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1.  META-GSA: Combining Findings from Gene-Set Analyses across Several Genome-Wide Association Studies 
PLoS ONE  2015;10(10):e0140179.
Gene-set analysis (GSA) methods are used as complementary approaches to genome-wide association studies (GWASs). The single marker association estimates of a predefined set of genes are either contrasted with those of all remaining genes or with a null non-associated background. To pool the p-values from several GSAs, it is important to take into account the concordance of the observed patterns resulting from single marker association point estimates across any given gene set. Here we propose an enhanced version of Fisher’s inverse χ2-method META-GSA, however weighting each study to account for imperfect correlation between association patterns.
Simulation and Power
We investigated the performance of META-GSA by simulating GWASs with 500 cases and 500 controls at 100 diallelic markers in 20 different scenarios, simulating different relative risks between 1 and 1.5 in gene sets of 10 genes. Wilcoxon’s rank sum test was applied as GSA for each study. We found that META-GSA has greater power to discover truly associated gene sets than simple pooling of the p-values, by e.g. 59% versus 37%, when the true relative risk for 5 of 10 genes was assume to be 1.5. Under the null hypothesis of no difference in the true association pattern between the gene set of interest and the set of remaining genes, the results of both approaches are almost uncorrelated. We recommend not relying on p-values alone when combining the results of independent GSAs.
We applied META-GSA to pool the results of four case-control GWASs of lung cancer risk (Central European Study and Toronto/Lunenfeld-Tanenbaum Research Institute Study; German Lung Cancer Study and MD Anderson Cancer Center Study), which had already been analyzed separately with four different GSA methods (EASE; SLAT, mSUMSTAT and GenGen). This application revealed the pathway GO0015291 “transmembrane transporter activity” as significantly enriched with associated genes (GSA-method: EASE, p = 0.0315 corrected for multiple testing). Similar results were found for GO0015464 “acetylcholine receptor activity” but only when not corrected for multiple testing (all GSA-methods applied; p≈0.02).
PMCID: PMC4621033  PMID: 26501144
2.  A Two-Dimensional Pooling Strategy for Rare Variant Detection on Next-Generation Sequencing Platforms 
PLoS ONE  2014;9(4):e93455.
We describe a method for pooling and sequencing DNA from a large number of individual samples while preserving information regarding sample identity. DNA from 576 individuals was arranged into four 12 row by 12 column matrices and then pooled by row and by column resulting in 96 total pools with 12 individuals in each pool. Pooling of DNA was carried out in a two-dimensional fashion, such that DNA from each individual is present in exactly one row pool and exactly one column pool. By considering the variants observed in the rows and columns of a matrix we are able to trace rare variants back to the specific individuals that carry them. The pooled DNA samples were enriched over a 250 kb region previously identified by GWAS to significantly predispose individuals to lung cancer. All 96 pools (12 row and 12 column pools from 4 matrices) were barcoded and sequenced on an Illumina HiSeq 2000 instrument with an average depth of coverage greater than 4,000×. Verification based on Ion PGM sequencing confirmed the presence of 91.4% of confidently classified SNVs assayed. In this way, each individual sample is sequenced in multiple pools providing more accurate variant calling than a single pool or a multiplexed approach. This provides a powerful method for rare variant detection in regions of interest at a reduced cost to the researcher.
PMCID: PMC3984111  PMID: 24728235
3.  Comparison of Pathway Analysis Approaches Using Lung Cancer GWAS Data Sets 
PLoS ONE  2012;7(2):e31816.
Pathway analysis has been proposed as a complement to single SNP analyses in GWAS. This study compared pathway analysis methods using two lung cancer GWAS data sets based on four studies: one a combined data set from Central Europe and Toronto (CETO); the other a combined data set from Germany and MD Anderson (GRMD). We searched the literature for pathway analysis methods that were widely used, representative of other methods, and had available software for performing analysis. We selected the programs EASE, which uses a modified Fishers Exact calculation to test for pathway associations, GenGen (a version of Gene Set Enrichment Analysis (GSEA)), which uses a Kolmogorov-Smirnov-like running sum statistic as the test statistic, and SLAT, which uses a p-value combination approach. We also included a modified version of the SUMSTAT method (mSUMSTAT), which tests for association by averaging χ2 statistics from genotype association tests. There were nearly 18000 genes available for analysis, following mapping of more than 300,000 SNPs from each data set. These were mapped to 421 GO level 4 gene sets for pathway analysis. Among the methods designed to be robust to biases related to gene size and pathway SNP correlation (GenGen, mSUMSTAT and SLAT), the mSUMSTAT approach identified the most significant pathways (8 in CETO and 1 in GRMD). This included a highly plausible association for the acetylcholine receptor activity pathway in both CETO (FDR≤0.001) and GRMD (FDR = 0.009), although two strong association signals at a single gene cluster (CHRNA3-CHRNA5-CHRNB4) drive this result, complicating its interpretation. Few other replicated associations were found using any of these methods. Difficulty in replicating associations hindered our comparison, but results suggest mSUMSTAT has advantages over the other approaches, and may be a useful pathway analysis tool to use alongside other methods such as the commonly used GSEA (GenGen) approach.
PMCID: PMC3283683  PMID: 22363742

Results 1-3 (3)