Large-Scale Identification, Mapping, and Genotyping of Single-Nucleotide Polymorphisms in the Human Genome David G. Wang, et al.

Science 280, 1077 (1998); DOI: 10.1126/science.280.5366.1077 The following resources related to this article are available online at www.sciencemag.org (this information is current as of October 29, 2007 ):
Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/cgi/content/full/280/5366/1077 This article cites 22 articles, 14 of which can be accessed for free: http://www.sciencemag.org/cgi/content/full/280/5366/1077#otherarticles This article has been cited by 946 article(s) on the ISI Web of Science. This article has been cited by 97 articles hosted by HighWire Press; see: http://www.sciencemag.org/cgi/content/full/280/5366/1077#otherarticles This article appears in the following subject collections: Genetics http://www.sciencemag.org/cgi/collection/genetics Information about obtaining reprints of this article or about obtaining permission to reproduce this article in whole or in part can be found at: http://www.sciencemag.org/about/permissions.dtl Downloaded from www.sciencemag.org on October 29, 2007

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env sequences in each specimen were examined by phylogenetic analysis. 11. The methods described by G. H. Learn et al. [J. Virol. 70, 5720 (1996)] were used to align DNA sequences (with the use of CLUSTALW plus manual adjustment), calculate genetic distances (with the use of DNADIST, using the maximum likelihood method), evaluate potential sample mixups, construct neighbor joining trees, and perform bootstrap analyses (1000 replicates). Sequence regions that could not be unambiguously aligned were removed from subsequent analyses. Each sequence was compared for phylogenetic relatedness to the entire set of published and available unpublished laboratory HIV database sequences. If after this analysis the viral sequences from a mother and an infant appeared as a monophyletic group on a phylogenetic tree, they were judged to be phylogenetically linked or to have a common ancestor not shared by sequences from any other individuals evaluated. Issues regarding the assignment of phylogenetic linkage are discussed in greater detail by Learn et al. L. M. Frenkel et al., at www.sciencemag.org/feature/ data/974996.shl. R. Liu et al., Cell 86, 367 (1996). L. M. Frenkel et al., unpublished data. M.-L. Newell et al., Lancet 347, 213 (1996). P. Palumbo, J. Skurnick, D. Lewis, M. Eisenberg, J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 10, 436 (1995). A. McMichael, R. Koup, A. J. Ammann, N. Eng. J. Med. 334, 801 (1996). E. C. Holmes et al., J. Infect. Dis. 167, 1411 (1993). T. Liu et al., J. Immunol. 154, 3147 (1995). A. Hoffenbach et al., ibid. 142, 452 (1989). G. Schochetman, S. Subbarao, M. L. Kalish, in Viral Genome Methods, K. W. Adolph, Ed. (CRC Press, Boca Raton, FL, 1996), pp. 25 41. 22. E. L. Delwart, M. P. Busch, M. L. Kalish, J. W. Mosley, J. I. Mullins, AIDS Res. Hum. Retrovir. 11, 1181 (1995). 23. C. H. Contag et al., J. Virol. 71, 1292 (1997). 24. We thank J. Conroy for performing PCR assays; E. Abrams, M. S. Orloff, R. C. Reichman, L. M. Demeter, J. S. Lambert, R. Dolin, R. Sperling, D. Shapiro, G. McSherry, and the Ariel Project and ACTG 076 investigators for critical patient specimens; D. Swofford for use of computer program PAUP*, version 4.0.0d63; and C. B. Wilson and K. K. Holmes for editorial contributions. This work was supported by grants from the Pediatric AIDS Foundation (500153 10-PGT, 50366 14-PGR, 55516-ARI, 55529-ARI, 55525-ARI, 55532-ARI, 55526-ARI, 55531-ARI, and 55522-ARI), the U.S. Public Health Service (UO127658, AI32910, AI27757, and AI35539), and the Foster Foundation. 22 December 1997; accepted 26 March 1998

12. 13. 14. 15. 16.

17. 18. 19. 20. 21.

David G. Wang, Jian-Bing Fan, Chia-Jen Siao, Anthony Berno, Peter Young, Ron Sapolsky, Ghassan Ghandour, Nancy Perkins, Ellen Winchester, Jessica Spencer, Leonid Kruglyak, Lincoln Stein, Linda Hsie, Thodoros Topaloglou, Earl Hubbell, Elizabeth Robinson, Michael Mittmann, Macdonald S. Morris, Naiping Shen, Dan Kilburn, John Rioux, Chad Nusbaum, Steve Rozen, Thomas J. Hudson, Robert Lipshutz,* Mark Chee, Eric S. Lander*
Single-nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome, and they provide powerful tools for a variety of medical genetic studies. In a large-scale survey for SNPs, 2.3 megabases of human genomic DNA was examined by a combination of gel-based sequencing and high-density variation-detection DNA chips. A total of 3241 candidate SNPs were identified. A genetic map was constructed showing the location of 2227 of these SNPs. Prototype genotyping chips were developed that allow simultaneous genotyping of 500 SNPs. The results provide a characterization of human diversity at the nucleotide level and demonstrate the feasibility of large-scale identification of human SNPs.

