A simple, bead-based approach for multi-SNP molecular haplotyping
http://www.100md.com
《核酸研究医学期刊》
PolyGenyx, Inc., 100 Barber Avenue, Worcester, MA 01606, USA
* To whom correspondence should be addressed. Tel: +1 508 459 6121; Fax: +1 508 459 6122; Email: jlanders@polygenyx.com
ABSTRACT
Single nucleotide polymorphisms (SNPs) within a gene region have often been studied to determine their effect on phenotype. Although a single base pair change can produce a phenotypic change, phenotype is often influenced by the presence of multiple polymorphisms and their relative positions within a given region. For example, if multiple changes occur in a promoter region, how they influence gene expression will depend on their cis/trans configuration. As such, it is essential to consider the haplotype, or the alignment of multiple SNP alleles on each chromosome when attempting to associate genomic changes with phenotype. Unfortunately, no method of high-throughput molecular haplotyping of multiple SNPs currently exists. In response to this unmet need, we have developed an inexpensive, reliable bead-based capture-based haplotyping (CBH) assay to determine the phase, or haplotype, of multiple SNP alleles in a high-throughput manner. The CBH assay requires minimal setup and handling, requires no centrifugation steps and can be performed in <1 h. Data collection is performed via flow cytometry and the assay yields plus/minus results allowing for automated calling by a simple computer application. We will present data demonstrating the molecular haplotyping of 11 SNPs within exon 2 of the N-acetyltransferase-2 (NAT2) gene, which expresses an important drug-metabolizing enzyme. This assay has applications in diagnostic testing, promoter analysis, association studies and pharmacogenetic analysis.
INTRODUCTION
Single nucleotide polymorphisms (SNPs) have been intensively studied in an attempt to establish the functional relationship between genotypic and phenotypic differences. Although many phenotypes are the result of a single base pair change, often multiple polymorphisms, as well as their phase, contribute to the expression or activity of a gene product (1–8). As such, genotypic data are not always sufficient to fully understand the genotypic/phenotypic relationship. One solution is to consider the haplotype or the alignment of multiple SNP alleles on each chromosome. Current methods of molecular haplotyping include DNA cloning (2), single molecule amplification (9,10), gel-based analysis (11,12) and allele-specific PCR approaches (13,14). These methods may be labor intensive requiring multiple handling/purification steps, gel electrophoresis and/or secondary DNA amplifications. Other non-molecular approaches such as applying pedigree genotypes are not always readily available and statistical analysis of genotyping experiments with different methodologies often produce conflicting data.
N-acetyltransferase 2 (NAT2) plays a major role in metabolizing aromatic and heterocyclic carcinogens found widely in cigarette smoke and cooked foods (15–17). The NAT2 transcript is encoded by two exons, where the gene product is encoded solely by the second exon (18). The gene is highly polymorphic, with exon 2 containing 11 SNPs (Figure 1A) with allele frequencies varying in different ethnic populations (19,20). Changes in NAT2 enzyme activity have been linked to cancer; however, attempts to directly correlate any particular genotype to cancer risk have yielded conflicting results (21–23). Since it has been proposed that a particular SNP haplotype may be responsible (22,24), the NAT2 gene was an ideal candidate to develop the capture-based haplotyping (CBH) assay. Our approach is two-stage; first, SNPs are genotyped in the population of interest to identify which samples require haplotyping (those samples with multiple heterozygous loci) and second, the heterozygous SNPs are phased using our CBH assay. This assay uses allele-specific oligonucleotide hybridizations in a bead-based format to establish the presence or absence of a given SNP haplotype. Furthermore, by using multiple fluorescently colored beads within each reaction, the phase of numerous SNPs can be assessed simultaneously.
Figure 1. Experimental outline for genotyping NAT2 polymorphisms. (A) Structure of exon 2 of the NAT2 gene. The position of the 11 SNPs found within exon 2 of the NAT2 gene and the different alleles observed at these positions are shown. The name of each SNP reflects the distance from the start of exon 2 and the upper allele shown for each SNP represents the common allele. (B) The assay is illustrated using a C/T and an A/G SNP and a genome heterozygous C/T at SNP 1 and homozygous A/A at SNP 2. (C) ASOs corresponding to each allele are attached to a different colored fluorescent bead and called Capture ASOs. (D) An amplicon encompassing both SNPs is generated from the test genome, and Capture ASOs for both SNPs are hybridized simultaneously to the amplicon. Included in this reaction is a biotinylated sequence-specific oligonucleotide (Detector SSO) that recognizes a sequence in that amplicon that is devoid of SNPs. Following this hybridization, the fluorescent reporter molecule, strepavidin–RPE, is hybridized to the Detector SSO annealed to the amplicon. The final complex is analyzed on a Luminex 100 instrument, which will report the RPE signal associated with each colored bead. Those Capture ASOs that have RPE signals associated with them will indicate the presence of that SNP allele in the genome. (E) SNP 1 is C/T and an RPE signal is detected in association with both the ‘C’ and ‘T’ Capture ASOs; however, since SNP 2 is homozygous, an RPE signal is only detected in association with the ‘A’ Capture ASO.
MATERIALS AND METHODS
NAT2 bead-based genotyping and haplotyping
Template generation
Anonymous genomic DNA samples, representing a diverse ethnic background were obtained from the Coriell Cell Repositories (Camden, NJ). Catalog numbers for the samples can be found as in Supplementary Table 1. All oligonucleotides and primers were obtained from Integrated DNA Technologies (Coralville, IA) (sequences can be found as in Supplementary Table 3). Owing to the high degree of sequence homology between the NAT1 and NAT2 (87%) genes, nested PCR was used to generate the template for typing. All amplifications were performed using PCR Mastermix (Abgene, Inc., Rochester, NY). Primary PCRs were carried out with 20 ng genomic DNA in 10 μl 1x PCR Mastermix, 0.2 μM NAT-R2 and NAT-InF1 primers, and 2 mM MgCl2 with the following cycling conditions: 95°C for 5 min; 40 cycles at 95°C for 30 s, 58°C for 30 s, 72°C for 2 min 30 s; 72°C for 10 min. The product was then diluted 1:500 in 1x TE and re-amplified using asymmetric PCR. Here, 5 μl primary PCR product was added to a 50 μl reaction containing 1x PCR Mastermix, 2 μM NAT2-R, 0.1 μM NAT2-InF2 and 1.5 mM MgCl2. The same cycling conditions were used as for the primary PCR. The amplified products were analyzed by gel electrophoresis and then used directly in the bead-based genotyping and haplotyping reactions.
Allele-specific hybridization
For genotyping and haplotyping, allele-specific oligonucleotides (ASOs), representing the 11 SNPs present within exon 2 of the NAT2 gene (see Supplementary Table 2), were synthesized. The dbSNP ID for each SNP is also given in Supplementary Table 2. It should be noted that of the 11 SNPs addressed in this publication, only 10 had been reported previously. The 11th SNP, designated #409, was discovered during analysis of the sequencing data. ASOs for this SNP were subsequently included in the genotyping reaction, which did confirm its presence. Each ASO was 19 nt long with a 5' Uni-Link amino modifier. Each ASO was attached to a different colored 5.5 μm carboxylated bead as described previously (25). Genotyping was performed in a 30 μl hybridization reaction containing 5 μl unpurified PCR product, 83 nM biotinylated sequence-specific oligonucleotide (NAT2–SSO) and 1000 beads corresponding to each allele of the 11 SNPs (i.e. 22 bead sets) in 1x TMAC buffer (4.5 M TMAC, 0.15% Sarkosyl, 75 mM Tris–HCl, pH 8.0 and 6 mM EDTA, pH 8.0). The reactions were denatured at 95°C for 2 min and incubated at 54°C for 30 min. An equal volume of 20 μg/ml streptavidin–R-phycoerythrin (RPE) (Molecular Probes, Inc., Eugene, OR) in 1x TMAC buffer was added and the reaction was incubated at 54°C for 20 min prior to analysis on a Luminex 100. The data collection software was set to analyze 100 beads from each set and the median relative fluorescent intensity was used for analysis. For haplotyping, 50 μl hybridization reactions were set up in duplicate; each containing either of the two biotinylated detector ASOs (30 nM) and template volume increased to 10 μl. Reaction conditions were identical to those for genotyping, except 25 μl of a 30 μg/ml streptavidin–RPE stock was added. For all analyses, non-specific background hybridization was determined by including a non-homologous oligonucleotide sequence attached to another colored bead in the reactions. Visual genotypes and haplotypes (26,27) were generated using the online software applications VG2 and VH1 (http://pga.gs.washington.edu), respectively.
