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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).
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2642662070
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note
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STSs with the largest sizes were used in the gel-based screen, and the remaining STSs, having somewhat smaller sizes, were used in the subsequent chip-based screen.
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18
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note
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The genomic sequence screened (279 kb) is the sum of the distances between the primer sites of the STSs successfully resequenced.
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19
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2642589604
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note
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The individuals surveyed were chosen from Centre d'Etude 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, K1331-1) 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).
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2642659992
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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 dye-primer 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.
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10244219858
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M. Chee et al., Science 274, 610 (1996); M. J. Kozal et al., Nature Med. 2, 753 (1996)
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M. Chee et al., Science 274, 610 (1996); M. J. Kozal et al., Nature Med. 2, 753 (1996)
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note
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4, 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 μg/ml), and 200 pM control oligomer] at 44°C for 15 hours on a rotisserie at 40 rpm. Chips were washed three times with 1x SSPET, 10 times with 6x SSPET at 22°C, and stained at room temperature with staining solution [streptavidin R-phycoerythrin (2 μg/ml) (Molecular Probes) and acetylated bovine serum albumin (0.5 mg/ml) in 6x SSPET] for 8 min. After they were stained, the chips were washed 10 times with 6x SSPET at 22°C on a fluidics workstation (Affymetrix). Hybridization to the chip was detected by using a confocal chip scanner (HP/Affymetrix) wilh a resolution of 40 to 80 pixels per feature and a 560-nm filter.
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ij 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.
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-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 chip-based survey). This agrees well with the observed increase of 38.8%.
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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.
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STSs were resequenced on both strands with dye-primer and dye-terminator chemistry.
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32
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2642597655
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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).
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33
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2642597656
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Confirmation was initially performed by multipass sequencing but is currently being done by using the clustering test on genotyping chips.
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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.
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2642664102
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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 57°C. Forward and reverse primers were synthesized with the T7 and T3 promoter sites (5′-TAATACGACTCACTATAGGGAGA-3′ and 5′-AATTAACCCTCACTAAAGGGAGA-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.
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2642697428
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2 and 0.001% gelatin. Thermocycling was performed with initial denaturation at 96°C for 10 min followed by 25 cycles of denaturation at 96°C for 30 s, primer annealing at 52°C for 1 min, and primer extension at 72°C for 1 min. After 25 cycles, a final extension reaction was carried out at 72°C 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).
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2642664101
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unpublished observations
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D. G. Wang, unpublished observations.
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Wang, D.G.1
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2642628297
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note
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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 MODECLUS 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).
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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.
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2642685162
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note
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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 Bristol-Meyers-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)].
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