Although the Human Genome Project still has tremendous work ahead to produce the first complete reference sequence of the human chromosomes, attention is already focusing on the challenge of large-scale characterization of the sequence variation
D. G. Wang, C.-J. Siao, P. Young, N. Perkins, E. Winchester, J. Spencer, L. Kruglyak, L. Stein, E. Robinson, D. Kilburn, J. Rioux, C. Nusbaum, S. Rozen, T. J. Hudson, Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA. J.-B. Fan, A. Berno, R. Sapolsky, G. Ghandour, L. Hsie, T. Topaloglou, E. Hubbell, M. Mittmann, M. S. Morris, N. Shen, R. Lipshutz, M. Chee, Affymetrix, Incorporated, 3380 Central Expressway, Santa Clara, CA 95051, USA. E. S. Lander, Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA, and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. * To whom correspondence should be addressed.

among individuals (1). This genetic diversity is of interest because it explains the basis of heritable variation in disease susceptibility, as well as harbors a record of human migrations. The most common type of human genetic variation is the SNP, a position at which two alternative bases occur at appreciable frequency ( 1%) in the human population. There has been growing recognition that large collections of mapped SNPs would provide a powerful tool for human genetic studies (1, 2). SNPs can serve as genetic markers for identifying disease genes by linkage studies in families, linkage disequilibrium in isolated populations, association analysis of patients and controls, and lossof-heterozygosity studies in tumors (1, 2).
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Large-Scale Identification, Mapping, and Genotyping of Single-Nucleotide Polymorphisms in the Human Genome

Although individual SNPs are less informative than currently used genetic markers (3), they are more abundant and have greater potential for automation (4, 5). We performed an initial survey to identify SNPs by using conventional gel-based DNA sequencing to examine sequencetagged sites (STSs) distributed across the human genome. STSs are short genomic sequences that can be amplified from DNA samples by means of a corresponding polymerase chain reaction (PCR) assay. From among 24,568 STSs used in the construction of a physical map of the human genome at the Whitehead Institute for Biomedical Research/MIT Center for Genome Research (6, 7), an initial collection of 1139 STSs was chosen (8). These STSs contained a total of 279 kb of genomic sequence (9), with one-third from random genomic sequence and two-thirds from 3 ends of expressed sequence tags (3 -ESTs) and primarily representing untranslated regions of genes. Each STS was amplified from four samples (10): three individual samples and a pool of 10 individuals (thereby permitting allele frequencies to be estimated among 20 chromosomes). The PCR products were subjected to single-pass DNA sequencing based on fluorescent-dye primers and gel electrophoresis; sequence traces were compared by a computer program followed by visual inspection (11). Candidate SNPs were declared when two alleles were seen among the three individuals, with both alleles present at a frequency greater than 30% in the pooled sample. The term candidate SNP is used because a subset of such apparent polymorphisms turn out to be sequencing artifacts, as discussed below. The survey identified 279 candidate SNPs, distributed across 239 of the STSs. This corresponds to a rate of one SNP per 1001 base pairs (bp) screened and an observed nucleotide heterozygosity of H 3.96 104 (Table 1). Expressed sequences (3 -ESTs) showed a lower polymorphism rate than random genomic sequence (with

Fig. 1. SNP screening on chips. (A) Small portion of a VDA for an STS hybridized with the expected target sequence. Chip features in each column are complementary to successive overlapping 25-nucleotide oligomer subsequences, with the central base substituted by A, C, G, or T in the four rows. Variations from the expected sequence can usually be detected by examination of the most intense signal in each column. (B) The same VDA was hybridized with sequence containing an SNP (A3 C) at position 19. The hybridization signal is now stronger at an alternative base at this position. It is also weaker at the surrounding positions (for example, positions 12 to 18 and 20 to 26), because probes at these positions are designed to be complementary to the A allele at the SNP and mismatch with the C allele.

Table 1. Results of SNP screening. Gel-based sequencing Variable All STSs No. of STSs screened Total bases screened No. of candidate SNPs found SNP frequency (K ) Heterozygosity (H ) ( 10 4) No. of STSs containing SNPs % transitions among SNPs % SNPs occurring within CpG , based on H , based on K 1078 1,139 279,165 279 1/1001 3.96 0.38 239 67% 24% 3.96 10 4 4.33 10 4 SCIENCE STSs from 3 -EST sequences 705 186,524 161 1/1159 3.42 0.43 137 67% 23% STSs from random genomic sequence 434 92,641 118 1/785 5.04 0.67 102 67% 25% Chip-based detection STSs from 3 -EST sequences 12,649 1,324,320 1,749 1/757 4.36 0.18 1,515 70% 25% STSs from random genomic sequence 4,076 656,710 999 1/657 5.02 0.28 784 71% 22%

All STSs 16,725 1,981,030 2,748 1/721 4.58 0.15 2,299 70% 24% 4.58 10 4 4.36 10 4