Haplotype determination by subcloning/sequencing
Initially, genotyping was confirmed by direct sequencing of PCR products. Primers NAT2InF2-M13F and NAT2R-M13R were used to amplify the NAT2 polymorphic region using the same conditions as the primary PCRs described above, except 100 ng genomic DNA was amplified in a 50 μl reaction. The PCR products were column purified and sequenced (ACGT Inc., Wheeling, IL). The genotype for each SNP was determined using PolyPhred sequence analysis software (28) and confirmed by visual inspection. Those samples that contained at least two heterozygotic polymorphisms were subcloned into the TA-TOPO vector (Invitrogen Life Technologies, Carlsbad, CA), and transformed into TOP10 F' bacteria according to the manufacturer's instructions. Three colonies were picked from each transformation and plasmid DNA was isolated using the Perfectprep Plasmid 96 Spin Kit (Brinkmann, Westbury, NY). Each colony, representing one of the two haplotypes for that individual, was sequenced. The opposite chromosomal haplotype was established by ‘subtracting’ the haplotype determined by plasmid sequencing from the genotype of each individual.
RESULTS
Genotype analysis of NAT2 gene
The genotyping assay is illustrated in Figure 1B–E using a C/T and an A/G SNP and a genome heterozygous C/T at SNP 1 and homozygous A/A at SNP 2 (Figure 1B). ASOs corresponding to each allele are attached to a different colored fluorescent bead and are called Capture ASOs (Figure 1C), based on the fact that the binding (or capturing) of the amplified product is to this ASO. An amplicon encompassing both SNPs is generated from the test genome, and Capture ASOs for both SNPs are hybridized simultaneously to the amplicon (Figure 1D). Included in this reaction is a biotinylated SSO (Detector SSO) that recognizes a sequence in that amplicon that is devoid of SNPs (Figure 1D). Following this hybridization, the fluorescent reporter molecule, Streptavidin–RPE, is hybridized to the Detector SSO attached to the amplicon (Figure 1D). The final reaction is analyzed on a Luminex 100 instrument, which will report the RPE signal associated with each colored bead. Those Capture ASOs that have RPE signals associated with them will indicate the presence of that SNP allele in the genome. In Figure 1E, SNP 1 is C/T and an RPE signal is detected in association with both the ‘C’ and ‘T’ Capture ASOs; however, since SNP 2 is A/A, an RPE signal is only detected in association with the ‘A’ Capture ASO.
To genotype all SNPs within the NAT2 gene in an ethnically diverse panel of 46 unrelated individuals, Capture ASOs, corresponding to each allele of the 11 SNPs, were attached to 22 different colored beads. Capture ASOs were designed such that the polymorphic base was positioned in the center (+10 position); however, this site was shifted, depending on the sequence context, to avoid polynucleotide runs, low/high GC-rich regions (<20 or >80%), duplex formation or secondary structures (as measured by delta G) and to minimize differences in the Tm and %GC content between the ASOs. All 22 Capture ASOs and a biotinylated Detector SSO were simultaneously hybridized to an amplicon encompassing all 11 SNPs. This reaction was hybridized with the strepavidin–RPE reporter molecule. The hybridization reactions were analyzed on a Luminex 100 instrument, and the RPE reporter signal associated with each colored bead was determined. From the initial set, Capture ASOs for 8 out of 11 SNPs demonstrated clear clustering patterns with good signal strength. The remaining three Capture ASOs were redesigned. Figure 2A shows that a clear clustering pattern was obtained for all 11 SNPs. This assay development success rate (72%) is well within the rate observed for other genotyping assays . The genotyping data, Figure 2A, demonstrate a clear clustering pattern for each SNP. All genotypes determined by our bead-based assay were in 100% accordance with those genotypes established by DNA sequencing. The rare allele was not observed for SNP #370 and #417; however, based on the previously established allele frequencies for these two SNPs (0.005 for each) (20), this is not surprising. The genotyping data indicate that 26 samples had at least one pair of heterozygotic SNPs requiring haplotype analysis (Figure 2B). Within the 26 samples analyzed, 142 double heterozygotic SNP pairings required phase resolution.
Figure 2. (A) Results of bead-based genotyping for all 11 SNPs of the NAT2 exon. The graphs shown represent the assay results for all 11 SNPs within the second exon of the NAT2 gene. Data are presented as the log10 versus total signal (32). Genotypes are represented by common homozygotes (blue diamonds), heterozyotes (pink squares) and rare homozygotes (yellow triangles). (B) Visual genotype representation of the NAT2 polymorphisms. The genotyping results are summarized through the use of a visual genotype representation. The genotype is represented at the intersection of a given polymorphism (top) with a given sample (left). In the graphic, genotypes are represented as common homozygotes (blue), rare homozygotes (yellow) and heterozyotes (red). White boxes reflect those samples containing at least one pair of double heterozygous SNPs thus requiring phase determination. The white box also reflects the Detector ASOs to be selected for haplotype determination (e.g. Detector ASOs for SNP #487 were chosen to phase sample D1, which was also heterozygous at positions #347 and #809).
Phase analysis of multiple heterozygous samples
Our multi-SNP haplotyping assay utilizes a similar ASO hybridization approach in a bead-based format and allows up to 50 SNPs to be phased simultaneously. To haplotype the SNPs, duplicate hybridization reactions were set up containing PCR product, Capture ASOs corresponding to each allele of all SNPs and biotinylated Detector ASOs. It should be noted that in the haplotyping assay, the biotinylated Detector ASOs are now allele-specific to any one heterozygotic SNP. This allows the linkage of the SNP alleles on each chromosome to be determined by association of the RPE signal with each Capture ASO signal. To illustrate, the haplotyping of two heterozygotic SNPs (SNP 1 T/C and SNP 2 A/G) is shown in Figure 3A and B. Capture ASOs for the T (green bead) and C (red bead) allele of SNP #1 are hybridized to an amplicon encompassing both SNP loci in two identical reactions. To each reaction tube, a biotinylated Detector ASO toward one or the other allele of a heterozygous SNP is also added (SNP 2 was chosen here, but any heterozygous SNP will suffice). In the illustration, the ‘G’ allele Detector ASO was added to tube #1 and the ‘A’ allele Detector ASO to tube #2. After hybridization of Capture and Detector ASOs, a strepavidin–RPE reporter molecule was hybridized to the mixture. Figure 3A and B shows the expected results for two samples that contain the same double heterozygotic genotype but have different haplotypes. Figure 3A shows a positive RPE signal associated with the green bead in tube #1 and with the red bead in tube #2, indicating a 1T-2G/1C-2A haplotype. Figure 3B shows a positive RPE signal associated with the red bead in tube #1 and with the green bead in tube #2, indicating the alternate 1T-2A/1C-2G haplotype. Since 100 different colored beads are currently available, this technique has the potential of phasing of up to 50 SNPs with a given Detector SNP. By analyzing each of these 2-SNP combinations, the multi-SNP haplotype can be established.
