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Method Detail
Submitter Method Handle: PERLEGEN
Submitter Method ID: AFD_CHIP_HYB
Submitted method description:
Long-range PCR assays were designed using Oligo primer design software
(Molecular Biology Insights). Primers were selected to have similar
stringency and to map uniquely to NCBI Build 34. From a collection of
all suitable candidate primers, we used custom software to select a
minimum spanning set having maximum coverage with minimal overlap
between adjacent amplicons.
Oligonucleotide arrays were designed so each SNP would be interrogated
by forty distinct 25 bp probes. These consisted of four sets of 10
features, corresponding to forward and reverse tilings of sequences
complementary to each SNP allele. A set of 10 features consisted of
five sets of two features where the location of the SNP within the
oligonucleotide varies from position 11 to position 15. For each
offset, we tiled one perfect-match feature and one feature having a
mismatch at the central position of the probe. Thus, for each allele,
we tiled ten perfect-match and ten mismatch probes. Mismatch probes
were used to measure background, and by comparison with the signal for
the perfect match probes, to detect the presence or absence of a
specific PCR product.
Light-directed chemical synthesis of oligonucleotide arrays was
carried out by Affymetrix, Inc. (Santa Clara, CA). Genomic DNA was
amplified by multiplex long-range PCR, then all PCR products destined
for a single genotyping array were pooled together and end-labeled
with biotinylated nucleotides. Each labeled DNA sample was hybridized
to a chip in hybridization buffer overnight at 50C. The hybridized
chips were washed several times in neutral buffer at RT, then
incubated with streptavidin for 15 min at RT. Following two washes at
35C in neutral buffer, chips were incubated with biotinylated
anti-streptavidin antibody for 15 min at RT, followed by another two
washes at 35C in neutral buffer. Then, chips were stained with
streptavidin-Cy-chrome conjugate for 15 min at RT, followed by two
washes with neutral buffer at 35C. Chips were incubated 30 min at 37C
in low-salt buffer, followed by a wash with neutral buffer at RT.
Hybridization of the sample to the array was detected using a confocal
laser scanner.
Individual genotypes for a SNP were determined by clustering
measurements from multiple scans in the two-dimensional space defined
by background-adjusted intensities of the perfect-match features for
the reference and alternate alleles. We used a K-means algorithm to
assign measurements to clusters representing distinct diploid
genotypes. We determined an optimal background value for each SNP
that minimized the variance within the assigned genotype clusters. The
K-means and background optimization steps were iterated until cluster
membership and background estimates converged. To determine the
appropriate number of genotype clusters, we repeated the analysis for
1, 2, and 3 clusters, and selected the most likely solution,
considering likelihoods of the data and the cluster parameters. The
data likelihood was determined using a normal mixture model for the
distribution of intensities around the cluster means. The model
likelihood was calculated using a prior distribution of expected
cluster positions.

This method was used in the following submission:

Submitter Handle Batch Type Submitter batch id Release build id
PERLEGEN Genotype 71_IND_CHR_1 123
PERLEGEN Genotype 71_IND_CHR_2 123
PERLEGEN Genotype 71_IND_CHR_3 123
PERLEGEN Genotype 71_IND_CHR_4 123
PERLEGEN Genotype 71_IND_CHR_5 123
PERLEGEN Genotype 71_IND_CHR_6 123
PERLEGEN Genotype 71_IND_CHR_7 123
PERLEGEN Genotype 71_IND_CHR_8 123
PERLEGEN Genotype 71_IND_CHR_9 123
PERLEGEN Genotype 71_IND_CHR_10 123
PERLEGEN Genotype 71_IND_CHR_11 123
PERLEGEN Genotype 71_IND_CHR_12 123
PERLEGEN Genotype 71_IND_CHR_13 123
PERLEGEN Genotype 71_IND_CHR_14 123
PERLEGEN Genotype 71_IND_CHR_15 123
PERLEGEN Genotype 71_IND_CHR_16 123
PERLEGEN Genotype 71_IND_CHR_17 123
PERLEGEN Genotype 71_IND_CHR_18 123
PERLEGEN Genotype 71_IND_CHR_19 123
PERLEGEN Genotype 71_IND_CHR_20 123
PERLEGEN Genotype 71_IND_CHR_21 123
PERLEGEN Genotype 71_IND_CHR_22 123
PERLEGEN Genotype 71_IND_CHR_X 123
PERLEGEN Genotype 71_IND_CHR_Y 123
PERLEGEN Genotype 71_IND_CHR_U 123
PERLEGEN Assay AFD_AFR_18-MAY-2004 123
PERLEGEN Assay AFD_CHN_18-MAY-2004 123
PERLEGEN Assay AFD_EUR_18-MAY-2004 123

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