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the difference falling just short of statistical significance at P 0.057, one-sided), consistent with greater constraint within genic sequences. The ratio of transitions to transversions was 2 :1. Although the dinucleotide CpG makes up only about 2% of the sequence surveyed, nearly 25% of the SNPs occurred at such sites with the substitution almost always being C7T. Cytosine residues within CpG dinucleotides are the most mutable sites within the human genome, because most are methylated and can spontaneously deaminate to yield a thymidine residue (12). In addition to the single-base substitutions, 23 insertion-deletion polymorphisms were also found (with all but eight involving a single base), corresponding to a frequency of one per 12 kb surveyed. Gel-based resequencing was satisfactory for the initial screen, but we sought a more streamlined approach for a larger scale SNP identification. One such approach involves hybridization to highdensity DNA probe arrays (13). Such DNA chips can be produced with parallel light-directed chemistry to synthesize specified oligonucleotide probes covalently bound at defined locations on a glass surface or chip (14). A target DNA se-

quence of length L can be screened for a polymorphism by hybridizing a biotin-labeled sample to a variant detector array (VDA) of size 8L (Fig. 1). For each position on both strands, the array has four 25-nucleotide oligomer probes complementary to the sequence centered at the position. The four differ only in that the central (13th) position is substituted by each of the four nucleotides. Homozygotes (AA) for the expected sequence should hybridize more strongly to the perfectly complementary probe than to the three probes containing a central mismatch. The presence of an SNP would be expected to give rise to a different hybridization pattern, with homozygotes (BB) showing strong hybridization to an alternative base and heterozygotes (AB) showing strong hybridization to two probes. The VDA thus signals the presence of a sequence variation (by a change in the hybridization pattern) and, in many cases, indicates the nature of the change (by a gain of signal at a specific mismatch probe). VDAs have been used for mutation detection of small, well-studied DNA targets [such as 387 bp from the human immunodeficiency virus1 genome, 3.5 kb from the breast cancerassociated BRCA1 gene, and 16.6 kb from the human

mitochondrion (13, 15)] in large numbers of samples. In this setting, the normal hybridization pattern can be characterized with precision and single-base substitutions detected with high accuracy. In this project, we used VDAs in a largescale survey. A total of 16,725 STSs covering 2 Mb of human DNA were selected, with one-third from random genomic sequence and two-thirds from 3 -ESTs. The survey used 149 distinct chip designs, each containing 150,000 to 300,000 features. The STSs were examined in seven individuals, representing about 14 Mb of genomic sequence. For each chip, the corresponding STSs were amplified from an individual, pooled together, labeled with biotin, hybridized, and stained (16), and the resulting hybridization patterns were compared by a computer program followed by visual inspection (17). At each position, samples were classified as homozygous for the expected sequence, homozygous for an alternative sequence, or heterozygous. A collection of 2748 candidate SNPs were identified, corresponding to a rate of one per 721 bp surveyed and an observed nucleotide heterozygosity of 4.58 104 (Table 1). The number of STSs containing SNPs was 2299. The SNPs had a mean heterozygosity of 33%, with the minor allele having a mean frequency of 25%. SNPs were found less often in 3 -ESTs than in random genomic sequence (P 0.023, onesided), consistent with greater constraint in genic regions. The nucleotide heterozygosity rate was indistinguishable from the estimate obtained from gel-based sequencing (P 0.12, two-sided test), as was the ratio of transitions to transversions and the proportion of SNPs occurring at CpG dinucleotides. SNPs were detected at a higher frequency in the chip-based survey because more samples were surveyed (seven versus three individuals). The observed increase of 38.8% (1/721 versus 1/1001) agreed closely

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with expectation under classical population genetic theory (18). This result has implications for the choice of sample size for an SNP survey (19). We estimated the error rates in the gelbased and chip-based surveys. The falsepositive rate was estimated by carefully confirming candidate SNPs found in each survey by using thorough multipass sequencing (20): 12% of 220 candidate SNPs found in the chip-based survey and 16% of 120 candidate SNPs found by single-pass gel-based sequencing were false positives. The falsenegative rate was estimated by considering a subset of STSs that had been included in both surveys: these STSs yielded 55 SNPs (all carefully confirmed to eliminate false positives), of which eight (15%) were missed by single-pass gel-based resequencing and seven (13%) were missed by the chip-based survey. Many of the errors were due to random factors, in that they were eliminated simply by repeating the original experiment. However, some were reproducible artifacts that could be eliminated only by changing the detection protocol (for example, by using dye terminators rather than dye primers in gel-based sequencing). The gel-based sequencing and chip-based analysis had similar rates of accuracywith a false positive and false negative being found roughly every 5000 to 10,000 bases, or about 10% of the true SNP frequency. The accuracy largely reflects the particular implementation of the technologies in a highthroughput setting and could be increased at the expense of assay optimization. Although the two surveys yielded comparable accuracy, the survey based on VDAs required considerably less laboratory work than gel-based resequencing. Both approaches required amplifying target loci. The gel-based approach then required a sequencing reaction and electrophoresis on each individual locus, whereas the chipbased approach allowed targets totaling 30 kb to be pooled into a single labeling reaction and hybridized (21). The SNP collection from the two surveys was supplemented by two directed approaches based on public databases. First, we collected reports from the literature of common variants in gene coding regions. We were able to confirm 120 of 143 cases tested by virtue of detecting two alleles in our screening panel; the remainder may be true polymorphisms but simply monomorphic in the individuals tested. Second, the GenBank database contains multiple entries for some ESTs. Such entries were compared to identify single-nucleotide differences, which might reflect either common polymorphisms or sequencing errors in single-pass EST sequencing. We tested 200 such apparent differences and confirmed
Fig. 2. A portion of the SNP genetic map (showing human chromosome 1). The full map is available on the Whitehead Institute Web site (www. genome.wi.mit.edu). Positions are based on genetic distances in centimorgans. Genetic positions of SNPs were inferred by localizing them relative to framework markers by RH mapping and then interpolating distances from centirays (on the RH map) to centimorgans (on the genetic map). Framework marker names are given in full. SNP names are named with the prefix WIAF (for example, WIAF-17), but the prefix is dropped and only the number is shown in the figure.