Figure 3. Haplotyping of all 11 SNPs within exon 2 of the NAT2 gene. (A and B) Strategy for CBH. An illustration of the phasing of just two heterozygotic SNPs by CBH is shown. ASOs for the T and C alleles of SNP 1 are attached to two different sets of colored beads (green and red). Duplicate hybridization reactions are set up, each containing both Capture ASOs, the single-stranded template and a biotinylated ASO (Detector ASO) for either the G or A allele of SNP 2. Streptavidin–RPE is then added to the mixture. After hybridization, the RPE reporter signal associated with each of the Capture ASOs in the two reactions is measured. When a Detector and Capture ASOs are aligned on the same template, an RPE signal will be associated with that Capture ASO, indicating the presence of that particular haplotype. The figure addresses two samples with the same genotype but different haplotypes, thus, showing two different outcomes. (C) The results of the assay tubes for one sample (D1) were analyzed and plotted as shown. SNP #487 was selected as the Detector ASO. Each Capture ASOs has four possible bars representing the four haplotypes possible in relation to the Detector SNP. The phase of each SNP, in relation to SNP #487, is determined by the bar heights. The 11 SNP haplotype for this sample is shown below the graph. The alignment of the SNP alleles above and below the horizontal line represents the haplotype on each chromosome. (D) Simplified data analysis to establish SNP haplotypes. The results for each Detector ASO and heterozygous Capture ASO were analyzed by plotting the log10 versus total signal (where CA–CA, RA–RA, CA–RA and RA–CA represent the four haplotype combinations of the common allele (CA) and rare allele (RA) for each Detector/Capture SNP pair). By this approach, the haplotype configuration of each SNP pair can easily be established by the log value for each point. Points with a positive log value represent the CA–CA/RA–RA haplotype, whereas points with a negative log value represent the CA–RA/RA–CA haplotype. The Capture ASO/Detector ASO pairings are as follows: 197/487 (blue crosses), 197/596 (blue circles), 288/487 (pink crosses), 347/487 (green crosses), 409/487 (blue on white crosses), 596/288 (brown squares), 596/487 (brown crosses), 809/288 (black squares), 809/487 (black crosses), 863/288 (yellow squares), 863/487 (yellow on black crosses), and 1027/487 (red crosses), respectively. (E) Visual haplotype representation of the NAT2 gene. Haplotypes for the 11 SNPs within exon 2 for the 26 individuals containing at least one pair of double heterozygotic SNPs are shown. For each sample, two rows (‘–1’ and ‘–2’) indicate alignment of the SNP alleles on the two chromosomes. The ‘color’ of each square at the intersection represents which SNP allele is present at that position on that given chromosome. Blue boxes represent the common SNP allele and yellow the rare SNP allele.
To determine the phase of all double heterozygotic SNP pairs in our samples, duplicate reactions for each genomic sample were set up containing Capture ASOs for all 11 SNPs and the appropriate pair of Detector ASOs. To select the appropriate Detector ASOs for each genomic sample, all heterozygous SNPs present in each sample were compared with each other and the minimum number of Detector ASOs required to haplotype all 26 samples were selected. From this comparison, it was determined that one of the three heterozygous SNPs (#288, #487 or #596) was present in each of the 26 samples (Figure 2B). As an internal negative control, Capture ASOs representing those SNPs already contained in the Detector ASOs were also included. If the Detector ASOs are hybridizing appropriately, then no RPE signal should be associated with the negative control Capture ASOs. Additionally, including Capture ASOs for all 11 SNPs, even those SNPs represented in the Detector ASOs, will allow the use of a single Capture ASO bead mixture for all samples throughout the study.
Figure 3C shows the haplotype results for all 11 SNPs for a single sample. The graph reflects the 10 different combinations expected using Detector ASOs for SNP #487. As expected, no significant RPE signal was associated with the internal negative control SNP #487 Capture ASOs and SNP #487 Detector ASOs (data not shown). The multi-SNP haplotype configuration displayed below the graph was determined based on data from each pairwise combination. For example, the results of SNP #487/SNP #347 combination show two peaks indicating that the ‘C’ allele for SNP #487 is linked to the common SNP allele ‘T’ of SNP #347 on one chromosome and that the ‘T’ allele of SNP #487 is linked to the rare SNP allele ‘C’ of SNP #347 on the opposite chromosome. Similar analysis of all other SNP combinations yielded haplotype configurations for all 11 SNPs for the given sample as shown.
To establish the phase of all double heterozygotic SNP pairs, we incorporated a simplified data analysis approach based on the fact that only two possible phases exist. For example, if a sample is heterozygous C/T at SNP #1 and heterozygous A/G at SNP #2, the individual either has a CA haplotype on one chromosome and a TG haplotype on the other or a CG haplotype on one chromosome and a TA haplotype on the other. Therefore, our data interpretation results from testing the likelihood of these two events and is represented as a plot of the log10 versus total signal (where 1A-2A, 1B-2B, 1A-2B, 1B-2A represent the four haplotype combinations for a given SNP pair). In our example, samples with values near +1 (10-fold more percentage total signal) will have a CA/TG haplotype, whereas values near –1 will indicate a CG/TA haplotype. Graphing the results for each double heterozygotic pairings within the 26 samples resulted in distinct clusters (Figure 3D). By using this two-stage approach to haplotyping, we have also reduced data analysis to a binary decision, thus making calls simpler and unambiguous. The haplotypes for all 26 samples, as determined by our graphing technique, are shown in Figure 3E and are in 100% agreement with the haplotypes derived by subcloning/sequencing (data not shown).
DISCUSSION
In this report, we have presented a simplified approach for directly determining the phase of multi-SNP alleles in a high-throughput manner. The CBH approach, as with other molecular-based haplotyping methods, is most applicable in situations requiring the highest level of accuracy, including diagnostic testing for clinically relevant haplotypes, association-based studies for disease-gene identification and in pharmacogenomic analysis. One-time optimization of conditions for hybridization and template labeling has reduced the labor-intensive handling normally associated with bead-based assays, such as centrifugation and bead washing. Additionally, by incorporating an initial genotyping step, we have reduced the number and complexity of experiments needed to haplotype a sample population. In the presented study, only 26 of the 46 samples contained SNP alleles requiring phase resolution.
Owing to its simplicity, no sophisticated robotics are needed to perform high-throughput levels of this assay. As indicated, the CBH assay can be performed in 1 h and no centrifugation, filtration, purification (both post-PCR and post-hybridization) or enzymatic steps are required. Using a low-end liquid handler, we have automated all aspects of the CBH assay including the cherry picking of samples that required phasing, the addition of all hybridization reagents and the addition of streptavidin–RPE. The throughput of this assay is approximately one 96-well plate (48 samples) in 1 h (nearly 400 samples per day). The number of SNPs analyzed within this period is directly related to the number of SNPs within each assay. For example, to analyze the NAT2 gene that contains 11 polymorphisms, over 4000 SNPs could be haplotyped per day. Given that our haplotyping assay can analyze up to 50 SNP haplotypes, over 19 000 polymorphisms could be haplotyped per day. This throughput is directly limited by the speed of the Luminex instrument in analyzing each well. However, given their low cost, higher throughput could easily be achieved with the purchase of additional instruments.
Recent experiments by our laboratory have shown that the CBH assay is reliable and robust. This has been demonstrated by the assay development for eight additional genes including MBL2 (17 SNPs and 1 ins/del polymorphism), ELAM (4 SNPs), IL10 (4 SNPs) and IL4 (3 SNPs). Furthermore, for each developed assay, we observed call rates and accuracy >99%. However, the CBH assay does have the limitation that it can only analyze SNPs within a PCR amplifiable region. Currently, we have analyzed haplotypes within an amplified region of up to 1500 bp; however, advances in long-range PCR protocols (30,31) should enable us to study larger loci. However, it is likely that as the amplicon size increases, the complexity of the secondary structure of the single-stranded PCR product will also increase, thus reducing hybridization efficiency. As a result, the addition of secondary structure inhibitors, or more intense fluorescent detection reagents, may be necessary to compensate for the reduced hybridization. Additionally, we are exploring other approaches to increase genomic coverage such as using overlapping amplicons and incorporating RT–PCR techniques.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
This work was supported by grants from the National Institutes of Health (1R43HG002247-01A2 and 2R44HG002247-02) to J.E.L.
REFERENCES
Martin,M.P., Dean,M., Smith,M.W., Winkler,C., Gerrard,B., Michael,N.L., Lee,B., Doms,R.W., Margolick,J., Buchbinder,S. et al. ( (1998) ) Genetic acceleration of AIDS progression by a promoter variant of CCR5. Science, , 282, , 1907–1911.
