| | 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. |