the presence of an SNP in 94 cases. These two directed approaches thus yielded an additional 214 SNPs. The project has thus identified 3241 candidate SNPs to date. Confirmation (22) has so far been obtained for 1477 SNPs and is expected to yield 2900 true SNPs. All information about the SNPs has been deposited on the Whitehead/MIT Center for Genome Research Web site (www.genome. wi.mit.edu) and will be updated with results of additional surveys and confirmation tests. The information is also being deposited in the GenBank database. For SNPs to be useful in human genetic studies, they must be assembled into maps showing their chromosomal location. To create a third-generation map based on SNPs, we used whole-genome radiation-hybrid (RH) mapping (6, 7, 23), which infers the position of loci based on co-retention in a panel of human-on-hamster cell lines; it has become a primary method for constructing maps of the human genome (6, 7). The current RH map of the human genome is anchored by a scaffold of 1036 genetic markers from an earlier genetic map consisting of simple sequence length polymorphisms (SSLPs) (7). SNPs can be integrated with respect to the earlier genetic map by determining their position on the RH map. We have localized 1880 STSs, containing 2227 of the 3241 candidate SNPs, on the RH map and thereby relative to the human genetic map (Fig. 2 and Table 2). SNPs are not evenly distributed among chromosomes or within chromosomes because most were derived from ESTs, which are known to have an uneven distribution (6, 7). SNP-containing STSs are present at a mean spacing of 2.0 centimorgans (cM) across the genome (24), and the map contains 58 intervals greater than 10 cM. The genetic distances on the map must be regarded as approximate because they are based on interpolation from distances in the RH map. It will be desirable to reestimate these distances on the basis of direct linkage analysis in the CEPH families, as highthroughput genotyping for the complete SNP collection becomes feasible.
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We next developed an efficient method for large-scale genotyping of SNPs based on extending the use of DNA chips from SNP

Fig. 3. Genotyping chips. (A) Schematic diagram of genotyping array for an SNP, consisting of two VDAs to study seven nucleotides centered around the SNP. The top and bottom arrays are designed to be complementary to the allelic sequences containing A and C, respectively. Probes perfectly matching the A and C alleles are shown in gray and black, respectively. A genotyping array for the complementary strand was also used but is not shown. (B) Hybridization signal for a genotyping array probed with samples from three individuals with respective genotypes AA, AC, and CC.

Table 2. Chromosomal distribution of genetic markers. No. of framework markers used from 5264-marker Genethon genetic map 88 91 78 54 65 71 57 45 40 53 58 43 23 31 29 36 23 29 22 34 18 12 36 1036 SCIENCE VOL. 280 Genetic distance in cM, on Genethon genetic map 293 277 233 213 198 201 184 166 167 182 156 169 118 129 110 131 129 124 110 97 60 58 191 3699 15 MAY 1998 No. of SNPs 236 177 160 98 86 158 119 135 106 85 105 108 57 83 76 70 94 52 58 58 29 31 46 2227 No. of STSs 201 149 133 87 72 118 94 108 88 78 92 91 45 64 67 64 87 43 53 49 26 28 43 1880 Avg. distance between STSs (cM) 1.5 1.9 1.8 2.4 2.8 1.7 2.0 1.5 1.9 2.3 1.7 1.9 2.6 2.0 1.6 2.0 1.5 2.9 2.1 2.0 2.3 2.1 4.4 2.0 No. of intervals 10 cM 3 2 1 4 4 4 1 3 3 1 1 3 4 3 0 0 2 4 2 2 2 1 8 58

Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Total 1080

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discovery to SNP genotyping (5). We synthesized genotyping chips containing genotyping arrays for each SNP to be tested. Each genotyping array consists of two short VDAs corresponding to the two alternative alleles (Fig. 3). The presence of an allele should be reflected in strong hybridization to the corresponding resequencing array. PCR assays were designed for the region containing each SNP (25), with the goal of being robust and mutually compatible: the amplification targets were all small (typically, a few nucleotides around the polymorphic site), the primers all had similar calculated melting temperatures, and constant sequences were added to the 5 ends of the forward and reverse primers to facilitate batch labeling of pooled PCR products. Each assay was tested to ensure that it amplified a single fragment from genomic DNA. The most complex genotyping chip tested contained genotyping arrays for 558 candidate SNPs identified in the chipbased survey. Initially, the 558 loci were separately amplified, pooled, labeled, and hybridized to the chip. To determine whether each locus could be reliably read, we defined a formal detection test: loci passed if, for each of three individuals tested, the expected DNA sequence could be successfully read on both strands for one or both alleles. In all, 98% of the loci passed this detection test (with the remaining 2% failing as a result of weak hybridization or cross-hybridization). We next sought to decrease substantially

the sample preparation required to genotype large numbers of SNPs, as required to perform a genome scan. We developed a protocol based on multiplex PCR in which primer pairs from many different loci are combined in a single reaction (26). Although it is typically difficult to combine many PCR assays, the approach worked well for our SNP assays: 92% of the 558 loci passed the detection test when amplification was performed in 24 sets of 23 loci; 90% passed when amplified in 12 sets of 46 loci; 85% passed when amplified in 6 sets of 92 loci; and 50% passed when amplified in a single set of 558 loci. The success appears to have resulted from a combination of factors, including the small size of the amplification targets, optimization of amplification conditions, and the presence of the constant sequence at the 5 -ends of the primers (27). It may be possible to salvage the unsuccessful assays by grouping them into additional multiplex sets or by redesigning the assays. Multiplex amplification of sets of 46 loci was used in subsequent experiments because it decreased the number of reactions by a factor of 46 while allowing the vast majority (512/558) of loci to be assayed. The procedure was further tested in 39 individuals and was quite consistent: 96% of the 512 loci could be successfully read in 100% of individuals tested and the remainder in nearly all individuals. We next developed a genotyping algorithm for each SNP. Loci were declared to pass a cluster test if the hybridization pat-