Drysdale,C.M., McGraw,D.W., Stack,C.B., Stephens,J.C., Judson,R.S., Nandabalan,K., Arnold,K., Ruano,G. and Liggett,S.B. ( (2000) ) Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc. Natl Acad. Sci. USA, , 97, , 10483–10488.
Hoehe,M.R., Kopke,K., Wendel,B., Rohde,K., Flachmeier,C., Kidd,K.K., Berrettini,W.H. and Church,G.M. ( (2000) ) Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence. Hum. Mol. Genet., , 9, , 2895–2908.
Mirel,D.B., Valdes,A.M., Lazzeroni,L.C., Reynolds,R.L., Erlich,H.A. and Noble,J.A. ( (2002) ) Association of IL4R haplotypes with type 1 diabetes. Diabetes, , 51, , 3336–3341.
Carew,J.A., Basso,F., Miller,G.J., Hawe,E., Jackson,A.A., Humphries,S.E. and Bauer,K.A. ( (2003) ) A functional haplotype in the 5' flanking region of the factor VII gene is associated with an increased risk of coronary heart disease. J. Thromb. Haemost., , 1, , 2179–2185.
Lambrechts,D., Storkebaum,E., Morimoto,M., Del-Favero,J., Desmet,F., Marklund,S.L., Wyns,S., Thijs,V., Andersson,J., van Marion,I. et al. ( (2003) ) VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nature Genet., , 34, , 383–394.
Knapp,S., Hennig,B.J., Frodsham,A.J., Zhang,L., Hellier,S., Wright,M., Goldin,R., Hill,A.V., Thomas,H.C. and Thursz,M.R. ( (2003) ) Interleukin-10 promoter polymorphisms and the outcome of hepatitis C virus infection. Immunogenetics, , 55, , 362–369.
Krajinovic,M., Lemieux-Blanchard,E., Chiasson,S., Primeau,M., Costea,I. and Moghrabi,A. ( (2004) ) Role of polymorphisms in MTHFR and MTHFD1 genes in the outcome of childhood acute lymphoblastic leukemia. Pharmacogenomics J., , 4, , 66–72.
Ruano,G., Kidd,K.K. and Stephens,J.C. ( (1990) ) Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules. Proc. Natl Acad. Sci. USA, , 87, , 6296–6300.
Ding,C. and Cantor,C.R. ( (2003) ) Direct molecular haplotyping of long-range genomic DNA with M1-PCR. Proc. Natl Acad. Sci. USA, , 100, , 7449–7453.
Prior,T.W., Wenger,G.D., Papp,A.C., Snyder,P.J., Sedra,M.S., Bartolo,C., Moore,J.W. and Highsmith,W.E. ( (1995) ) Rapid DNA haplotyping using a multiplex heteroduplex approach: application to Duchenne muscular dystrophy carrier testing. Hum. Mutat., , 5, , 263–268.
Veenstra-VanderWeele,J., Kim,S.J., Gonen,D., Hanna,G.L., Leventhal,B.L. and Cook,E.H.,Jr ( (2001) ) Genomic organization of the SLC1A1/EAAC1 gene and mutation screening in early-onset obsessive-compulsive disorder. Mol. Psychiatry, , 6, , 160–167.
Eitan,Y. and Kashi,Y. ( (2002) ) Direct micro-haplotyping by multiple double PCR amplifications of specific alleles (MD-PASA). Nucleic Acids Res., , 30, , e62.
Pettersson,M., Bylund,M. and Alderborn,A. ( (2003) ) Molecular haplotype determination using allele-specific PCR and pyrosequencing technology. Genomics, , 82, , 390–396.
Minchin,R.F., Reeves,P.T., Teitel,C.H., McManus,M.E., Mojarrabi,B., Ilett,K.F. and Kadlubar,F.F. ( (1992) ) N- and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells. Biochem. Biophys. Res. Commun., , 185, , 839–844.
Hein,D.W., Doll,M.A., Rustan,T.D., Gray,K., Feng,Y., Ferguson,R.J. and Grant,D.M. ( (1993) ) Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases. Carcinogenesis, , 14, , 1633–1638.
Zenser,T.V., Lakshmi,V.M., Rustan,T.D., Doll,M.A., Deitz,A.C., Davis,B.B. and Hein,D.W. ( (1996) ) Human N-acetylation of benzidine: role of NAT1 and NAT2. Cancer Res., , 56, , 3941–3947.
Blum,M., Grant,D.M., McBride,W., Heim,M. and Meyer,U.A. ( (1990) ) Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression. DNA Cell Biol., , 9, , 193–203.
Grant,D.M., Hughes,N.C., Janezic,S.A., Goodfellow,G.H., Chen,H.J., Gaedigk,A., Yu,V.L. and Grewal,R. ( (1997) ) Human acetyltransferase polymorphisms. Mutat. Res., , 376, , 61–70.
Packer,B.R., Yeager,M., Staats,B., Welch,R., Crenshaw,A., Kiley,M., Eckert,A., Beerman,M., Miller,E., Bergen,A. et al. ( (2004) ) SNP500Cancer: a public resource for sequence validation and assay development for genetic variation in candidate genes. Nucleic Acids Res., , 32, , D528–D532.
Brockton,N., Little,J., Sharp,L. and Cotton,S.C. ( (2000) ) N-acetyltransferase polymorphisms and colorectal cancer: a HuGE review. Am. J. Epidemiol., , 151, , 846–861.
Hein,D.W., Doll,M.A., Fretland,A.J., Leff,M.A., Webb,S.J., Xiao,G.H., Devanaboyina,U.S., Nangju,N.A. and Feng,Y. ( (2000) ) Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol. Biomarkers Prev., , 9, , 29–42.
Butcher,N.J., Boukouvala,S., Sim,E. and Minchin,R.F. ( (2002) ) Pharmacogenetics of the arylamine N-acetyltransferases. Pharmacogenomics J., , 2, , 30–42.
Cascorbi,I. and Roots,I. ( (1999) ) Pitfalls in N-acetyltransferase 2 genotyping. Pharmacogenetics, , 9, , 123–127.
Iannone,M.A., Taylor,J.D., Chen,J., Li,M.S., Rivers,P., Slentz-Kesler,K.A. and Weiner,M.P. ( (2000) ) Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry. Cytometry, , 39, , 131–140.
Nickerson,D.A., Taylor,S.L., Weiss,K.M., Clark,A.G., Hutchinson,R.G., Stengard,J., Salomaa,V., Vartiainen,E., Boerwinkle,E. and Sing,C.F. ( (1998) ) DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene. Nature Genet., , 19, , 233–240.
Rieder,M.J., Taylor,S.L., Clark,A.G. and Nickerson,D.A. ( (1999) ) Sequence variation in the human angiotensin converting enzyme. Nature Genet., , 22, , 59–62.
Nickerson,D.A., Tobe,V.O. and Taylor,S.L. ( (1997) ) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res., , 25, , 2745–2751.
Wiltshire,T., Pletcher,M.T., Batalov,S., Barnes,S.W., Tarantino,L.M., Cooke,M.P., Wu,H., Smylie,K., Santrosyan,A., Copeland,N.G. et al. ( (2003) ) Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse. Proc. Natl Acad. Sci. USA, , 100, , 3380–3385.
Barnes,W.M. ( (1994) ) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl Acad. Sci. USA, , 91, , 2216–2220.
Cheng,S., Fockler,C., Barnes,W.M. and Higuchi,R. ( (1994) ) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl Acad. Sci. USA, , 91, , 5695–5699.
