Computational protocol: Identification of Genes with Allelic Imbalance on 6p Associated with Nasopharyngeal Carcinoma in Southern Chinese

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Protocol publication

[…] The SNPs in this study were selected based on the candidate gene in combination with PLINK and Haploview tag SNP selection algorithms. We used DDD study subjects as controls; the DDD study already has the whole genome scan data with 17,313 SNPs genotyped on chromosome 6. For quality control, the genotype missingness frequency (GENO) was set to GENO <0.05 and the minor allele frequency (MAF) to MAF >0.05 when we selected SNPs from the DDD study. After frequency and genotype pruning, 11,772 SNPs were left. Focusing solely on the genes located in the candidate region identified by the meta-analysis of the top candidate genes (PLINK filter from chromosome 6: 6–31 Mb), 2,730 SNPs remained, and among these only tag SNPs were selected using the HapMap tag SNP picking tool Tagger. The R-square cut-off value was set to 0.8. Only 233 tag SNPs were ultimately selected for genotyping. [...] The association analyses in this study were performed by PLINK (available online at: http://pngu.mgh.harvard.edu/purcell/plink/) . Single SNP association analyses on allelic and genotypic associations include determining the p value, odds ratio (OR), and 95% confidence interval (CI). The Hardy-Weinberg equilibrium (HWE) and MAF were estimated by PLINK; for all tested SNPs, the HWE p-value was >0.001 and MAF was >0.01. Logistic regression analyses introduced age and gender as covariates. Linkage disequilibrium (LD) analysis was conducted using both PLINK and Haploview 4.2 . The panel of SNPs was entered into the Haploview software, and the individual haplotype frequency was estimated by the expectation-maximization (EM) algorithm. The haplotype structure was also analyzed by PLINK using the three-SNP sliding window option. Multiple testing was performed with 10,000 permutations and/or by Bonferroni correction. LocusZoom was used to generate the association plot (http://csg.sph.umich.edu/locuszoom/).The detection of runs of homozygosity (ROH) analysis was done by PLINK. The algorithm involves taking a window of SNPs and sliding it across the region. The homozygosity is determined if the window looks “homozygous” enough at each window position. For each SNP, the proportion of “homozygous” windows that overlaps that position is calculated. The default window size and homozygous segment criteria were set to the following: 1,000 kb length, 100 SNPs, 50 kb/SNP density, and 1,000 kb for the largest gap .The SPSS software standard version 13.0 (SPSS Inc., Chicago, IL) was used to perform the non-parametric t-tests, χ2 tests, and logistic regression tests to analyze the real-time PCR results quantitatively. […]

Pipeline specifications

Software tools PLINK, Haploview, LocusZoom
Application GWAS
Diseases Carcinoma, Neoplasms, Genetic Diseases, Inborn
Chemicals gamma-Aminobutyric Acid