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terns seen in a test set of 39 individuals fell into distinct clusters, corresponding to the possible genotypes (28). These clusters could then be used to assign genotypes for further samples (29). The cluster test was applied to the 500 candidate SNPs that worked well under multiplex amplification conditions: 75% passed the cluster test, and careful resequencing demonstrated that all such loci were true polymorphisms. The cluster test thus provides reliable confirmation of an SNP. The remaining 25% failed the cluster test, and resequencing revealed that half were false positives in the SNP screen and half were true polymorphisms (with the poor discrimination on the chip typically due to one allele hybridizing more weakly than the other). Thus, 88% of the candidate SNPs proved to be true polymorphisms, and 86% of true SNPs passed the cluster test. To test the reproducibility and accuracy of the genotyping method, we genotyped a set of 91 loci (passing the cluster test) in three individuals by performing chip-based genotyping on six separate occasions over a 2-month period. The correct genotypes were independently determined by thorough gel-based resequencing. The genotyping-chip assay assigned a genotype in 98% of cases (1613/1638), and this assignment proved correct in 99.9% (1611/1613) of these cases. The loci were also genotyped in two complete CEPH families. The genotypes were not independently confirmed, but they were fully consistent with mendelian segregation. For SNPs passing the cluster test, highly accurate genotypes could thus be obtained with the simple design used here. For the remaining SNPs (14%), similar accuracy can likely be obtained but may require optimization of the genotyping array design, depending on the locus [as shown in (5)]. The SNP surveys provide data about human genetic diversity. Two classical measures of diversity (30) are H, the average heterozygosity per nucleotide, and K, the proportion of sites harboring a variation. H does not depend on sample size, whereas K increases with the number of genomes surveyed. For a population at equilibrium, the neutral theory of evolution relates H and K to the classical population genetic parameter 4Ne , where Ne is the effective population size and is the mutation rate per nucleotide. ( can be thought of as twice the number of new mutations per generation arising in a population with size Ne.) Specifically, H and K [1 1 2 1 3 1 ... (n 1) 1], provided that is small. From these equations, one can estimate based on H or K. The human population is not at equilibrium, but rather underwent a rapid population expansion in the last 100,000 to 200,000 years. Such population explosions tend to suppress the effects of genetic drift and thus preserve the distribution of common alleles and the value of . Accordingly, the value of is relevant to the ancestral human population before its recent expansion. The four estimates of derived from H and K for the two surveys are all roughly 4 104 (Table 1). Assuming a mutation frequency of 108 to 109, this would suggest an effective population size of Ne 104 to 105, which seems reasonable for the ancestral population preceding the explosion in the last 100,000 years (31). Strictly speaking, these estimates apply only to the European population, from which all samples were drawn. However, a preliminary survey of a more diverse sample of 31 individuals representing all major racial groups yielded a value of that is only 30% larger (26), consistent with the idea that human variation occurs primarily within rather than between racial groups (32). The resources reported here represent only a first step toward a dense SNP map of the human genome. The genetic map should already be useful for family-based linkage studies, given the average spacing (2 cM) and average heterozygosity (34%) of the markers. (The heterozygosity applies to the European-derived samples studied here, but a preliminary survey of 180 of the SNPs shows that most are also polymorphic in other groups.) It still remains to develop a suitable genotyping system, such as a 2000-SNP genotyping chip. Large-scale screening for human variation is clearly feasible. Someday it may become possible to screen entire human genomes. In the nearer term, a key goal will be to extend SNP discovery to the protein coding regions of all human genes (roughly 120 Mb of sequence, only about 40 times more than the current study) in order to catalog the common variants that may explain susceptibility to common genetic traits and diseases (1).
REFERENCES AND NOTES ___________________________
1. N. Risch and K. Merikangas, Science 273, 1516 (1996); E. S. Lander, ibid. 274, 536 (1996); F. S. Collins, M. S. Guyer, A. Chakravarti, ibid. 278, 1580 (1997). 2. L. Kruglyak, Nature Genet. 17, 21 (1997). 3. SNPs have only two alleles and are less informative than typical multi-allelic simple sequence length polymorphisms (SSLPs). This disadvantage can be offset by using a greater density of SNPs: a genome scan with 1000 well-spaced SNPs, for example, will extract about the same linkage information as the current standard of 400 well-spaced SSLPs (2). 4. B. J. Conner et al., Proc. Natl. Acad. Sci. U.S.A. 80, 278 (1983); U. Landegren, R. Kaiser, J. Sanders, L. Hood, Science 241, 1077 (1988); D. Y. Wu et al., Proc. Natl. Acad. Sci. U.S.A. 86, 2757 (1989); R. K. Saiki et al., ibid., p. 6230; A.-C. Syvanen et al., Genomics 8, 684 (1990); D. A. Nickerson et al., Proc. Natl. Acad. Sci. U.S.A. 87, 8923 (1990); K. J. Livak et al., Nature Genet. 9, 341 (1995); M. T. Roskey et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4724 (1996). M. T. Cronin et al., Hum. Mutat. 7, 244 (1996). T. J. Hudson et al., Science 270, 1945 (1995). G. D. Schuler et al., ibid. 274, 540 (1996). STSs with the largest sizes were used in the gelbased screen, and the remaining STSs, having somewhat smaller sizes, were used in the subsequent chip-based screen. The genomic sequence screened (279 kb) is the sum of the distances between the primer sites of the STSs successfully resequenced. The individuals surveyed were chosen from Centre dEtude du Polymorphisme Humain (CEPH) pedigrees K104, K884, and K1331 from the Amish, Venezuelan, and Utah populations, respectively. The SNP survey by gel-based sequencing examined three unrelated individuals (K104-1, K884-2, K13311) and a pool of 10 individuals (K104-13, -14, -15, -16; K884-15, -16; K1331-12, -13, -14, -15). The SNP survey by chip-based analysis examined seven unrelated individuals (K104-1, -16; K884-2, -15, -16; K1331-12, -13). STSs were amplified with their corresponding PCR primers as described (6), except that the forward primer was modified to include the M13 21 primer site (5 -TGTAAAACGACGGCCAGT-3 ) at its 5 -end. The resulting PCR products were subjected to dyeprimer sequencing (33), with products detected on an ABI377 or ABI373 fluorescence sequence detector. Possible sequence variations were detected by the ABI Sequence Navigator software package, which suggests potential heterozygotes by identifying nucleotide positions at which a secondary peak exceeds a selected threshold (50%). Such apparent variations were then visually inspected to compare the patterns seen among the several individuals. D. N. Cooper and M. Karwczak, Hum. Genet. 85, 55 (1990). M. Chee et al., Science 274, 610 (1996); M. J. Kozal et al., Nature Med. 2, 753 (1996). S. P. A. Fodor et al., Science 251, 767 (1991); A.-C. Pease et al., Proc. Natl. Acad. Sci. U.S.A. 91, 5022 (1994). The current generation of technology allows fabrication of 1.28 cm by 1.28 cm arrays of 320,000 distinct oligonucleotides, each residing in a feature of 20 m by 25 m and containing 107 copies of the probe. J. G. Hacia et al., Nature Genet. 14, 441 (1996). STSs were amplified with their corresponding PCR primers as described (6). PCR products intended for hybridization to the same chip (typically 100 to 200 STSs from a single individual) were pooled together for subsequent processing. About 1 to 2 g of the pooled PCR product was purified with Qiaquick purification kit (Qiagen), fragmented with deoxyribonuclease (DNase) I (Promega) and labeled with biotin with terminal deoxynucleotidyl transferase ( TdT, GibcoBRL Life Technology). The purification was performed according to the manufacturers instructions. The fragmentation was performed in a 40- l reaction with 0.2 unit of DNase I, 10 mM tris-acetate (pH 7.5), 10 mM magnesium acetate, and 50 mM potassium acetate at 37C for 15 min, after which the reaction was stopped by heat inactivation at 96oC for 15 min. The terminal transferase reaction was performed by adding 15 units of TdT and 12.5 M biotin-N6-ddATP (DuPont NEN) to the preceding reaction mixture, incubating it at 37C for 1 hour, and then heat-inactivating it at 96C for 15 min. The labeled samples were hybridized to the chip as follows. Samples were denatured at 96C for 5 to 6 min and cooled on ice for 2 to 5 min. Chips were first hybridized with 6 SSPET [0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA (pH 7.4), 0.005% Triton X-100] for 5 min and then hybridized with the denatured sample in hybridization buffer [3M tetramethylammonium chloride, 10 mM tris-HCl (pH 7.8), 1 mM EDTA, 0.01% Triton X-100, herring sperm DNA (100 g/ml), and 200 pM control oligomer] at 44C for 15 hours on a rotisserie at 40 rpm. Chips were washed three times with 1 SSPET, 10 times

5. 6. 7. 8.