Hirschhorn,J.N., Sklar,P., Lindblad-Toh,K., Lim,Y.M., Ruiz-Gutierrez,M., Bolk,S., Langhorst,B., Schaffner,S., Winchester,E. and Lander,E.S. ( (2000) ) SBE-TAGS: an array-based method for efficient single-nucleotide polymorphism genotyping. Proc. Natl Acad. Sci. USA, , 97, , 12164–12169.(James D. Hurley, Linda J. Engle, Jesse T)
* To whom correspondence should be addressed. Tel: +1 508 459 6121; Fax: +1 508 459 6122; Email: jlanders@polygenyx.com
ABSTRACT
Single nucleotide polymorphisms (SNPs) within a gene region have often been studied to determine their effect on phenotype. Although a single base pair change can produce a phenotypic change, phenotype is often influenced by the presence of multiple polymorphisms and their relative positions within a given region. For example, if multiple changes occur in a promoter region, how they influence gene expression will depend on their cis/trans configuration. As such, it is essential to consider the haplotype, or the alignment of multiple SNP alleles on each chromosome when attempting to associate genomic changes with phenotype. Unfortunately, no method of high-throughput molecular haplotyping of multiple SNPs currently exists. In response to this unmet need, we have developed an inexpensive, reliable bead-based capture-based haplotyping (CBH) assay to determine the phase, or haplotype, of multiple SNP alleles in a high-throughput manner. The CBH assay requires minimal setup and handling, requires no centrifugation steps and can be performed in <1 h. Data collection is performed via flow cytometry and the assay yields plus/minus results allowing for automated calling by a simple computer application. We will present data demonstrating the molecular haplotyping of 11 SNPs within exon 2 of the N-acetyltransferase-2 (NAT2) gene, which expresses an important drug-metabolizing enzyme. This assay has applications in diagnostic testing, promoter analysis, association studies and pharmacogenetic analysis.
INTRODUCTION
Single nucleotide polymorphisms (SNPs) have been intensively studied in an attempt to establish the functional relationship between genotypic and phenotypic differences. Although many phenotypes are the result of a single base pair change, often multiple polymorphisms, as well as their phase, contribute to the expression or activity of a gene product (1–8). As such, genotypic data are not always sufficient to fully understand the genotypic/phenotypic relationship. One solution is to consider the haplotype or the alignment of multiple SNP alleles on each chromosome. Current methods of molecular haplotyping include DNA cloning (2), single molecule amplification (9,10), gel-based analysis (11,12) and allele-specific PCR approaches (13,14). These methods may be labor intensive requiring multiple handling/purification steps, gel electrophoresis and/or secondary DNA amplifications. Other non-molecular approaches such as applying pedigree genotypes are not always readily available and statistical analysis of genotyping experiments with different methodologies often produce conflicting data.
N-acetyltransferase 2 (NAT2) plays a major role in metabolizing aromatic and heterocyclic carcinogens found widely in cigarette smoke and cooked foods (15–17). The NAT2 transcript is encoded by two exons, where the gene product is encoded solely by the second exon (18). The gene is highly polymorphic, with exon 2 containing 11 SNPs (Figure 1A) with allele frequencies varying in different ethnic populations (19,20). Changes in NAT2 enzyme activity have been linked to cancer; however, attempts to directly correlate any particular genotype to cancer risk have yielded conflicting results (21–23). Since it has been proposed that a particular SNP haplotype may be responsible (22,24), the NAT2 gene was an ideal candidate to develop the capture-based haplotyping (CBH) assay. Our approach is two-stage; first, SNPs are genotyped in the population of interest to identify which samples require haplotyping (those samples with multiple heterozygous loci) and second, the heterozygous SNPs are phased using our CBH assay. This assay uses allele-specific oligonucleotide hybridizations in a bead-based format to establish the presence or absence of a given SNP haplotype. Furthermore, by using multiple fluorescently colored beads within each reaction, the phase of numerous SNPs can be assessed simultaneously.
Figure 1. Experimental outline for genotyping NAT2 polymorphisms. (A) Structure of exon 2 of the NAT2 gene. The position of the 11 SNPs found within exon 2 of the NAT2 gene and the different alleles observed at these positions are shown. The name of each SNP reflects the distance from the start of exon 2 and the upper allele shown for each SNP represents the common allele. (B) The assay is illustrated using a C/T and an A/G SNP and a genome heterozygous C/T at SNP 1 and homozygous A/A at SNP 2. (C) ASOs corresponding to each allele are attached to a different colored fluorescent bead and called Capture ASOs. (D) An amplicon encompassing both SNPs is generated from the test genome, and Capture ASOs for both SNPs are hybridized simultaneously to the amplicon. Included in this reaction is a biotinylated sequence-specific oligonucleotide (Detector SSO) that recognizes a sequence in that amplicon that is devoid of SNPs. Following this hybridization, the fluorescent reporter molecule, strepavidin–RPE, is hybridized to the Detector SSO annealed to the amplicon. The final complex is analyzed on a Luminex 100 instrument, which will report the RPE signal associated with each colored bead. Those Capture ASOs that have RPE signals associated with them will indicate the presence of that SNP allele in the genome. (E) SNP 1 is C/T and an RPE signal is detected in association with both the ‘C’ and ‘T’ Capture ASOs; however, since SNP 2 is homozygous, an RPE signal is only detected in association with the ‘A’ Capture ASO.
MATERIALS AND METHODS
NAT2 bead-based genotyping and haplotyping
Template generation
Anonymous genomic DNA samples, representing a diverse ethnic background were obtained from the Coriell Cell Repositories (Camden, NJ). Catalog numbers for the samples can be found as in Supplementary Table 1. All oligonucleotides and primers were obtained from Integrated DNA Technologies (Coralville, IA) (sequences can be found as in Supplementary Table 3). Owing to the high degree of sequence homology between the NAT1 and NAT2 (87%) genes, nested PCR was used to generate the template for typing. All amplifications were performed using PCR Mastermix (Abgene, Inc., Rochester, NY). Primary PCRs were carried out with 20 ng genomic DNA in 10 μl 1x PCR Mastermix, 0.2 μM NAT-R2 and NAT-InF1 primers, and 2 mM MgCl2 with the following cycling conditions: 95°C for 5 min; 40 cycles at 95°C for 30 s, 58°C for 30 s, 72°C for 2 min 30 s; 72°C for 10 min. The product was then diluted 1:500 in 1x TE and re-amplified using asymmetric PCR. Here, 5 μl primary PCR product was added to a 50 μl reaction containing 1x PCR Mastermix, 2 μM NAT2-R, 0.1 μM NAT2-InF2 and 1.5 mM MgCl2. The same cycling conditions were used as for the primary PCR. The amplified products were analyzed by gel electrophoresis and then used directly in the bead-based genotyping and haplotyping reactions.
Allele-specific hybridization
For genotyping and haplotyping, allele-specific oligonucleotides (ASOs), representing the 11 SNPs present within exon 2 of the NAT2 gene (see Supplementary Table 2), were synthesized. The dbSNP ID for each SNP is also given in Supplementary Table 2. It should be noted that of the 11 SNPs addressed in this publication, only 10 had been reported previously. The 11th SNP, designated #409, was discovered during analysis of the sequencing data. ASOs for this SNP were subsequently included in the genotyping reaction, which did confirm its presence. Each ASO was 19 nt long with a 5' Uni-Link amino modifier. Each ASO was attached to a different colored 5.5 μm carboxylated bead as described previously (25). Genotyping was performed in a 30 μl hybridization reaction containing 5 μl unpurified PCR product, 83 nM biotinylated sequence-specific oligonucleotide (NAT2–SSO) and 1000 beads corresponding to each allele of the 11 SNPs (i.e. 22 bead sets) in 1x TMAC buffer (4.5 M TMAC, 0.15% Sarkosyl, 75 mM Tris–HCl, pH 8.0 and 6 mM EDTA, pH 8.0). The reactions were denatured at 95°C for 2 min and incubated at 54°C for 30 min. An equal volume of 20 μg/ml streptavidin–R-phycoerythrin (RPE) (Molecular Probes, Inc., Eugene, OR) in 1x TMAC buffer was added and the reaction was incubated at 54°C for 20 min prior to analysis on a Luminex 100. The data collection software was set to analyze 100 beads from each set and the median relative fluorescent intensity was used for analysis. For haplotyping, 50 μl hybridization reactions were set up in duplicate; each containing either of the two biotinylated detector ASOs (30 nM) and template volume increased to 10 μl. Reaction conditions were identical to those for genotyping, except 25 μl of a 30 μg/ml streptavidin–RPE stock was added. For all analyses, non-specific background hybridization was determined by including a non-homologous oligonucleotide sequence attached to another colored bead in the reactions. Visual genotypes and haplotypes (26,27) were generated using the online software applications VG2 and VH1 (http://pga.gs.washington.edu), respectively.
