mixture when synthetic, in silico-designed DNA sequences unrelated to genomic DNA are used as sorting tools during hybridization. Of the possible 9900 mismatch hybridization events that could have occurred when each of the 100 tags was hybridized to the array, only 6 and 5 events were observed in runs 1 and 2, respectively, with the highest nonspecific signal observed being only 3.7% of the signal observed for the corresponding perfectly matched pair. These results (Fig. 2
) clearly demonstrate the high specificity attainable in a multiplexed system that uses the Tag-It universal array. By empirically validating each sequence comprising the universal array, we have established a solid foundation on which to build any multiplex genotyping assay.
With respect to PCR, the gel image shown in Fig. 3
illustrates the high specificity of the PCR primers used in the multiplex reaction under optimized conditions. Because PCR represents the first component of the Tag-It platform, it is essential that the reaction be as specific as possible to minimize potential complications in the downstream components of the Tag-It platform.
Specificity was also assessed for the multiplexed ASPE reaction, where nonspecific interactions may potentially occur between (a) the universal tag sequence and the target alleles and/or (b) the allele-specific primer region and the target alleles. To address this issue of specificity, each allele comprising the thrombophilia panel was PCR-amplified individually and then subjected to ASPE with all 12 universally tagged ASPE primers present. The ASPE reactions were then sorted on the universal array (i.e., all 12 anti-tags present). The data (Fig. 4
) clearly demonstrated that the allele-specific primer sequences were highly specific for their intended targets because each of the 12 amplimers representative of a single allele reacted only with its designated ASPE primer. Two levels of discrimination are built into primer extension genotyping chemistry, with one occurring at the hybridization level and the other at the enzymatic extension of a mismatched primer. Additionally, this experiment reconfirmed the specificity of the universal tags under the conditions used in the fully integrated thrombophilia genotyping assay. Because of the higher complexity of the fully integrated genotyping assay, hybridization was performed for 1 h at 37 °C instead of 30 min at 37 °C, which was used for universal array validation. In addition, a filtration wash step was added to the genotyping assay to remove any unbound biotin-dCTP, which would sequester the streptavidin–phycoerythrin reporter. This wash step also removed unbound tagged sequences, which allowed the posthybridization steps (reporter binding and detection) to be performed at room temperature. Despite the differences in assay conditions resulting from the higher complexity of the fully integrated Tag-It genotyping assay, mismatch hybridization between the universal tag sequences and/or any other sequences present in the hybridization reaction was not observed, supporting the high specificity built into the design of the universal tag sequence set.
The concordance between the Tag-It assay and DNA dideoxy sequencing was 100% after resequencing of 14 results that were initially discordant. The 13 no calls obtained with the Tag-It assay (excluded from the accuracy determination) resulted from only 3 samples, whereas the 43 no calls obtained by sequencing (also excluded from accuracy determination) were distributed over 38 samples. Compared with manual bidirectional dideoxy sequencing, manually genotyping 132 patient samples with the Tag-It mutation detection assay offers shorter analysis time and potentially lower cost. Although the study was carried out over 3 days, results for all 132 genomic DNA samples could have been generated in a single day. Sequencing the same number of samples is somewhat more tedious because multiple PCR reactions are required per genomic sample being analyzed (i.e., six PCR reactions per sample are required, followed by sequencing reaction set-ups). Analysis of the sequencing data also requires additional time. Even with automated sequencing, manual set-up of the Tag-It assay would compete favorably.
The patient study also illustrated the ability of the Tag-It assay to discriminate wild-type, mutant, and heterozygous samples (Fig. 5
). The large separation between signals generated for a wild-type, mutant, or heterozygous sample greatly reduces the risk of miscalls. Although two MTHFR 1298 calls and one MTHFR 677 call in Fig. 5
appear as outliers, the calculated mutant allelic ratios for these samples fell within the defined ranges for heterozygous calls. The variation in signal intensities among the different SNPs varied and was likely attributable to the different priming efficiencies of the ASPE primers and/or the number of incorporated biotin-dCTP nucleotides. The variation observed between samples with the same call for any given SNP was attributable to differences in sample preparations, whether it was the quality of the DNA or the presence of underlying polymorphisms.
Recommendations as to who should be tested for inherited thrombophilia have not yet been developed, in part because not all of the genetic risk factors involved have been defined, nor have additional interacting factors been identified (24). To date, the diagnostic tools required for cost-effective SNP panel testing have been unavailable, thus limiting the number of SNPs being analyzed in combination. It may be that the polymorphisms believed to have no effect on venous thrombosis were characterized as such simply because each SNP was studied in isolation and not as part of a comprehensive panel. We believe that use of the Tag-It microsphere-based universal array genotyping platform to study complex diseases is clinically and economically advantageous because it could provide physicians with an improved ability to associate genotype with phenotype. Any diagnostic test designed for multiplex analysis of the gene variations associated with thrombophilia must be flexible enough to change as more clinical information is gathered. The Tag-It platform allows for the entry of new SNPs and/or the removal of unnecessary SNPs in existing assays. Population-wide screening for thrombophilia-associated mutations will likely be dependent on the availability of multiplexed genetic tests that can be performed with high throughput at relatively low cost. The Tag-It platform represents one step in this direction.
Several alternative molecular methods are available for the individual detection of sequence variations in factor V Leiden and factor II, including the Invader® Assay (Third Wave), NanoChip® (Nanogen), the LightCyclerTM (Roche), and direct sequencing (Pyrosequencing) (25)(26)(27)(28)(29)(30)(31). Although the accuracy attainable with most of the aforementioned systems is comparable, the multiplexing potential of each system appears to be far less than that of the Tag-It universal microsphere-based genotyping platform described here. The Tag-It genotyping platform represents a valuable tool in the rapid, accurate, and cost-effective detection of multiple alleles associated with the risk of thrombophilia. In addition, the multiplexed nature of the Tag-It platform makes it well suited to improve the productivity of the clinical laboratory for any test requiring analysis of multiple genetic markers for a given sample. By analyzing larger SNP panels, the platform could permit newly identified potential risk factors to be studied in the context of established risk factors, allowing for more comprehensive understanding of the interactions among the various risk factors and their overall contribution to venous thromboembolism.
In summary, we have demonstrated the use of the Tag-It microsphere-based universal array genotyping platform for the multiplex detection of six SNPs believed to be associated with venous thromboembolism. Although only 12 universal sequences were used for this assay, empirical validation of the full 100-plex array established its potential as a highly specific sorting tool for multiplex analysis of up to 50 biallelic SNPs or 100 analytes.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T transition in the human tissue factor pathway inhibitor (TFPI) gene is statistically associated with a higher risk of venous thrombosis. Thromb Haemost 1999;82:1-5.
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, wild-type; 
, mutant; +, heterozygote.