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12. 13. 14.

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11.

17.

18.

19.

20. 21.

22.

23. 24.

25.

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with 6 SSPET at 22C, and stained at room temperature with staining solution [streptavidin R-phycoerythrin (2 g/ml) (Molecular Probes) and acetylated bovine serum albumin (0.5 mg/ml) in 6 SSPET] for 8 min. After they were stained, the chips were washed 10 times with 6 SSPET at 22C on a fluidics workstation (Affymetrix). Hybridization to the chip was detected by using a confocal chip scanner (HP/Affymetrix) with a resolution of 40 to 80 pixels per feature and a 560-nm filter. Candidate SNPs were identified by using a combination of four algorithms followed by a visual inspection. At each position, the VDA contains one expected probe (corresponding to the sequence from which the chip was designed) and three variant probes (containing a substitution in the central position). The first algorithm (base-calling) looked for positions at which, in some individuals, a variant probe gave a stronger signal than the expected probe. The second algorithm (clustering) considered the signal vector sij from the eight probes at position i (four base substitutions on both strands) in individual j and looked for positions i at which the vectors sij fell into multiple clusters. The third algorithm (mutant fraction) was similar but focused only on the expected probe and a single variant probe at a time (rather than all three variant probes). The fourth algorithm (footprint detection) looked for the loss of signal that occurs at the expected probes in the neighborhood of an SNP (13, 15). The algorithms have different sensitivities for detecting heterozygous and homozygous variations. As discussed in the text below, the proportion K of polymorphic sites is expected to be proportional to [11 21 31 . . . (n 1) 1], where n is the number of genomes sampled. The proportion of polymorphic sites is thus expected to increase by 39.3% when the number of genomes is increased from 6 (in the gel-based survey) to 14 (in the chipbased survey). This agrees well with the observed increase of 38.8%. A relatively small sample size suffices to capture much of the common variation. The sample size of 14 has a 50% chance of detecting an allele with a frequency of 5%. Doubling the proportion of variant sites identified would require increasing the number of genomes surveyed from 14 to 325, on the basis of the formula for K. The larger sample size will tend to identify polymorphisms with lower heterozygosity. STSs were resequenced on both strands with dyeprimer and dye-terminator chemistry. The chip-based approach has the further advantage that long STSs can be analyzed, whereas gel-based sequencing is limited to about 600 bp. It is thus possible to use fewer PCR products to analyze a region. The current study did not take advantage of this feature because we used short STSs already available from our previous work (6, 7). Confirmation was initially performed by multipass sequencing but is currently being done by using the clustering test on genotyping chips. M. A. Walter et al., Nature Genet. 7, 22 (1994). The lowest density occurs on chromosome X, which has the lowest density of STSs and which was screened in fewer total genomes in as much as the screening panel included three males. For each SNP, PCR primers were chosen with the PRIMER software package (6) to closely flank the polymorphic base and to have a predicted melting temperature of 57C. Forward and reverse primers were synthesized with the T 7 and T3 promoter sites (5 -TAATACGACTCACTATAGGGAGA-3 and 5 AAT TAACCCTCACTAAAGGGAGA-3 ) at their respective 5 -ends. Each PCR primer pair was individually tested to determine if it produced a single clear fragment visible by agarose gel electrophoresis and ethidium-bromide staining, as described (6). PCR assays passing this test were further classified as being strong or weak according to the yield of the fragment produced. Primer pairs were grouped into multiplex sets, with the sets chosen to consist of either strong assays or weak assays. Multiplex PCR was performed by using multiple PCR primer pairs in a single reaction. Specifically, multiplex PCR reactions were performed in a 50- l vol-

33. 34. 35.

5 February 1998; accepted 31 March 1998

RasGRP, a Ras Guanyl Nucleotide Releasing Protein with Calcium- and Diacylglycerol-Binding Motifs
Julius O. Ebinu, Drell A. Bottorff, Edmond Y. W. Chan, Stacey L. Stang, Robert J. Dunn, James C. Stone*
RasGRP, a guanyl nucleotidereleasing protein for the small guanosine triphosphatase Ras, was characterized. Besides the catalytic domain, RasGRP has an atypical pair of EF hands that bind calcium and a diacylglycerol (DAG)-binding domain. RasGRP activated Ras and caused transformation in fibroblasts. A DAG analog caused sustained activation of Ras-Erk signaling and changes in cell morphology. Signaling was associated with partitioning of RasGRP protein into the membrane fraction. Sustained ligandinduced signaling and membrane partitioning were absent when the DAG-binding domain was deleted. RasGRP is expressed in the nervous system, where it may couple changes in DAG and possibly calcium concentrations to Ras activation.