Haplotype determination by subcloning/sequencing
Initially, genotyping was confirmed by direct sequencing of PCR products. Primers NAT2InF2-M13F and NAT2R-M13R were used to amplify the NAT2 polymorphic region using the same conditions as the primary PCRs described above, except 100 ng genomic DNA was amplified in a 50 μl reaction. The PCR products were column purified and sequenced (ACGT Inc., Wheeling, IL). The genotype for each SNP was determined using PolyPhred sequence analysis software (28) and confirmed by visual inspection. Those samples that contained at least two heterozygotic polymorphisms were subcloned into the TA-TOPO vector (Invitrogen Life Technologies, Carlsbad, CA), and transformed into TOP10 F' bacteria according to the manufacturer's instructions. Three colonies were picked from each transformation and plasmid DNA was isolated using the Perfectprep Plasmid 96 Spin Kit (Brinkmann, Westbury, NY). Each colony, representing one of the two haplotypes for that individual, was sequenced. The opposite chromosomal haplotype was established by ‘subtracting’ the haplotype determined by plasmid sequencing from the genotype of each individual.
RESULTS
Genotype analysis of NAT2 gene
The genotyping assay is illustrated in Figure 1B–E using a C/T and an A/G SNP and a genome heterozygous C/T at SNP 1 and homozygous A/A at SNP 2 (Figure 1B). ASOs corresponding to each allele are attached to a different colored fluorescent bead and are called Capture ASOs (Figure 1C), based on the fact that the binding (or capturing) of the amplified product is to this ASO. An amplicon encompassing both SNPs is generated from the test genome, and Capture ASOs for both SNPs are hybridized simultaneously to the amplicon (Figure 1D). Included in this reaction is a biotinylated SSO (Detector SSO) that recognizes a sequence in that amplicon that is devoid of SNPs (Figure 1D). Following this hybridization, the fluorescent reporter molecule, Streptavidin–RPE, is hybridized to the Detector SSO attached to the amplicon (Figure 1D). The final reaction is analyzed on a Luminex 100 instrument, which will report the RPE signal associated with each colored bead. Those Capture ASOs that have RPE signals associated with them will indicate the presence of that SNP allele in the genome. In Figure 1E, SNP 1 is C/T and an RPE signal is detected in association with both the ‘C’ and ‘T’ Capture ASOs; however, since SNP 2 is A/A, an RPE signal is only detected in association with the ‘A’ Capture ASO.
To genotype all SNPs within the NAT2 gene in an ethnically diverse panel of 46 unrelated individuals, Capture ASOs, corresponding to each allele of the 11 SNPs, were attached to 22 different colored beads. Capture ASOs were designed such that the polymorphic base was positioned in the center (+10 position); however, this site was shifted, depending on the sequence context, to avoid polynucleotide runs, low/high GC-rich regions (<20 or >80%), duplex formation or secondary structures (as measured by delta G) and to minimize differences in the Tm and %GC content between the ASOs. All 22 Capture ASOs and a biotinylated Detector SSO were simultaneously hybridized to an amplicon encompassing all 11 SNPs. This reaction was hybridized with the strepavidin–RPE reporter molecule. The hybridization reactions were analyzed on a Luminex 100 instrument, and the RPE reporter signal associated with each colored bead was determined. From the initial set, Capture ASOs for 8 out of 11 SNPs demonstrated clear clustering patterns with good signal strength. The remaining three Capture ASOs were redesigned. Figure 2A shows that a clear clustering pattern was obtained for all 11 SNPs. This assay development success rate (72%) is well within the rate observed for other genotyping assays . The genotyping data, Figure 2A, demonstrate a clear clustering pattern for each SNP. All genotypes determined by our bead-based assay were in 100% accordance with those genotypes established by DNA sequencing. The rare allele was not observed for SNP #370 and #417; however, based on the previously established allele frequencies for these two SNPs (0.005 for each) (20), this is not surprising. The genotyping data indicate that 26 samples had at least one pair of heterozygotic SNPs requiring haplotype analysis (Figure 2B). Within the 26 samples analyzed, 142 double heterozygotic SNP pairings required phase resolution.
Figure 2. (A) Results of bead-based genotyping for all 11 SNPs of the NAT2 exon. The graphs shown represent the assay results for all 11 SNPs within the second exon of the NAT2 gene. Data are presented as the log10 versus total signal (32). Genotypes are represented by common homozygotes (blue diamonds), heterozyotes (pink squares) and rare homozygotes (yellow triangles). (B) Visual genotype representation of the NAT2 polymorphisms. The genotyping results are summarized through the use of a visual genotype representation. The genotype is represented at the intersection of a given polymorphism (top) with a given sample (left). In the graphic, genotypes are represented as common homozygotes (blue), rare homozygotes (yellow) and heterozyotes (red). White boxes reflect those samples containing at least one pair of double heterozygous SNPs thus requiring phase determination. The white box also reflects the Detector ASOs to be selected for haplotype determination (e.g. Detector ASOs for SNP #487 were chosen to phase sample D1, which was also heterozygous at positions #347 and #809).
Phase analysis of multiple heterozygous samples
Our multi-SNP haplotyping assay utilizes a similar ASO hybridization approach in a bead-based format and allows up to 50 SNPs to be phased simultaneously. To haplotype the SNPs, duplicate hybridization reactions were set up containing PCR product, Capture ASOs corresponding to each allele of all SNPs and biotinylated Detector ASOs. It should be noted that in the haplotyping assay, the biotinylated Detector ASOs are now allele-specific to any one heterozygotic SNP. This allows the linkage of the SNP alleles on each chromosome to be determined by association of the RPE signal with each Capture ASO signal. To illustrate, the haplotyping of two heterozygotic SNPs (SNP 1 T/C and SNP 2 A/G) is shown in Figure 3A and B. Capture ASOs for the T (green bead) and C (red bead) allele of SNP #1 are hybridized to an amplicon encompassing both SNP loci in two identical reactions. To each reaction tube, a biotinylated Detector ASO toward one or the other allele of a heterozygous SNP is also added (SNP 2 was chosen here, but any heterozygous SNP will suffice). In the illustration, the ‘G’ allele Detector ASO was added to tube #1 and the ‘A’ allele Detector ASO to tube #2. After hybridization of Capture and Detector ASOs, a strepavidin–RPE reporter molecule was hybridized to the mixture. Figure 3A and B shows the expected results for two samples that contain the same double heterozygotic genotype but have different haplotypes. Figure 3A shows a positive RPE signal associated with the green bead in tube #1 and with the red bead in tube #2, indicating a 1T-2G/1C-2A haplotype. Figure 3B shows a positive RPE signal associated with the red bead in tube #1 and with the green bead in tube #2, indicating the alternate 1T-2A/1C-2G haplotype. Since 100 different colored beads are currently available, this technique has the potential of phasing of up to 50 SNPs with a given Detector SNP. By analyzing each of these 2-SNP combinations, the multi-SNP haplotype can be established.