The

cellular properties of neurons are modulated by a number of extrinsic signals, including synaptic activity, neurotrophic factors, and hormones. These signaling systems alter the intracellular concentrations of second messengers such as calcium and cyclic nucleotides, and these small moleVOL. 280 15 MAY 1998

cules can regulate the activities of protein kinases (1). As the mechanisms linking Ras signaling to nerve function are not completely understood, we developed a cDNA cloning approach to identify proteins that enhance Ras signaling in the brain. From rat brain mRNA, we derived cDNAs that

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ume containing 100 ng of human genomic DNA, 0.1 to 0.2 M of each primer, 1 unit of AmpliTaq Gold (Perkin-Elmer), 1 mM deoxynucleotide triphosphates (dNTPs), 10 mM tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, and 0.001% gelatin. Thermocycling was performed on a Tetrad (MJ Research), with initial denaturation at 96C for 10 min followed by 30 cycles of denaturation at 96C for 30 s, primer annealing at 55C for 2 min, and primer extension at 65C for 2 min. After 30 cycles, a final extension reaction was carried out at 65C for 5 min. Because the resulting PCR products were small, it was unnecessary to fragment them (as was done for the STSs in the SNP screen). The PCR products were then labeled with biotin in a standard PCR reaction, by using T 7 and T3 primers with biotin labels at their 5 -ends. The reaction was performed with 1 l of template DNA, 0.1 to 0.2 M labeled primer, 1 unit of AmpliTaq Gold (Perkin-Elmer), 100 M dNTPs, 10 mM tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin. Thermocycling was performed with initial denaturation at 96C for 10 min followed by 25 cycles of denaturation at 96C for 30 s, primer annealing at 52C for 1 min, and primer extension at 72C for 1 min. After 25 cycles, a final extension reaction was carried out at 72C for 5 min. The PCR products from the various multiplex reactions for an individual were then pooled together. One-tenth of the pooled sample was denatured and used for chip hybridization. Chips were hybridized, washed, stained and scanned, as above (16). 27. D. G. Wang, unpublished observations. 28. A classification procedure for assigning genotypes was derived for each locus on the basis of the hybridization results observed in a test population of 39 individuals. The proportions of the two alleles present in the i-th sample, denoted A,i and B,i, 1) were estimated essentially by (with A,i B,i comparing the observed hybridization signal to the expected signals for the two VDAs. The values A,i for the 39 individuals lie in the interval [0,1] and should ideally cluster near 0, 0.5, and 1.0, but other patterns might occur because of differences in hybridization intensity between the two alleles. The values were optimally clustered (33) with the MOD-

29.

30.

31. 32.

ECLUS procedure of the SAS software package (SAS Institute). A maximum of three nonoverlapping clusters was permitted, defined by points with a minimum separation of 0.12. A locus failed the cluster test if all the samples fell into a single cluster, if the samples gave rise to two clusters but neither corresponded to the heterozygous genotype (AB), or if too many samples (more than 9 of 39) fell outside the three optimal clusters. A locus passing the cluster test gave rise to either three clusters (genotypes AA, AB, BB) or two clusters (genotypes AA, AB or BB, AB). Subsequent samples were genotyped according to the cluster in which the hybridization pattern fell, with no genotype being called for samples falling outside these predefined clusters. W.-H. Li, Molecular Evolution (Sinauer, Sunderland, MA, 1997); N. Takahata and Y. Satta, Proc. Natl. Acad. Sci. U.S.A. 94, 4811 (1997); M. Nei and D. Graur, in Evolutionary Biology, M. K. Hecht, B. Wallace, G. T. Prance, Eds. (Plenum, New York, 1984), vol. 17, pp. 73118. F. J. Ayala, Science 270, 1930 (1995). R. C. Lewontin, in Evolutionary Biology, T. H. Dobzhansky, M. K. Hecht, W. C. Steere, Eds. (Appleton-Century-Crofts, New York, 1972), vol. 6, pp. 381398. M. M. DeAngelis, D. G. Wang, T. L. Hawkins, Nucleic Acids Res. 23, 4742 (1995). W. L. G. Koontz and K. Fukunaga, IEEE Trans. Comp. C-21, 171 (1972). We thank D. Stern for construction of chip scanners used in the project, C. Chen-Cheng for computation work related to the polymorphisms among EST sequences in GenBank, T. Hawkins for sequencing of some STSs, and D. Lockhart for helpful comments on the manuscript. Supported in part by grants from Affymetrix, Millennium Pharmaceuticals and BristolMeyers-Squibb (to Whitehead Institute), from the National Human Genome Research Institute [to Whitehead Institute (HG00098) and Affymetrix (HG01323)] and from the National Institute of Standards and Technology [to Affymetrix (70NANB5H1031)].