Figure 3. Haplotyping of all 11 SNPs within exon 2 of the NAT2 gene. (A and B) Strategy for CBH. An illustration of the phasing of just two heterozygotic SNPs by CBH is shown. ASOs for the T and C alleles of SNP 1 are attached to two different sets of colored beads (green and red). Duplicate hybridization reactions are set up, each containing both Capture ASOs, the single-stranded template and a biotinylated ASO (Detector ASO) for either the G or A allele of SNP 2. Streptavidin–RPE is then added to the mixture. After hybridization, the RPE reporter signal associated with each of the Capture ASOs in the two reactions is measured. When a Detector and Capture ASOs are aligned on the same template, an RPE signal will be associated with that Capture ASO, indicating the presence of that particular haplotype. The figure addresses two samples with the same genotype but different haplotypes, thus, showing two different outcomes. (C) The results of the assay tubes for one sample (D1) were analyzed and plotted as shown. SNP #487 was selected as the Detector ASO. Each Capture ASOs has four possible bars representing the four haplotypes possible in relation to the Detector SNP. The phase of each SNP, in relation to SNP #487, is determined by the bar heights. The 11 SNP haplotype for this sample is shown below the graph. The alignment of the SNP alleles above and below the horizontal line represents the haplotype on each chromosome. (D) Simplified data analysis to establish SNP haplotypes. The results for each Detector ASO and heterozygous Capture ASO were analyzed by plotting the log10 versus total signal (where CA–CA, RA–RA, CA–RA and RA–CA represent the four haplotype combinations of the common allele (CA) and rare allele (RA) for each Detector/Capture SNP pair). By this approach, the haplotype configuration of each SNP pair can easily be established by the log value for each point. Points with a positive log value represent the CA–CA/RA–RA haplotype, whereas points with a negative log value represent the CA–RA/RA–CA haplotype. The Capture ASO/Detector ASO pairings are as follows: 197/487 (blue crosses), 197/596 (blue circles), 288/487 (pink crosses), 347/487 (green crosses), 409/487 (blue on white crosses), 596/288 (brown squares), 596/487 (brown crosses), 809/288 (black squares), 809/487 (black crosses), 863/288 (yellow squares), 863/487 (yellow on black crosses), and 1027/487 (red crosses), respectively. (E) Visual haplotype representation of the NAT2 gene. Haplotypes for the 11 SNPs within exon 2 for the 26 individuals containing at least one pair of double heterozygotic SNPs are shown. For each sample, two rows (‘–1’ and ‘–2’) indicate alignment of the SNP alleles on the two chromosomes. The ‘color’ of each square at the intersection represents which SNP allele is present at that position on that given chromosome. Blue boxes represent the common SNP allele and yellow the rare SNP allele.
To determine the phase of all double heterozygotic SNP pairs in our samples, duplicate reactions for each genomic sample were set up containing Capture ASOs for all 11 SNPs and the appropriate pair of Detector ASOs. To select the appropriate Detector ASOs for each genomic sample, all heterozygous SNPs present in each sample were compared with each other and the minimum number of Detector ASOs required to haplotype all 26 samples were selected. From this comparison, it was determined that one of the three heterozygous SNPs (#288, #487 or #596) was present in each of the 26 samples (Figure 2B). As an internal negative control, Capture ASOs representing those SNPs already contained in the Detector ASOs were also included. If the Detector ASOs are hybridizing appropriately, then no RPE signal should be associated with the negative control Capture ASOs. Additionally, including Capture ASOs for all 11 SNPs, even those SNPs represented in the Detector ASOs, will allow the use of a single Capture ASO bead mixture for all samples throughout the study.
Figure 3C shows the haplotype results for all 11 SNPs for a single sample. The graph reflects the 10 different combinations expected using Detector ASOs for SNP #487. As expected, no significant RPE signal was associated with the internal negative control SNP #487 Capture ASOs and SNP #487 Detector ASOs (data not shown). The multi-SNP haplotype configuration displayed below the graph was determined based on data from each pairwise combination. For example, the results of SNP #487/SNP #347 combination show two peaks indicating that the ‘C’ allele for SNP #487 is linked to the common SNP allele ‘T’ of SNP #347 on one chromosome and that the ‘T’ allele of SNP #487 is linked to the rare SNP allele ‘C’ of SNP #347 on the opposite chromosome. Similar analysis of all other SNP combinations yielded haplotype configurations for all 11 SNPs for the given sample as shown.
To establish the phase of all double heterozygotic SNP pairs, we incorporated a simplified data analysis approach based on the fact that only two possible phases exist. For example, if a sample is heterozygous C/T at SNP #1 and heterozygous A/G at SNP #2, the individual either has a CA haplotype on one chromosome and a TG haplotype on the other or a CG haplotype on one chromosome and a TA haplotype on the other. Therefore, our data interpretation results from testing the likelihood of these two events and is represented as a plot of the log10 versus total signal (where 1A-2A, 1B-2B, 1A-2B, 1B-2A represent the four haplotype combinations for a given SNP pair). In our example, samples with values near +1 (10-fold more percentage total signal) will have a CA/TG haplotype, whereas values near –1 will indicate a CG/TA haplotype. Graphing the results for each double heterozygotic pairings within the 26 samples resulted in distinct clusters (Figure 3D). By using this two-stage approach to haplotyping, we have also reduced data analysis to a binary decision, thus making calls simpler and unambiguous. The haplotypes for all 26 samples, as determined by our graphing technique, are shown in Figure 3E and are in 100% agreement with the haplotypes derived by subcloning/sequencing (data not shown).
DISCUSSION
In this report, we have presented a simplified approach for directly determining the phase of multi-SNP alleles in a high-throughput manner. The CBH approach, as with other molecular-based haplotyping methods, is most applicable in situations requiring the highest level of accuracy, including diagnostic testing for clinically relevant haplotypes, association-based studies for disease-gene identification and in pharmacogenomic analysis. One-time optimization of conditions for hybridization and template labeling has reduced the labor-intensive handling normally associated with bead-based assays, such as centrifugation and bead washing. Additionally, by incorporating an initial genotyping step, we have reduced the number and complexity of experiments needed to haplotype a sample population. In the presented study, only 26 of the 46 samples contained SNP alleles requiring phase resolution.
Owing to its simplicity, no sophisticated robotics are needed to perform high-throughput levels of this assay. As indicated, the CBH assay can be performed in 1 h and no centrifugation, filtration, purification (both post-PCR and post-hybridization) or enzymatic steps are required. Using a low-end liquid handler, we have automated all aspects of the CBH assay including the cherry picking of samples that required phasing, the addition of all hybridization reagents and the addition of streptavidin–RPE. The throughput of this assay is approximately one 96-well plate (48 samples) in 1 h (nearly 400 samples per day). The number of SNPs analyzed within this period is directly related to the number of SNPs within each assay. For example, to analyze the NAT2 gene that contains 11 polymorphisms, over 4000 SNPs could be haplotyped per day. Given that our haplotyping assay can analyze up to 50 SNP haplotypes, over 19 000 polymorphisms could be haplotyped per day. This throughput is directly limited by the speed of the Luminex instrument in analyzing each well. However, given their low cost, higher throughput could easily be achieved with the purchase of additional instruments.
Recent experiments by our laboratory have shown that the CBH assay is reliable and robust. This has been demonstrated by the assay development for eight additional genes including MBL2 (17 SNPs and 1 ins/del polymorphism), ELAM (4 SNPs), IL10 (4 SNPs) and IL4 (3 SNPs). Furthermore, for each developed assay, we observed call rates and accuracy >99%. However, the CBH assay does have the limitation that it can only analyze SNPs within a PCR amplifiable region. Currently, we have analyzed haplotypes within an amplified region of up to 1500 bp; however, advances in long-range PCR protocols (30,31) should enable us to study larger loci. However, it is likely that as the amplicon size increases, the complexity of the secondary structure of the single-stranded PCR product will also increase, thus reducing hybridization efficiency. As a result, the addition of secondary structure inhibitors, or more intense fluorescent detection reagents, may be necessary to compensate for the reduced hybridization. Additionally, we are exploring other approaches to increase genomic coverage such as using overlapping amplicons and incorporating RT–PCR techniques.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
This work was supported by grants from the National Institutes of Health (1R43HG002247-01A2 and 2R44HG002247-02) to J.E.L.
REFERENCES
Martin,M.P., Dean,M., Smith,M.W., Winkler,C., Gerrard,B., Michael,N.L., Lee,B., Doms,R.W., Margolick,J., Buchbinder,S. et al. ( (1998) ) Genetic acceleration of AIDS progression by a promoter variant of CCR5. Science, , 282, , 1907–1911.
Drysdale,C.M., McGraw,D.W., Stack,C.B., Stephens,J.C., Judson,R.S., Nandabalan,K., Arnold,K., Ruano,G. and Liggett,S.B. ( (2000) ) Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc. Natl Acad. Sci. USA, , 97, , 10483–10488.
Hoehe,M.R., Kopke,K., Wendel,B., Rohde,K., Flachmeier,C., Kidd,K.K., Berrettini,W.H. and Church,G.M. ( (2000) ) Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence. Hum. Mol. Genet., , 9, , 2895–2908.
Mirel,D.B., Valdes,A.M., Lazzeroni,L.C., Reynolds,R.L., Erlich,H.A. and Noble,J.A. ( (2002) ) Association of IL4R haplotypes with type 1 diabetes. Diabetes, , 51, , 3336–3341.
Carew,J.A., Basso,F., Miller,G.J., Hawe,E., Jackson,A.A., Humphries,S.E. and Bauer,K.A. ( (2003) ) A functional haplotype in the 5' flanking region of the factor VII gene is associated with an increased risk of coronary heart disease. J. Thromb. Haemost., , 1, , 2179–2185.
Lambrechts,D., Storkebaum,E., Morimoto,M., Del-Favero,J., Desmet,F., Marklund,S.L., Wyns,S., Thijs,V., Andersson,J., van Marion,I. et al. ( (2003) ) VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nature Genet., , 34, , 383–394.
Knapp,S., Hennig,B.J., Frodsham,A.J., Zhang,L., Hellier,S., Wright,M., Goldin,R., Hill,A.V., Thomas,H.C. and Thursz,M.R. ( (2003) ) Interleukin-10 promoter polymorphisms and the outcome of hepatitis C virus infection. Immunogenetics, , 55, , 362–369.
Krajinovic,M., Lemieux-Blanchard,E., Chiasson,S., Primeau,M., Costea,I. and Moghrabi,A. ( (2004) ) Role of polymorphisms in MTHFR and MTHFD1 genes in the outcome of childhood acute lymphoblastic leukemia. Pharmacogenomics J., , 4, , 66–72.
Ruano,G., Kidd,K.K. and Stephens,J.C. ( (1990) ) Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules. Proc. Natl Acad. Sci. USA, , 87, , 6296–6300.
Ding,C. and Cantor,C.R. ( (2003) ) Direct molecular haplotyping of long-range genomic DNA with M1-PCR. Proc. Natl Acad. Sci. USA, , 100, , 7449–7453.
Prior,T.W., Wenger,G.D., Papp,A.C., Snyder,P.J., Sedra,M.S., Bartolo,C., Moore,J.W. and Highsmith,W.E. ( (1995) ) Rapid DNA haplotyping using a multiplex heteroduplex approach: application to Duchenne muscular dystrophy carrier testing. Hum. Mutat., , 5, , 263–268.
Veenstra-VanderWeele,J., Kim,S.J., Gonen,D., Hanna,G.L., Leventhal,B.L. and Cook,E.H.,Jr ( (2001) ) Genomic organization of the SLC1A1/EAAC1 gene and mutation screening in early-onset obsessive-compulsive disorder. Mol. Psychiatry, , 6, , 160–167.
Eitan,Y. and Kashi,Y. ( (2002) ) Direct micro-haplotyping by multiple double PCR amplifications of specific alleles (MD-PASA). Nucleic Acids Res., , 30, , e62.
Pettersson,M., Bylund,M. and Alderborn,A. ( (2003) ) Molecular haplotype determination using allele-specific PCR and pyrosequencing technology. Genomics, , 82, , 390–396.
Minchin,R.F., Reeves,P.T., Teitel,C.H., McManus,M.E., Mojarrabi,B., Ilett,K.F. and Kadlubar,F.F. ( (1992) ) N- and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells. Biochem. Biophys. Res. Commun., , 185, , 839–844.
Hein,D.W., Doll,M.A., Rustan,T.D., Gray,K., Feng,Y., Ferguson,R.J. and Grant,D.M. ( (1993) ) Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases. Carcinogenesis, , 14, , 1633–1638.
Zenser,T.V., Lakshmi,V.M., Rustan,T.D., Doll,M.A., Deitz,A.C., Davis,B.B. and Hein,D.W. ( (1996) ) Human N-acetylation of benzidine: role of NAT1 and NAT2. Cancer Res., , 56, , 3941–3947.
Blum,M., Grant,D.M., McBride,W., Heim,M. and Meyer,U.A. ( (1990) ) Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression. DNA Cell Biol., , 9, , 193–203.
Grant,D.M., Hughes,N.C., Janezic,S.A., Goodfellow,G.H., Chen,H.J., Gaedigk,A., Yu,V.L. and Grewal,R. ( (1997) ) Human acetyltransferase polymorphisms. Mutat. Res., , 376, , 61–70.
Packer,B.R., Yeager,M., Staats,B., Welch,R., Crenshaw,A., Kiley,M., Eckert,A., Beerman,M., Miller,E., Bergen,A. et al. ( (2004) ) SNP500Cancer: a public resource for sequence validation and assay development for genetic variation in candidate genes. Nucleic Acids Res., , 32, , D528–D532.
Brockton,N., Little,J., Sharp,L. and Cotton,S.C. ( (2000) ) N-acetyltransferase polymorphisms and colorectal cancer: a HuGE review. Am. J. Epidemiol., , 151, , 846–861.
Hein,D.W., Doll,M.A., Fretland,A.J., Leff,M.A., Webb,S.J., Xiao,G.H., Devanaboyina,U.S., Nangju,N.A. and Feng,Y. ( (2000) ) Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol. Biomarkers Prev., , 9, , 29–42.
Butcher,N.J., Boukouvala,S., Sim,E. and Minchin,R.F. ( (2002) ) Pharmacogenetics of the arylamine N-acetyltransferases. Pharmacogenomics J., , 2, , 30–42.
Cascorbi,I. and Roots,I. ( (1999) ) Pitfalls in N-acetyltransferase 2 genotyping. Pharmacogenetics, , 9, , 123–127.
Iannone,M.A., Taylor,J.D., Chen,J., Li,M.S., Rivers,P., Slentz-Kesler,K.A. and Weiner,M.P. ( (2000) ) Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry. Cytometry, , 39, , 131–140.
Nickerson,D.A., Taylor,S.L., Weiss,K.M., Clark,A.G., Hutchinson,R.G., Stengard,J., Salomaa,V., Vartiainen,E., Boerwinkle,E. and Sing,C.F. ( (1998) ) DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene. Nature Genet., , 19, , 233–240.
Rieder,M.J., Taylor,S.L., Clark,A.G. and Nickerson,D.A. ( (1999) ) Sequence variation in the human angiotensin converting enzyme. Nature Genet., , 22, , 59–62.
Nickerson,D.A., Tobe,V.O. and Taylor,S.L. ( (1997) ) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res., , 25, , 2745–2751.
Wiltshire,T., Pletcher,M.T., Batalov,S., Barnes,S.W., Tarantino,L.M., Cooke,M.P., Wu,H., Smylie,K., Santrosyan,A., Copeland,N.G. et al. ( (2003) ) Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse. Proc. Natl Acad. Sci. USA, , 100, , 3380–3385.
Barnes,W.M. ( (1994) ) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl Acad. Sci. USA, , 91, , 2216–2220.
Cheng,S., Fockler,C., Barnes,W.M. and Higuchi,R. ( (1994) ) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl Acad. Sci. USA, , 91, , 5695–5699.
Hirschhorn,J.N., Sklar,P., Lindblad-Toh,K., Lim,Y.M., Ruiz-Gutierrez,M., Bolk,S., Langhorst,B., Schaffner,S., Winchester,E. and Lander,E.S. ( (2000) ) SBE-TAGS: an array-based method for efficient single-nucleotide polymorphism genotyping. Proc. Natl Acad. Sci. USA, , 97, , 12164–12169.(James D. Hurley, Linda J. Engle, Jesse T)